The ash compound found on the heavily corroded steel in biomass combustion and gasification plants is KCl, but the mechanism, which triggers the breakdown of the protective Cr2O3 layer under the KCl salt is not known. We studied the chemical reactions involved with furnace exposure of KCl and KOH with Cr2O3 and identified the formed reaction products with XRD analysis. The amount of reaction products was analyzed from the leachates of the salt-oxide mixtures by UV/VIS spectroscopy. We also used thermodynamic Gibbs energy minimization calculations to evaluate the evolution of reactions as a function of temperature. The results suggests that the reaction of KCl with Cr2O3 involves a KOH reaction intermediate that forms before K2CrO4 is formed. The amount of reacted potassium as a function of temperature follows the trend of KCl decomposition to KOH and HCl(g) as predicted by thermodynamics calculations. Therefore, we argue that the suggested overall reaction of KCl with Cr2O3 as found in the corrosion literature: , starting with the initiation step: KCl + H2O(g) ⇒ KOH + HCl(g) and then the formed KOH reacts with Cr2O3 to form K2CrO4. This explains the initial breakdown of the protective Cr2O3 under KCl salt in water containing high temperature atmospheres. The result is essential for the development of new alloys for biomass fired combustors.Biomass is considered a replacement fuel over fossil fuels to mitigate climate change. The switch to biomass in the combustors changes the inorganic chemistry of the flue gases and leads to more severe corrosion of the construction materials of the combustors. The integrity of most high temperature steels relies on the formation of a protective Cr 2O3 on high temperature steels under KCl containing deposits in biomass combustion.New chemical mechanism explaining the breakdown of protective Cr The largest use of coal as a fuel is in electricity and heat production, which accounts for about 16% of the total CO2 emissions.1 Replacing coal with sustainable biomass as the fuel in power boilers would lead to substantial reductions in the net CO2 emission. Also, SO2 emissions would be diminished due to the generally much lower sulphur content of biomass based fuels compared to coal. However, biomass combustion leads to more severe fouling and corrosion issues of the heat transfer surfaces in boilers.2–5 One way to minimize these problems is to operate the boilers with lower steam temperatures, but that lowers the efficiency of the Rankine cycle leading to higher emission factors per unit of electricity produced.Burning coal is responsible for approximately 25% of anthropogenic CO6 Potassium is found associated in the fuel moisture as water soluble salts, or reacted with the organic functional groups  of the lignocellulosic matrix.7 During combustion, potassium is released in the gas phase as elemental potassium, potassium chloride or potassium hydroxide as measured by mass spectrometric or optical methods.8–10 Due to its extreme reactivity, elemental potassium is expected to react to compounds in the vicinity of the biomass particle it is released from. Therefore, the major gas phase potassium compounds in the biomass boiler flue gases are KCl(g) and KOH(g). These compounds may react further with other fuel elements, such as silicon or sulphur to form potassium silicates or potassium sulphate. K2O–SiO2 silicate formation plays a major role in deposit formation in the furnace (slagging) and in bed agglomeration issues in fluidized bed boilers, where silica containing sand is often used as the bed material.11–13 Formation of K2SO4 plays a major role in aerosol and deposit formation further downstream of the boiler.14,15 K2SO4 formation may decrease the corrosion rate of the heat exchangers forming a less corrosive deposit than KCl or KOH/K2CO3. Its formation may however, also increase fouling rate of the heat exchangers forming tenuous deposits that are difficult to be removed by soot blowing, even though the corrosion rate under the deposits may still be low.16,17 During their path through the boiler, KCl(g) and KOH(g) in the flue gases condense out when the gas temperature is decreased below their dew points. Condensation can happen directly on the heat exchanger surfaces or on the ash particles present in the flue gas flow. Purely homogeneous nucleation is also possible in conditions where the flue gas does not contain enough foreign surfaces that can act as nucleation sites for heterogeneous nucleation.18,19 After condensation KCl can react heterogeneously further to K2SO4 and KOH can react to K2SO4, KCl or K2CO3 as predicted by thermodynamic stabilities of the compounds.20,21Potassium behaviour in the boiler has been identified as the major cause of slagging, fouling and corrosion in biomass fired boilers.22–28 Recently, the effect of K2CO3 on the high temperature corrosion has gained more interest. It appears both potassium and chlorine are important in the corrosion reactions with steels. It has been suggested that potassium can initiate the destruction of the protective oxide, but chlorine is needed to sustain the corrosion.29,30 KOH induced corrosion has not been extensively studied in the context of high temperature corrosion in biomass fired boilers, but earlier studies have shown that Fe–Cr alloys are unsuitable for service in KOH containing high temperature environments.31–33 Earlier work of the author on the elemental balances of the deposit forming elements in biomass based fuels suggest that KOH(g) condensation may be more important in fouling and corrosion than has previously been thought. The details of K2CO3 formation on the heat exchanger surfaces has not been clarified yet, but its formation has been predicted by thermodynamics when the (Cl(g) + 2S(g)) molar content in the flue gases is lower than the molar K(g) content. K2CO3 is formed on the heat exchanger surfaces likely via a surface reaction of adsorbed KOH(ads.) with CO2(g). Homogeneous formation of K2CO3(g) in the gas phase, followed by condensation of K2CO3(g) is less likely, because of the thermodynamic instability of K2CO3(g).34,35 K2CO3 has also been directly detected in some boiler deposits.17 In this work we studied in detail the reactivity of KCl and KOH towards Cr2O3 and Fe2O3, the protective oxide components formed on the Fe–Cr alloys in high temperature oxidizing service conditions. The results may also be of interest for chromite ore roasting by KOH and for understanding corrosion of Cr2O3 containing refractory bricks in potassium and chlorine containing environments.36–38KCl induced corrosion of Fe–Cr steels has been studied extensively in the scientific literature.2.2.12O3, KCl–Cr2O3, KOH–Fe2O3, KCl–Fe2O3 and K2CrO4–Cr2O3 mixtures were prepared by mixing known amounts of powders in a 10 ml glass bottle and manually shaking the bottles for approximately 30 s. KOH was from Sigma Aldrich technical grade ≥ 85%, KCl from Alfa Aesar, ACS grade 99–100.5%, K2CrO4 from Merck, EMSURE®, ACS grade ≥ 99.5%, Cr2O3 from E. Merck, grade unknown and Fe2O3 from Johnson Matthey Chemicals, Specpure® grade. All powders were weighted with Sartorius CPA225D analytical balance. Larger agglomerates of KCl, KOH and K2CrO4 were grinded manually in a mortar before mixing with the Cr2O3 or Fe2O3 powder. Cr2O3 and Fe2O3 powders seemed visually homogeneous without agglomerates and were used without any pre-grinding. KCl/Cr2O3, KCl/Fe2O3 or KOH/Cr2O3, KOH/Fe2O3 molar ratios were 1 and K2CrO4/Cr2O3 molar ratio was 0.5 in order to have the same K/metal molar ratio in all of the mixtures. A 10 g batch of each mixture was prepared in one go in a screw cap sealed bottle. From the 10.0 g batch bottles, 1.00 g samples were weighted to a 10 ml sintered Al2O3 crucible and then loaded immediately in a muffle furnace (Nabertherm P330) that was at the isothermal exposure temperature (100–800 °C). The samples were exposed in the furnace for 2 hours in ambient air atmosphere. After furnace exposures, the samples were cooled in ambient air so that handling of the crucible was possible with nitrile gloves (5–10 min) and then transferred to glass bottles that were sealed with screw caps. Then the samples were placed in a desiccator cabin for storage. During the analyses, the sample exposure times to ambient air before starting the analyses were minimized by opening the cap and preparing the sample from the bottle only just before starting the analysis. However, the XRD analysis took approximately an hour per sample, therefore possible reactions during the analysis with ambient air could not be completely eliminated. KOH especially is known to be highly hygroscopic and reactive towards CO2 during exposure to ambient air.KOH–Cr2.242− ion started to appear immediately after adding the sample powder and the intensity of the colour increased with time. The sample bottles were then photographed to record the characteristic yellow colour of CrO42− and the orange colour of Cr2O72−.20 ml glass bottles were filled almost full with ion exchanged water. Then 0.10 g of each sample was added to the already water filled bottle. The bottles were not agitated by any means in order to let the sample powder sediment to the bottom of the bottle by gravity. The samples were left to stand overnight. The characteristic yellow colour of the CrO2.33 (yellow to orange) or K2FeO4 (FeO42− ion is purple) in the samples in concentrations high enough to be visible to the naked eye.20 ml glass bottles were filled almost full with ion exchanged water. Then 0.10 g of each sample was added to the already water filled bottle. The bottles were not agitated by any means in order to let the sample powder sediment to the bottom of the bottle by gravity. The samples were let to stand still overnight. All the solutions were colourless indicating no signs of water soluble FeCl2.4α = 1.5406 Å). Samples were first grinded manually in a mortar to make a visually homogeneous powder. Then the sample holder was filled evenly with the powder, pressed against a flat surface to level the sample surface with the sample holder top surface and then XRD θ–2θ scans were recorded from 10–90°. The automatic phase identification algorithm of the X'Pert HighScore Plus program was used for preliminary identification of the phases. Then the results were checked manually and the most likely crystalline phases where manually identified using the JCPDS cards. The XRD cards used for phase identifications were 00-021-0645 for KOH, 00-011-0655 for K2CO3·1.5H2O, 00-015-0365 for K2CrO4, 04-007-3113 for KCl, 01-078-5435 for Cr2O3, 04-012-4476 for K2Cr2O7, 01-085-0599 for Fe2O3, 00-039-0892 for KFeO2, 00-039-1106 for K2Fe4O7 and 01-078-6089 for K1.75Fe1.25O4.XRD analysis was done with PANalytical X'pert Pro PW 3040/60 powder diffraction spectrometer with monochromated Cu K-alpha X-ray source . All the KCl based samples had pH = 7 and KOH based samples had pH = 7 in samples exposed at ≥ 500 °C and pH 8–12 in samples exposed at ≤400 °C. The higher pH with low temperature samples in case of KOH mixtures was caused by the KOH that was not reacted to K2CrO4, but formed K2CO3·1.5H2O during the furnace/ambient exposures. When dissolving in water, K2CO3·1.5H2O results in a basic solution. Concentration standards were prepared by dissolving known amounts of K2CrO4 powder to ion exchanged water in 50 ml volumetric flasks , the residue washed two times with 10 ml of room temperature ion exchanged water. Then the filtrates were transferred to 50 ml volumetric flasks and the flask was filled to the mark with ion exchanged water and mixed. The CrOlasks ESI. Then th2CrO4 determination was done by running a few duplicate runs with KOH + Cr2O3 mixtures (2 at 300 and 400 °C exposure temperatures). Because of the small number of duplicate samples, error was estimated with the range rather than with statistical methods. The error range (max–min) of the method was found to be about 0.08 (or 8%-points). This error value was assumed to be similar at other exposure temperatures and also for the leaching tests described below for Fe3+.Error estimation of the above described method for K2.62.6.13+ concentrations of the filtrates were determined by UV/VIS spectroscopy using Shimadzu UV-2600 spectrophotometer. Absorbance at 225 nm was used for Fe3+. Fe3+ ion in a water solution is present as Fe3+, Fe(OH)2+ and Fe(OH)2+ ions or as non-water soluble Fe(OH)3 precipitate, depending on the pH of the solution.40 The pH values of the filtrates were determined with a pH indicator sticks (Fisher Scientific number 10642751). All the KCl based samples had pH = 7 and all the KOH based samples had pH = 12. The high pH value over the entire exposure temperature in case of KOH based samples indicate that they may have contained some unreacted KOH/K2CO3 or then the higher pH at firing temperatures ≥ 500 °C  was originated from the dissolution of KFeO2 (KFeO2 + 2H2O = K+ + Fe3+ + 4OH−). In order to shift the equilibrium so that all the dissolved Fe(iii) was in the Fe3+ state, the filtrates were buffered to pH = 1 by mixing 1 : 1 ratio of sample with a solution to 1 mol l−1 HCl (1 : 2 dilution). This pH stabilized solution was then used to fill the quartz cuvette in the UV/VIS absorption measurements. The added Cl-ion can also form complex ions with Fe in the form Fe(H2O)x6−(Cl)xx3−. Concentration standards were prepared by dissolving known amounts of FeCl3·6H2O powder to ion exchanged water and diluting , the residue washed two times with 10 ml of room temperature ion exchanged water. Then the filtrates were transferred to 50 ml volumetric flasks and the flask was filled to the mark with ion exchanged water and mixed. The Feuting ESI. The pH 2.6.22O soluble Fe3+ determination, except that the 15 min leaching steps were done in 20 ml of 1 mol l−1 HCl in water solutions instead of pure water. Also the washing of the filtration residue was done with HCl containing water  instead of pure water. This prevented the precipitation of Fe(OH)3 that was possible in the water leaching steps, where the pH values of the filtrates were ≥7. In case of 1 M HCl leaching, some filtrates had a yellow colour visible already to a naked eye, indicating that indeed the acid leaching resulted in much higher dissolution of the reaction products compared to H2O leaching. All the filtrates had a pH value of 1, measured with pH indicator sticks (Fisher Scientific number 10642751). Because the pH of the filtrates were stabilized to one already during the leaching step, they were used directly to fill the quartz cuvette in the UV/VIS absorption measurements. In cases where the sample had Fe3+ concentration so high that the absorbance was higher than the standards, the sample was diluted in volumetric flasks with 1 : 2, 1 : 50 or 1 : 100 dilution ratios, which ever was suitable to bring the absorbance value in between the standards. In the 1 : 50 and 1 : 100 dilution cases, the dilution was done by adding 1 ml of sample, then 5 ml of 1 mol l−1 HCl, and then filling the volumetric flask with pure H2O.The analysis was done in a similar way as has been discussed for the H2.741 The input files for the calculations are presented in 42 with ≈0.99 mol% H2O vapor (RH ≈ 32% at 25 °C) was used to simulate the humid ambient gas phase in the muffle furnace. In case of KCl based systems, HCl(g) was added as the possible gas phase Cl-compound released in the reactions. Note that each gas phase component in the atmosphere has at least several times higher absolute amounts than any of the solid phase components. This assures that the amount of the formed products was never limited by the amount of the gas phase component, thus simulating an open ambient system. The calculated systems were kept as simple as possible. Therefore, in addition to the reactants, only the phases identified in the XRD analysis were added to the solid phase input file. In the case of Fe2O3 systems, K2Fe4O7 was detected by XRD in the sample with the highest exposure temperature with KOH, but unfortunately it was not found in the HSC v6.12 database and could not be included in the thermodynamic calculation.HSC v6.12 Gibbs energy minimization software was used in the thermodynamic calculations.3.3.12CO3·1.5 H2O in samples exposed to furnace temperatures below 500 °C. Samples exposed to 500 °C or higher firing temperatures did not contain any residual K2CO3·1.5 H2O or KOH. This was also reflected by the pH of the water soluble filtrates as explained in the experimental section. It seems that the reaction of KOH with Cr2O3, forming K2CrO4 is not fast enough below 500 °C and competes with K2CO3·1.5 H2O formation in the experimental conditions used. According to XRD analysis, K2CrO4 formation starts already with a solid–solid reaction at 200 °C. Melting point of KOH is 406 °C, so it appears that there is no need for molten phase to form in the system before K2CrO4 formation proceeds. The photograph in 42− ion already appearing in the water soluble fraction of the products at room temperature exposures. However, this CrO42− may form from the dissolution of Cr2O3 in the basic water solution and therefore it is considered that the XRD analysis provides a better estimation of the onset temperature where K2CrO4 formation starts. The characteristic orange colour of the Cr2O72− ion was detected in samples fired at 700 °C and 800 °C. K2Cr2O7 was also detected in the XRD analysis at these firing temperatures. Thermodynamic calculations predict K2Cr2O7 to be the most stable reaction product throughout the temperature range used as shown in 2CrO4 formation is kinetically preferred at the studied temperature range and exposure time used. Like explained above, K2CrO4 formation itself seemed also to be kinetically controlled at temperatures lower than 500 °C. The maximum CrO42− amount  in the reaction products was measured at 500 °C furnace exposure, after which the amount decreased slightly at higher temperatures. Taking into account the experimental errors associated with the CrO42− analysis, it is suggested that starting at 500 °C, practically all the KOH had reacted to K2CrO4 and that the slightly lower amounts of CrO42− detected at higher exposure temperatures can be explained by slight loss of KOH by evaporation, competing with the reaction with Cr2O3.The powder XRD analysis of the system after furnace exposures is shown in 3.22Cr2O7 formation at higher firing temperatures proceeds directly from the reaction with KOH or via first formation of K2CrO4 and then further reaction with Cr2O3, the K2CrO4–Cr2O3 system was studied with XRD analysis. Results are presented in 2CrO4 can react with Cr2O3 and form K2Cr2O7 at exposure temperatures ≥ 500 °C. Therefore, it is suggested that direct reaction of KOH with Cr2O3 forms K2CrO4 and that the formation of K2Cr2O7 requires always the K2CrO4 reaction intermediate to form first.In order to determine if the K3.32O3. As shown in the XRD analysis in 2O3 until the highest firing temperature, 800 °C. At 800 °C, an additional peak was detected at 26.95° (2θ). This peak coincides with the (021) reflection of K2Cr2O7. However, using only one peak in phase identification is not reliable, but based on the analogy with results from the KOH–Cr2O3 and K2CrO4–Cr2O3 systems, K2Cr2O7 was considered to be the most likely reaction product at 800 °C. CrO42− was detected qualitatively and quantitatively also at lower firing temperatures as shown in 2Cr2O7 and K2CrO4 as the major products, but now the discrepancy between the thermodynamic prediction and experimental results was higher than with KOH. CrO42− was detected only in samples exposed to firing temperatures ≥ 400 °C and even at 800 °C the CrO42− amount was only ≈16% of the theoretical maximum.Compared to KOH, KCl is substantially less reactive with Cr3.42O3 system. Reaction with Fe2O3 started at 200 °C firing temperature, forming KFeO2 as the reaction product. KFeO2 stayed as the major reaction product throughout the temperature range. At 500 °C an additional peak at 27.95° (2θ) was detected that could not be assigned to KFeO2. This peak was assigned to potassium containing magnetite, K1.75Fe1.25O4 that has the maximum intensity powder XRD peak at this position, but with one peak only, the identification remains doubtful. At 800 °C firing temperature, K2Fe4O7 could also be identified as the reaction product in addition to KFeO2. There was no indication of higher oxidation state than +3 reaction products of Fe, such as K2FeO4. This was consistent with the lack of any colour of the water solutions containing the dissolved reaction products shown in 3+ amounts with the thermodynamic prediction are presented in 3+ was found, but the amount of acid soluble Fe3+ was very close to the thermodynamically predicted amount of KFeO2. This difference in the filtrates as a function of pH is caused by the fact that either KFeO2 is not soluble in water, or that after initial dissolution of KFeO2, rapid formation of insoluble Fe(OH)3 takes place in the basic solution. The high OH− concentration precipitates the initially dissolved iron and captures it in the filtration residue. In acidic conditions, Fe(OH)3 formation is prevented and KFeO2 was dissolved completely in the 15 min leaching step and the dissolved iron stayed in the solution phase during the filtration step.3.52O3 system, KCl turned out to be much less reactive towards Fe2O3 compared to KOH. There was no sign of any reaction in the XRD analysis or in the visual qualitative analysis of the water soluble reaction products as shown in 2O3 case, no water soluble iron was found, but now there was only traces of Fe3+ detected even in the acid soluble filtrate. The results agree with thermodynamic equilibrium calculations, which predicted only slight reaction in the KCl–Fe2O3 system at temperatures > 600 °C to form KFeO2 as shown in As with the KCl–Cr4.2O3 is initiated by K2CrO4 formation as found in the corrosion literature.22,23,43 However, we argue that the suggested overall reaction of KCl with Cr2O3:2(g) or HCl(g) as the corrosive substances and the effect of KOH has been neglected.44,45 However, combustion environments contain inevitably also H2O(g) in the flue gases and therefore KOH formation should not be neglected. Furthermore, many virgin biomass fuels contain very little Cl, so there is simply not much Cl2(g) or HCl(g) available in the flue gases when combusting these types of fuels. Still KCl layer has been found on the heat exchanger surfaces and previous work has argued that the corroding Cl2(g) or HCl(g) must form from the KCl layer, but the fate of the K when the Cl2(g) or HCl(g) forms from KCl has not been studied in detail. In waste incineration, the situation is different, because the feedstock contains typically large amounts of Cl and therefore the direct gas phase attack by Cl2(g) or HCl(g) may be more relevant. It may be that the chlorine corrosion mechanisms taking place in high chlorine content feedstock combustion cannot be applied to explain the corrosion in virgin biomass combustion.Our results are consistent with the suggestion that the reaction of KCl with Cr4.12O3, reacting already at 200 °C:KOH will react readily with Cr2CrO4 can react further to K2Cr2O7 at high temperatures. Direct formation of K2Cr2O7 without the K2CrO4 intermediate is always kinetically hindered:The formed K4.22O3, reacting already at 200 °C:KOH will react readily with Fe2O3 case, the formed KFeO2 can react further to K2Fe4O7 at high temperatures. It is suggested that K2Fe4O7 formation without the KFeO2 intermediate is kinetically hindered as the K2Cr2O7 formation in the Cr2O3 case:Although not experimentally verified in this study, we propose that in analogy to KOH–Cr4.32O(g) containing environment can simply be explained by the thermodynamics of the decomposition reaction 2O3, but did detect K2CrO4 formation with Cr2O3 at >500 °C can be explained by the higher thermodynamic stability of K2CrO4 compared to KFeO2 as shown in 2CrO4 is much more stable than KFeO2, therefore it is suggested that the KOH formed in the decomposition of KCl at >500 °C does not form KFeO2 as readily as K2CrO4 and most of the formed KOH is lost in the gas phase by vaporization before reacting with Fe2O3. That is why KFeO2 was not detected in the KCl–Fe2O3 system.Based on the results in this study, it is proposed that the reactivity of KCl towards both oxides in HIn case of Fe–Cr steel corrosion, once the protective oxide is destroyed by KOH and if the bare metals are exposed to the atmosphere, HCl(g) may take part in the continuation of corrosion:If KCl diffuses through the oxide scale (grain boundary diffusion), the formation of the reactive chlorine species can also take place at the metal-scale interface:46 However, it is somewhat arbitrary to speculate about the corrosion reactions with elemental metals, because these reactive metals in the elemental form will thermodynamically favour reaction with practically any reactant. For example, a chlorine free corrosion mechanism involving only KOH may also be suggested:The formed chlorine compounds can further attack the metal leading to the active oxidation mechanism by chlorine.2(g) diffusing through the oxide scale can further oxidize the underlying metal and form Cr2O3 or Fe2O3 oxides, which are then consumed by KOH according to reactions + and O2− ions diffusing to the metal-scale interphase:The formed water or the OThe schematic image in 5.2O3 and Fe2O3 than KCl in ambient air environment. Thermodynamic calculations predict the difference in reactivity quite nicely above 400 °C. KOH reacts with both oxides at temperatures higher than 200 °C while KCl reacts with Cr2O3 at temperatures > 400 °C, but no reaction with Fe2O3 was detected in the temperature range from room temperature to 800 °C. Both KOH and KCl form K2CrO4 as the reaction product when reacting with Cr2O3. K2CrO4 can further react with Cr2O3 and form K2Cr2O7 at temperatures > 400 °C. K2CrO4 and K2Cr2O7 reaction products can be easily leached from the mixture with water at room temperature. KOH forms KFeO2 as the reaction product when reacting with Fe2O3. KFeO2 cannot be leached with pure water, but requires an acidic media. In 1 M HCl solution, KFeO2 can be easily leached at room temperature. The reactivity of KCl towards the protective oxides of Fe–Cr steels in water containing high temperature environments can be explained by its decomposition to KOH and HCl(g) and the subsequent reaction of the formed KOH with the protective oxide. These results are very valuable for the development of materials for boilers, gasifiers, furnaces and gas turbines utilizing biomass derived fuels as their energy source.KOH is much more reactive towards CrThere are no conflicts of interest to declare.RA-009-C9RA00582J-s001


G protein‐coupled receptors (GPCRs) participate in most physiological processes and are important drug targets in many therapeutic areas. Recently, many GPCR X‐ray structures became available, facilitating detailed studies of their sequence‐structure‐mobility‐function relations. We show that the functional role of many conserved GPCR sequence motifs is to create weak spots in the transmembrane helices that provide the structural plasticity necessary for ligand binding and signaling. Different receptor families use different conserved sequence motifs to obtain similar helix irregularities that allow for the same motions upon GPCR activation. These conserved motions come together to facilitate the timely release of the conserved sodium ion to the cytosol. Most GPCR crystal structures could be determined only after stabilization of the transmembrane helices by mutations that remove weak spots. These mutations often lead to diminished binding of agonists, but not antagonists, which logically agrees with the fact that large helix rearrangements occur only upon agonist binding. Upon activation, six of the seven TM helices in GPCRs undergo helix motions and/or deformations facilitated by weak spots in these helices. The location of these weak spots is much more conserved than the sequence motifs that cause them. Knowledge about these weak spots helps understand the activation process of GPCRs and thus helps design medicines. GPCRs are—certainly in vitro—rather unstable molecules, which makes it hard to crystallize them. GPCR structures have been solved after stabilization by, for example, protein fusions, the use of exotic cocktails of lipids and metal ions, or by binding ligands, nanobodies, antibodies, or other peptides, normally combined with several mutations.www.gpcrdb.orgThe X‐ray structure determination of the first GPCR  is not involved in helix weakening.Most GPCR helices contain Pro, Gly, Asp, Asn, Ser, and Thr (PGDNST) residues that one would not expect abundantly present in a regular α‐helix. Proline weakens the helix because the backbone nitrogen atom does not have a proton so that it always lacks one hydrogen bond. Glycine has a very flexible backbone that can facilitate local deformations by accepting φ,ψ torsion angles that are energetically unfavorable for the other residue types. Asparagine, aspartic acid, serine, and threonine have small side chains that do not lose much rotameric entropy when a hydrogen bond is formed with the local backbone. When such a hydrogen bond is formed, the backbone and side chain exert a force on each other that leads to backbone atom displacements away from the ideal positions observed in regular α‐helices. PGDNST, especially proline, have often been associated with functionally important kinks in GPCR helicesOur finding that different GPCRs use different combinations of residues to allow for the same molecular rearrangements upon activation can only be understood in the context of the four‐state model depicted in Figure 3.2The GPCRdb mutation collection5x43A mutation in the 5‐HT1A receptor5x44A mutation in the 5‐HT2A receptor also results in a decrease in affinity for serotonin and other agonists, while, again, binding to antagonists is not affected. The activity of this mutant does not decrease significantly.5x50A mutation in the M3 receptor2‐adrenoceptor has been mutated to threonine,7x41A mutation in the M1 receptorThe S4x53S in several receptors , and C7x45A and C7x45N in the 5‐HT2C receptor. The C7x41S mutation in the M1 receptor results in a lower affinity for its agonist carbachol and a higher affinity for its antagonist atropine,6x47A mutation in the α1B‐adrenoceptor results in an increased agonist affinity,4x59A mutations in the M134x59A is so large that the affinity for all ligands is diminished.The GPCRdb lists an absence of significant effects on ligand binding for the mutations X3.3Crystallographers want a homogeneous GPCR sample for crystallization attempts. They want all GPCRs in their crystallization experiment to be in the same state, and thus must stabilize that one particular state relative to all other states. To obtain a homogeneous sample they often introduce mutations that reduce potential helix mobility. These mutations tend to stabilize the R and Rest states and thus are expected to reduce the binding affinity of agonists (as agonists bind to a state near SE). Antagonist binding is not associated with the same small (activating) motions and therefore stabilizing mutations—that reduce these motions—are not expected to influence antagonist binding. Many combinations of stabilizing mutations can be observed in PDB files for GPCR structures and many mutations can be observed in more than one receptor. Table 1B and 5‐HT2B receptor structures always have the mutation L3x41W or M3x41W, respectively. This X3x41W mutation is also found in four out of 20 β2‐adrenoceptor structures, one of the 18 β1‐adrenoceptor structures, all of the CXCR4 structures and the D3 receptor structure. This mutation was first used in a β2‐adrenoceptor structure. The rationale for the introduction of this mutation was that tryptophan was highly conserved at 3x41 in rhodopsin where it contacts both TM4 and TM5, and thus influences the dynamics of the receptor profoundly. The β2‐adrenoceptor is in vitro much less stable than rhodopsin and this tryptophan residue has been suggested to be one of the reasons.6x36P mutation. This mutation ensures that the helix ends at this position, thus inhibiting any length change of the helix in the activation process. A T3x36A mutation is often introduced in the A2A receptor to ease crystallization. This threonine forms a hydrogen bond with the local backbone that destabilizes the helix. Mutating it is therefore likely to stabilize the receptor. Together with A2x52L, R3x55A, K4x43A, L5x63A, L6x37A, V6x41A, and S7x41A, this mutation gives the receptor a higher affinity for the inverse agonists ZM241385, XAC, and for caffeine.2x46A, Q3x37A, A2x52L, and T2x62A is said to be in an intermediate state between active and inactive.1x59S, M2x53V, Y5x58A, A6x27L, F7x37A, and F7x48M stabilize the structure of the β1‐adrenoceptor with a bound antagonist.4x60N improves antagonist binding, the A6x33D stabilizes the environment of the DRY motif in TM3, and the C1x60Y stabilizes the TM7 ‐ helix 8 corner through several favorable interactions. Neither the structure, nor the associated article8x49E stabilizes the receptor. We lack information to draw strong conclusions, but it might well be that the three mutations, leaving out K8x49E, would have worked even better than the combination of four mutations used. L2x40A, F3x34A, G3x49A and Y5x58F increase thermostability and expression of the FFA1 receptor.E. coli.1x54L, E3x49A, L6x37A, F7x42A, and V7x44A and a glycine to alanine mutation in ECL2) that disabled G protein activation.3x49A, L6x37A, and F7x42A mutations were reverted, the receptor was able to catalyze nucleotide exchange at the G protein,The 5‐HT3.4Mutations can influence the binding of all types of ligands by modifying the mobility that is needed to reach the state to which they can bind. Consequently, ligand binding can be influenced by mutating residues that do not have a direct atomic contact with that ligand. Many such allosteric mutations are known. Figure This same reasoning can explain how the lipid composition and the cholesterol concentration can influence the apparent binding constant of ligands.3.52 muscarinic acetylcholine receptor allows the sodium ion to egress from this binding site into the cytosol. It was shown2 receptor  and a serine at position 5x44 in the 5‐HT2B receptor. TM6 with TM7: The cysteine 6x47 of the CWXP motif in helix 6 forms a hydrogen bond with the local backbone (left panel). Such hydrogen bonds are generally not very strong, but in this case the cysteine is “helped” by the side chain NH2 group of asparagine 7x45. Muscarinic receptors have a threonine at position 6x47 that can form this same hydrogen bond in a more normal way. The 5‐HT2 receptors have a methionine at position 6x47. In these receptors a hydrogen bond is formed between TM7 (serine 7x45) and the carbonyl of residue 6x44. TM7 with TM6: TM7 of most aminergic receptors is weakened at the cytosolic side of glycine 7x41. Surprisingly, a cysteine at this position can create the same instability in the muscarinic acetylcholine receptors when “helped” by a glycine at position 7x37. The cysteine distorts the helix because it is pushed into the helix backbone by the highly conserved tryptophan 6x48 of TM6. This is shown in the muscarinic receptor 4.In this paragraph we discuss the panels of Figure In some cases the residues that combine into the mobility‐supporting sequence motif are not located at the same position in the helix. Figure 4Residues that are important for a protein's function tend to remain conserved during evolution, with residues important for the primary function remaining most conserved. Residues associated with auxiliary functions may vary over evolutionary time between sub‐families.absence of hydrogen bonds that is conserved) provides radically new input into the study of this very large protein family. It is likely that similar effects play a role in many protein families in which structure deformation is part of the activation process, which would make our finding a novel concept in the study of protein families in general.It has often been stated that structures are more conserved than sequences, and it is also true that the location of functional residues is even more conserved.We hope that this study will help in the design of new GPCR stabilization strategies so that many more GPCRs can be crystallized and have their coordinates determined so that they can contribute to research in which GPCR structures play a crucial role, especially in the area of drug design. We also hope that it will help make structures available with different combinations of unmutated key residues. These will be important for deepening our understanding of the GPCR (in‐)activation process, which in turn will be crucial for the design of medicines that can be better tailored to the intended biological effect, be more specific, and have fewer side effects.https://publons.com/publon/10.1002/prot.26179.The peer review history for this article is available at AppendixS1: Supporting Information.Click here for additional data file.


ACE2TMPRSS2 and FURIN,ACE2 and its co‐factors in colorectal cancer, as well as their effects on SARS‐CoV‐2 infection.SARS‐CoV‐2 is the novel coronavirus leading to COVID‐19.ACE2 and two co‐factors in colorectal cancer, we performed RNA‐sequencing and proteomics analysis in both colorectal cancer tissues and adjacent normal tissues. ACE2 was higher in colorectal tumour tissues than in normal tissues .We further explored the association of genetic variants in  Figures  and S10. Figures . HaplotyACE2 and its co‐factors, we extracted mutational signatures of these genes in colorectal cancer. Missense mutations in ACE2 (73.33%), FURIN (75.00%), and TMPRSS2 (83.33%) were common in colorectal cancer tissues (Figure ACE2 in the early‐onset colorectal cancer group compared to the late‐onset group (Table ACE2 expression in patients with ACE2 mutations (Figure ACE2 mutations and expression. TMB was higher in individuals with ACE2 mutations compared to patients without ACE2 mutations (Figure ACE2 mRNA expression (Figure To identify somatic mutation patterns in s Figure . Notablyup Table . We alsos Figure . Tumour s Figure  and was n Figure .ACE2 was upregulated and positively correlated with TMPRSS2 expression in colorectal cancer tissues. SARS‐CoV‐2 infection was significantly higher in ACE2‐positive colon cells. The mRNA expressions of ACE2 and TMPRSS2 were associated with the immune infiltration level of colorectal cancer. Our results suggest that ACE2 and its co‐factors participate in the mechanisms underlying the association of colorectal cancer with COVID‐19 (Figure In conclusion, we identified that 9 Figure .The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.ACE2 expression includes data from colorectal adenoma tissues from the GEO databases.Figure S1 The box plot of Figure S2 Linear correlation between ACE2 and its co‐factors.ACE2 and its co‐factors in subgroups.Figure S3 Stratification analyses of the expression of ACE2 and TMPRSS2/FURIN using single‐cell RNA‐Seq in colorectal cancer cell clusters.Figure S4 Expression analysis of Figure S5 The expression patterns of ACE2 and its co‐factors in colorectal cancer cells.ACE2 and its co‐factors in different tumour tissues based on the HPA database.Figure S6 Protein expression of ACE2 and its co‐factors in different tumour tissues based on the TCGA database.Figure S7 mRNA expression of ACE2 and co‐factor expression with immune infiltration in colorectal cancer tissues.Figure S8 The correlation of Figure S9 Summary of the design and workflow in the association study of genetic variants with colorectal risk.ACE2, FURIN, and TMPRSS2 corresponding to the regional plot.Figure S10 The linkage disequilibrium plot exhibits the partition of pairwise variants in Figure S11 Schematic representation of SARS‐CoV‐2 infection in colorectal cancer patients.Table S1 The protein expressions of ACE2, TMPRSS2, and FURIN in different colorectal cell lines.ACE2, TMPRSS2, and FURIN relates genes and markers of immune cells in TIMER.Table S2 Correlation analysis between ACE2 and the co‐factors with colorectal cancer risk.Table S3 The association of genetic variants in Table S4 Estimated frequency of haplotypes and the association with colorectal cancer risk.ACE2 and the co‐factors in colorectal cancer tissues by TCGA database.Table S5 The mutation frequencies of Table S6 Stratification analyses for the association between mutation frequencies of three genes and different subgroups.Click here for additional data file.


Mutagenesis and electrophysiology supported the functional relevance of both interfaces, and extensive additional data validated the primary interface. However, ITC evidence suggesting that binding via the tripartite interface occurs in solution was called into question by subsequent NMR data. Here, we describe joint efforts to address this apparent contradiction. Using the same ITC approach with the same C2B domain mutant used previously (C2BKA‐Q) but including ion exchange chromatography to purify it, which is crucial to remove polyacidic contaminants, we were unable to observe the substantial endothermic ITC signal that was previously attributed to binding of this mutant to the complexin‐1‐SNARE complex through the tripartite interface. We were also unable to detect substantial populations of the tripartite interface in NMR analyses of the ITC samples or in measurements of paramagnetic relaxation effects, despite the high sensitivity of this method to detect weak protein complexes. However, these experiments do not rule out the possibility of very low affinity (KD > 1 mm) binding through this interface. These results emphasize the need to develop methods to characterize the structure of synaptotagmin‐1‐SNARE complexes between two membranes and to perform further structure–function analyses to establish the physiological relevance of the tripartite interface.Characterizing interactions of Synaptotagmin‐1 with the SNARE complex is crucial to understand the mechanism of neurotransmitter release. X‐ray crystallography revealed how the Synaptotagmin‐1 C 2+ sensor Synaptotagmin‐1 through a so‐called tripartite interface. Using ITC and highly sensitive NMR methods, we were unable to detect this interaction in solution, indicating that establishing the physiological relevance of the tripartite interface will require further research.Contradictory data were reported regarding the binding of the neuronal SNARE complex involved in neurotransmitter release to the Ca Exocytosis involves several steps, which include tethering of synaptic vesicles to specialized sites of the presynaptic plasma membrane called active zones, priming of the vesicle to a state(s) that is ready for release, and very fast (< 0.1 ms) fusion of the vesicle and plasma membranes upon Ca2+ influx 13C‐methyl alpha‐ketobutyric acid (80 mg·L−1) and [3‐2H]13C‐dimethyl alpha‐ketoisovaleric acid (80 mg·L−1)  to the cell cultures 30 min before isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) induction.For the PRE experiments of Fig. eviously , 57. TheQ)]; a pGEX‐6P‐1 vector to express rat complexin‐1 residue 48–73; a pETDuet construct to express rat complexin‐1 residues 26–83; pGEX‐6P‐1 constructs to express rat synaptotagmin‐1 residues 271–421 bearing the R281A/E295A/K326/K327A/Y338W/R398A/R399A mutations (referred to as C2BKA‐Q) or the R281A/E295A/K326/K327A/Y338W/L387Q/L394Q/R398A/R399A mutations (referred to as C2BKA‐Q‐LLQQ); pGEX‐KG constructs to express rat synaptotagmin‐1 residues 270–421 bearing the R322E/K325E mutations (referred to as C2BRK‐EE) or the R322E/K325E/R398Q/R399Q mutations (referred to as C2BRKRR‐EEQQ). Duet constructs for co‐expression of the four SNARE motifs but bearing the D44C mutation in synaptobrevin‐2 (28–89) or the E27C in SNAP‐25 (7–83) for MTSL tagging were prepared using the QuickChange II Site‐Directed Mutagenesis kit . Constructs prepared in the Brunger lab were shared with the Rizo laboratory, and vice versa, to ensure that the proteins expressed in both labs had identical amino acid sequences.The following constructs, which were described previously , 31, 33,2B domain mutants used in this study were expressed and purified by both laboratories following the procedures described below, following those previously reported  was ≤100 mm. Cation exchange was performed on a HiTrap SP 5 mL column (GE Life Sciences) using a linear gradient from 0 to 1 m NaCl over at least 12 column volumes (> 60 mL). It should be noted that the cation‐exchange chromatography step gave at least two distinct peaks, both corresponding to protein of the correct molecular weight when run on SDS/PAGE gel. Only the later fractions contained C2B domain devoid of polyacidic contaminants. The previous steps (protamine sulfate and nuclease treatments) minimized the presence of polyacidic contaminants in the preparation, significantly increasing the yield of the pure fractions. Collected fractions were concentrated to 100–1200 μm depending on the next experimental steps. The concentration of all purified protein was estimated by measuring UV absorbance at 280 nm unless specified otherwise.All synaptotagmin‐1 Creported . 15 N laQ) used for ITC experiments was expressed and purified as follows. BL21 (DE3) cells were co‐transformed with plasmids containing decimal‐histidine‐tagged, synaptobrevin‐2 (28–89), syntaxin‐1A (191–256), SNAP‐25 , and SNAP‐25 . Cells were grown using an auto‐inducing LB medium at 37 °C for 8 h, followed by 16 h at 30 °C. Cells were harvested by centrifugation and resuspended in lysis buffer  supplemented with lysozyme (Sigma), DNAse I (Sigma), and protease inhibitor tablets . Cells were lysed by sonication and subjected to centrifugation to clear cell debris. The cleared cell lysate was mixed with Ni‐NTA agarose beads  previously equilibrated with lysis buffer and incubated for 2 h at 4 °C. After incubation, the beads were harvested by centrifugation and poured into a column. The beads were subsequently washed with 8 column volumes (CV) of lysis buffer (without TX‐100), Urea wash buffer , and Wash buffer . The complex was eluted with wash buffer supplemented with 370 mm Imidazole. All washes, as well as the elution, were done at RT. A Bradford assay was used to detect the fractions containing the complex. These fractions were combined, supplemented with tobacco etch virus protease, and dialyzed against cleavage buffer  overnight at RT to remove the Histidine tag. After cleavage and dialysis, the NaCl concentration was adjusted to 50 mm using cleavage buffer without NaCl in preparation for anion exchange chromatography . The complex was purified using a linear NaCl gradient (50–500 mm). Peak fractions were pooled, concentrated to ~ 350 μm, flash‐frozen in liquid nitrogen, and stored at −80 °C.The quintuple mutant of the SNARE complex . The protein complex was expressed and purified in the same manner as the ones used for ITC experiments, with the addition of a final buffer exchange: after anion exchange chromatography, the pooled fractions were concentrated and loaded onto a Superdex 75 16/600 column  equilibrated with 50 mm Tris pH 8.0, 200 mm NaCl. After buffer exchange, the complex was flash frozen and stored at −80 °C.Two versions of the SNAREm DTT, and the DTT was then removed by gel filtration. The protein was concentrated to 40–60 μm and a 10‐fold excess of MTSL was added from a 40‐mm stock in dimethyl sulfoxide. The mixture was rotated overnight at 4 °C to allow complete cysteine labeling and the excess MTSL was removed by buffer exchange in 25 mm Tris pH 7.4 (pD 7.8), 125 mm NaCl and 1 mm EDTA. The MTSL‐labeled proteins were mixed with the other three SNARE motifs to form the SNARE and the unassembled fragments were removed by concentration dilution through a filter with a 10 kDa cutoff as described  from SNARE complexes anchored on supported lipid bilayers in the presence of Ca2+  from of Ca2+ . Mutagenomplexes , 55. Basomplexes , we antivery low , 69. We domains CB could domplexes .2B domain were strongly broadened, some beyond detection. Substantial PREs were also observed for many C2B domain cross‐peaks when MTSL was placed on residues 61 or 72 of synaptobrevin, or residue 239 of syntaxin‐1, whereas less extensive broadening was observed with MTSL on residue 187 or 197 of SNAP‐25  accessory helix.Importantly, the Cerformed , 31, 54 x bundle , rather 1H‐15 N TROSY‐HSQC spectra of the Syt1 C2B domain acquired in the presence of increasing concentrations of SNARE complex bound to Cpx1(26‐83) revealed shifts in multiple cross‐peaks corresponding to residues from the polybasic region or the primary interface, showing that these two surfaces of the C2B domain are responsible for binding to the Cpx1(26‐83)‐SNARE complex ; and (d) Syt1 C2B domain with the same seven mutations plus an additional L387Q/L394Q mutation (LLQQ) designed to disrupt the tripartite interface (referred to as C2BKA‐Q‐LLQQ) , which proved crucial to remove polyacidic contaminants that have a high tendency to bind to the C2B domain [2B domain mutants were uniformly 15 N‐labeled such that the same samples used in the ITC experiments could be examined by NMR spectroscopy. 1H‐15 N TROSY‐HSQC spectra of C2BKA‐Q and C2BKA‐Q‐LLQQ acquired in the absence and presence of Ca2+ confirmed that both proteins are properly folded and bind to Ca2+  Fig. B. The prB domain , 71. Mora2+ Figs .2B constructs with KA‐Q mutations in ref. [2B constructs with KA‐Q mutations for the experiments described here contained 20 mm Ca2+ and the proteins bound to the ion exchange resin under these conditions. We note that all other Syt1 C2B constructs studied in ref. [2B and the C2BKA mutant, included an ion exchange step. Hence, the absence of an ion exchange chromatography step for purification applies only to the ITC studies of the C2B mutants with KA‐Q mutations, which provided the key evidence for binding through the tripartite interface in solution in ref. [2BKA‐Q was titrated onto the SNAREQ‐Cpx1(48‐73) complex that was not observed in the absence of Cpx1(48‐73) and was strongly decreased by the LLQQ mutation [2BKA‐Q to the SNAREQ‐Cpx1(48‐73) complex through the tripartite interface, these results indicated that such binding required Cxp1(48‐73).It is worth noting that the ion exchange step was not used for the purification of the C in ref.  because  in ref. , includi in ref. . In thesmutation . In addi2BKA‐Q on SNAREQ either in the absence or presence of Cpx1(48‐73) using the same conditions as in [2BKA‐Q‐LLQQ was titrated onto SNAREQ   complex on C2BKA‐Q‐LLQQ was hindered by the additional shifts caused by the LLQQ mutation but, overall, the perturbations  Fig. . Since trum Fig. , we assitra Fig. . The croace Fig. . These r complex , possiblons Fig.  were comA‐Q Fig. . These r2BKA‐Q purified by ion exchange chromatography did not detect an interaction of Syt1 C2BKA‐Q with SNAREQ through the tripartite interface in the absence or presence of Cpx1(48‐73). These results suggest that binding of the Syt1 C2B domain to the SNARE complex through the tripartite interface is very weak even when the SNARE complex is bound to complexin‐1, consistent with previous experiments in which no binding of the Cpx1(26‐83)‐SNARE complex to the quadruple R322E/K325E/R398Q/R399Q Syt1 C2B domain mutant (below referred to as C2BRKRR‐EEQQ) was detected by NMR spectroscopy [1H‐15 N HSQC spectra  residue 44 of synaptobrevin, which is expected to be close to helix HA of the C2B domain on one side of the tripartite interface . Below we refer to the assemblies of the MTSL‐tagged SNARE complexes bound to Cpx1(26‐83) as SCCpx D44C and SCCpx E27C, respectively.Our ITC and NMR experiments using Syt1 Ctroscopy . Since ma , we useace Fig. C. Before1H‐15 N TROSY‐HSQC spectra acquired on samples containing 20 μm C2BRKRR‐EEQQ that was 15 N,2H–labeled, in the presence of 110 μm MTSL‐labeled SCCpx D44C or SCCpx E27C , shows that there is no substantial binding through the tripartite interface under the conditions of these experiments.This conclusion is further supported by plots of the observed PREs versus the approximate distance between the corresponding NH and the MTSL group predicted if the Cdistance  . Thus, this mutant still binds to the SNARE complex through the primary interface [m SCCpx D44C or SCCpx E27C, we observed a considerable number of moderate PREs below 0.8 and a strong PRE with the W404 side chain NH induced by SCCpx E27C . Moreover, the PREs observed for other nearby NH groups that are expected to be within 11–12 Å of the MTSL group of SCCpx E27C  were 0.89, 1.10, and 1.07, respectively. Assuming that the PREs observed for the NH groups of residues 350 and 354 actually arose from binding through the tripartite interface and that their distances to the MTSL in the tripartite complex are 11.6 and 10.6 Å, respectively, the populations of the tripartite complex that can be calculated from these PREs are 0.7% and 0.9%, which correspond to KDs of 16 and 12 mm, respectively. There is an uncertainty in these affinity calculations given the assumptions made . However, the overall PRE data obtained with the C2BRK‐EE mutant does provide strong evidence that, if the tripartite complex forms in solution, it has a very low affinity, with a KD in the low millimolar range or higher. This is corroborated by molecular dynamics simulations of both the primary and tripartite interfaces [Plots of the PREs induced by SCCpx D44C and SCCpx E27C on the Cved Fig. . The NH terfaces , which s2BRK‐EE mutant might have been hindered by the interaction involving the primary interface, we performed additional PRE measurements with this mutant and an assembly of Cpx1(26‐83) with SNARE complex that contained the quintuple K40A/D51A/E52A/E55A/D166A mutation designed to disrupt the primary interface (SNAREQ) and was again tagged with MTSL at residue 44 of synaptobrevin or residue 27 of SNAP‐25 . The PRE patterns observed in these experiments  might suggest that there is a small population of tripartite complex because this NH is expected to be close to the MTSL probe (at 10.4 Å) in this complex, the PREs observed for nearby NH groups of the HA helix did not confirm this conclusion. It is plausible that the PRE observed for G384 NH arises because of its proximity to the β‐strand containing W404, which also exhibits PREs below 0.8. Note also that the PRE induced by SCQCpx E27C on G384 NH is similar (0.56) even though this NH group is expected to be at 23 Å from the MTSL probe in the tripartite complex with SCQCpx E27C.To address the possibility that binding to the tripartite interface in the PRE experiments with the Cnts Figs  exhibite2B domain‐SNARE‐complexin‐1 complexes involving the tripartite interface, although they do not rule out the possibility that these complexes are formed with a very low affinity (KD >1 mm). The PRE data do corroborate the notion that there are several binding modes between the Syt1 C2B domain and the SNARE complex that are distinct from the primary complex, but some of these complexes involve the β‐sheet involved in the primary interface, as multiple PREs below 0.8 were observed in this region in the different data sets. Most of these binding modes likely involve the arginine‐rich region containing R398 and R399, as fewer PREs below 0.8 were observed in the experiments performed with the quadruple C2BRKRR‐EEQQ mutant than in those involving the double C2BRK‐EE mutant. Since the experiment with the latter mutant and SCQCpx E27C exhibited somewhat stronger PREs than those performed with SCCpx E27C, we investigated whether the additional binding modes that are visited with the SNAREQ complex depend on the presence of complexin‐1 using ITC. Interestingly, titrations of SNAREQ with the C2BRK‐EE mutant in the absence of Cpx1(48‐73) yielded an endothermic ITC signal that, although weak, was consistently stronger than that observed in the presence of Cpx1(48‐73)  to SNAREQ and might be related to the binding modes observed in our PRE experiments monitored by 1H‐13C HMQC spectra  Fig. . These r2BKA‐Q to SNAREQ‐Cpx1(48‐73) while the TGQQ mutation did not [2BKA‐Q to SNAREQ‐Cpx1(48‐73) or to SNAREQ through the tripartite interface by ITC or NMR spectroscopy, and the interaction between C2BKA‐Q and SNAREQ‐Cpx1(48‐73) through the primary interface that we did observe was not affected by the LLQQ mutation  and has well‐defined densities for most side chains, which is not typical of interactions that merely constitute crystal contacts. Moreover, the fact that the tripartite interface involves interactions of the Syt1 C2B domain with both the SNARE complex and complexin‐1 provides attractive explanations for the cooperation between Syt1 and complexin‐1 in inhibiting neurotransmitter release and for the finding that complexins are required for the dominant negative effect observed upon overexpression of Syt1 bearing mutations that abolish Ca2+ binding to the C2B domain (referred to as Syt1DA) [2B domain to the SNARE complex through this interface and to the plasma membrane through the polybasic region dictates that the accessory helix of complexin (bound to the SNARE complex) has steric clashes with the vesicles that are expected to hinder final C‐terminal assembly of the SNARE four‐helix bundle [The stoichiometry of Syt1 and the complexin‐1‐SNARE complex in the crystals that revealed the tripartite interface was 1 : 1, and Syt1 also interacted with the SNARE complex through the primary interface in these crystals . Since t Syt1DA) . Note, hx bundle , 34.2+‐evoked neurotransmitter release and the dominant negative effects of Syt1DA. Here, we show that the Syt1 LLQQ mutant is properly localized to the synapse  by the LLQQ mutation but not the TGQQ mutation. However, we could not reproduce these results when C2BKA‐Q was purified by ion exchange chromatography  through the tripartite interface in the ITC samples by NMR spectroscopy  complex or the SNAREQ‐Cpx1(48‐73) complex via the tripartite interface through analysis of 1H‐15 N TROSY‐HSQC perturbations or PREs even at SNARE complex concentrations on the 100 μm range  from membrane‐anchored SNARE complexes in the presence of Ca2+ [2+ influx, binding of Syt1 to the C‐terminus of the SNARE complex releases the inhibition of neurotransmitter release caused by the complexin accessory helix [2+‐dependent binding of Syt1 to PIP2‐containing membranes. Moreover, there is evidence that Syt1 C2AB does not bind to SNARE complexes anchored on PIP2‐containing membranes in the presence of Ca2+, ATP, and physiological salt concentrations [Our PRE data clearly show that there are multiple interaction modes between Syt1 and the SNARE complex beyond those involving the primary and tripartite interfaces Figs . Unfortu neurons . Howeverractions . The Sytlace Cpx1‐83 from  of Ca2+ . These iry helix . Howevertrations , 75.1H‐15 N TROSY‐HSQC spectrum of C2BRKRR‐EEQQ mutant [2B domain to the SNARE complex is practically abolished by this quadruple mutation that impairs binding through the polybasic region and the arginine‐rich region of the primary interface. Hence, C2B domain‐SNARE complex binding is largely governed by ionic interactions involving these two regions. It is noteworthy that SCQCpx D44C and SCQCpx E27C still caused sizeable PREs on the 1H‐15 N TROSY‐HSQC spectrum of C2BRK‐EE  causes no marked perturbations on the Q mutant  and that‐EE Figs  and 6E,F3 causes REQ Fig. , even th2+‐triggered synaptic vesicle fusion with multiple components are available [in silico [In summary, it is well established that binding of Syt1 to the SNARE complex through the primary interface plays a key function in neurotransmitter release, but further research will be necessary to determine whether the tripartite interface is physiologically relevant and whether other Syt1‐SNARE complex interactions that have not been well characterized might also play important roles. The diversity of such interactions that can be detected in solution arises in part because of the absence of phospholipids, the natural targets of the promiscuous polybasic region. Thus, further progress in this field will likely require the use of trans‐SNARE complexes bridging two membranes so that the configuration existing in primed vesicles can be mimicked as closely as possible. Such studies are currently feasible, as methods to prepare stable trans‐SNARE complexes bridging reconstituted proteoliposomes have been described e.g., , 76, 77), 7776, 7vailable , 4, 5. Cvailable , 79, 80,n silico  and compThe authors declare no conflict of interest.KJ, QZ, ETK, ATB, and JR conceptualized and supervised the study, and designed the experiments. KJ, LE, RAP, BA, and KDB performed the experiments. KJ, QZ, ATB, and JR wrote the manuscript, and LE, RAP, BA, KDB, and ETK revised the manuscript.Fig. S1. Summary of mutants of the various presynaptic proteins used in this study.Fig. S2. PRE analysis of Syt1 C2AB binding to the C‐terminal half of the SNARE complex.Fig. S3. The C2BKA‐Q mutant is properly folded and binds Ca2+.Fig. S4. The C2BKA‐Q‐LLQQ mutant is properly folded and binds Ca2+.Fig. S5. ITC analysis of interactions between C2BKA‐Q and SNAREQ.Fig. S6. ITC analysis of interactions of Cpx1(48‐73) with SNAREQ and C2BKA‐Q.Fig. S7. Cross‐peaks assignments of the 1H‐15 N HSQC spectrum of C2BKA‐Q.Fig. S8. Analysis of potential tripartite Syt1‐SNARE‐complexin‐1 complexes using PREs.Fig. S9. ITC analysis of interactions between C2BRK‐EE and SNAREQ.Fig. S10. Analysis of the localization of the Syt1 LLQQ and TGQQ mutants.Click here for additional data file.


We treat the solvent implicitly by solving the linearized Poisson–Boltzmann equation and evaluate the electrochemical potential and energetics of ions with the computational hydrogen electrode approach. We find that Cl− alone is hardly adsorbed at sufficiently negative electrochemical potentials μCl but stable phases with half and full Cl− coverage was observed as μCl is made more positive. For Cl− and Cu2+ coadsorption, we identified five stable phases for electrode biases between −2V < USHE < 2V, with two being Cl− adsorption phases, two being Cl− + Cu2+ coadsorption phases and one being a pure Cu2+ adsorption phase. In general, the free energy of adsorption for the most stable phases at larger |USHE| are dominated by the energy required to move electrons between the system and the Fermi level of the electrode, while that at smaller |USHE| are largely dictated by the binding strength between Cl− and Cu2+ adsorbates on the Cu (110) substrate. In addition, by studying the free energy of adsorption of Cu2+ onto pristine and Cl− covered Cu (110), we conclude that the introduction of Cl− ion does not improve the energetics of Cu2+ adsorption onto Cu (110).Motivated by the importance of Cl Free energy of adsorption for the most stable phases predicted by DFT calculations as a function of electrode potential. Apart from the suppressor and accelerator, the next most important additive is the chloride ion (Cl−), as it is one of the most common constituents of commercial acid-copper plating baths. A small amount of Cl− is essential as it affects the surface appearance, structure, and crystallographic orientation of the deposits, and it is also known to exert a synergetic effect with additives such as bis(3-sulfopropyl) disulfide [SPS] and polyethylene glycol [PEG].10–15 In the past, extensive experimental and theoretical studies were carried out to uncover the working mechanism of PEG and SPS in the presence of Cl−.16,17 There also exists a variety of experimental and theoretical studies18–29 for Cl− adsorption on copper due to other industry applications; but less work was reported on the understanding of the Cl− effect under copper electroplating environments. In view of the importance of Cl− addition in the industrial plating process, a detailed study of chloride adsorption, especially ones that consider solvent and applied electrode potential effects, is necessary as it will serve as a good foundation for further studies into more complex situations including plating with multiple additives. Therefore, in this research, we perform density functional calculations to investigate the adsorption behaviour of Cl− and Cu2+ on Cu(110) surface. We model solvent effects by solving the linearized Poisson–Boltzmann equation, and tune the electrode potential by adding or removing electrons to the simulation system. In this way, the energetics of Cl− adsorption and Cl− and Cu2+ coadsorption on Cu(110) surface are evaluated. We choose Cu(110) as this surface is commonly observed during acid-copper plating but was less commonly investigated in previous literature.Copper is a ubiquitous metal of great importance due to its excellent physical properties such as mechanical strength, chemical stability as well as high electrical and thermal conductivities. This allows copper to be utilized in numerous applications such as catalysis, heat exchange and electrical wiring. In particular, copper is used extensively as interconnects in modern electronic circuits where the copper metallization is achieved through electrodeposition.Gsurf,ads and Gsurf,0 are the Gibbs free energies of the ion adsorbed and pristine surfaces, ni is the number of ith ion adsorbed, μi is the electrochemical potential of the ith ion in solution, and AS is the surface area of the calculation cell.28 Since the terms TS and pV are rather small for solids, it is reasonable to neglect these dependencies and equate Gsurf,ads and Gsurf,0 to the total energies obtained from DFT calculations, i.e. Gsurf,ads = Esurf,ads and Gsurf,0 = Esurf,0. To compute the electrochemical potentials of the ions μi, we use the concept of the computational hydrogen electrode generalized to arbitrary ions.29 Under standard conditions, the free energy of a singly charged positive ion plus that of an electron at the Fermi level differs from the free energy of its bulk phase by eU0, where U0 is the standard reduction potential. This relation allows us to obtain μi without having to calculate the solvation energy of ions, but instead derive it from the bulk energy of the material in question. To obtain μi under general thermodynamic conditions, we just correct for the electrode potential with a eUSHE term and the ion concentration with a kBT ln(a) term, where USHE is the electrode potential relative to the standard hydrogen electrode and a is the ion activity. Thus, we haveECu and E2Cl are the energies of a Cu atom in bulk Cu and Cl2 molecule respectively, both of which are obtained from DFT calculations.  and  are the standard reduction potentials and a2+Cu and a−Cl are the activities of the Cu2+ and Cl− ions respectively. We can also rewrite From the thermodynamic point of view, the most stable adsorption configuration can be predicted by identifying the structure that minimizes the Gibbs free energy of adsorption per unit surface areaThis way, we separate quantities obtained by first-principles calculations from those that are defined by external conditions.Esurf,ads and Esurf,0, we perform DFT calculations on atomic slabs with 40.0 Å of vacuum perpendicular to the surface normal. Adsorbates are introduced on both sides of the surface to ensure that the interface dipole is symmetric on both faces. In addition, the electrode potential can be tuned in our simulation by adding or removing electrons to the system, as this becomes surface charge that creates an electric field near the slab which changes the work function. The corresponding electrode bias is then USHE = W − 4.44V, where 4.44V is the standard potential of the hydrogen electrode and W is the work function of the system. We also model the electrolyte solution with an implicit solvent method.30,31 Here, we solve the linearized Poisson–Boltzmann equation in place of the standard Poisson equation to account for screening effects of the ions and solvent. This effectively imposes a dielectric constant that varies from 1.0 in the solute to the bulk value of water in the vacuum region of the calculation. Also, there is an ionic charge term that varies linearly with potential and is parametrized by the Debye length. This addition enables the electric double layer to be represented more realistically and balances any excess surface charge.To compute the total energies 32 and the exchange correlation is described using the GGA-PBE density functional.33 Wavefunctions have been expanded using a plane wave basis with an energy cutoff of 500 eV and dispersion interactions are included using the DFT-D2 scheme.34 The ionic potentials are represented using the PAW formalism35 and the Brillouin zone is sampled with the spacing between k-points set to 0.3 Å−1. Geometry was optimized for the neutral system and convergence is reached when each force component is less than 0.01 eV Å−1. Electronic self-consistency is reached when the total energy is converged to within 10−6 eV. The implicit solvent dielectric permittivity is set to the bulk water value of 78.4 and the Debye screening length is determined by the ionic concentration and calculated using eqn (15) of Our calculations have been performed using the VASP package2+ and Cl− coadsorption on the Cu (110) surface, let us first look at the simpler case of Cl− adsorption. The starting point of our calculation is the Cu (110) surface, which we construct from a slab of 7 atomic layers thick as shown in x and y axes respectively. The in-plane lattice constants are obtained from DFT calculations on bulk Cu to give 2.57 Å and 3.64 Å for the (1 × 1) surface unit cell. We consider multiple adsorption configurations and adsorption sites for Cl on Cu (110) as shown in Before we consider CuEads of Cl on Cu (110). For a given type of adsorption site i.e. top, hollow, long or short bridge, the magnitude of Eads decreases with increasing coverage due to electrostatic repulsion between negatively charged Cl ions.23 At low coverage, the repulsion is weaker due to larger distances between Cl adsorbates and screening by the Cu metal, as a result |Eads| decreases slowly. As the coverage increases, the repulsion energy becomes dominant and this can be seen in the dramatic decrease of |Eads| from 1/2ML to full coverage. At half coverage, we also see stark differences in the adsorption energy between the (1 × 2) 1/2ML, (2 × 1) 1/2ML, (2 × 2) 1/2ML configurations, which we similarly attribute to repulsion between Cl adsorbates. The adsorption energy |Eads| is weakest for the (1 × 2) 1/2ML system while that of (2 × 2) 1/2ML and (2 × 1) 1/2ML configurations are close to each other. This is because the nearest neighbour Cl–Cl distance for the (1 × 2) 1/2ML system is 2.57 Å, which leads to significantly larger electrostatic repulsion than in the (2 × 1) 1/2ML and (2 × 2) 1/2ML configurations, which have the nearest Cl–Cl distances of 3.64 Å and 4.46 Å respectively.We show in e for the short bridge and hollow sites, 0.27e for the long bridge site and 0.24e for the top site. Thus we see that the magnitude of adsorption energy increases with decreasing charge transfer to Cl.Another trend that is clear from γ using γ for the (2 × 1) 1/2ML, (2 × 2) 1/2ML, (2 × 2) 1/4ML, (3 × 3) 1/9ML, (4 × 4) 1/16ML structures with Cl adsorbed on the short bridge site and (1 × 1) 1ML, (1 × 2) 1/2ML configurations with Cl adsorbed on the top site. The effects of the applied electrode bias and ion activity are represented through the electrochemical potential Δμ−Cl and Eads is obtained from our previous neutral slab calculations. Also, since the slope of Δγ vs. Δμ−Cl is only dependent on the coverage, we only need to consider the lowest adsorption energy site for a given coverage to identify the state with the lowest Δγ. Using this procedure, we generate a plot of Δγ as shown in μ−Cl. If we focus on the region near Δμ−Cl ∼ 0 eV, we have Δγ = Eads/AS and we find that the (1 × 1) 1ML, (2 × 2) 1/2ML and (2 × 1) 1/2ML configurations almost equally stable with the lowest free energies. However as one increases the electrochemical potential Δμ−Cl, the (1 × 1) configuration becomes most favourable as the slope −ni/AS, which is the negative of the areal density of Cl adsorbates, is most negative for the (1 × 1) system. For the intermediate region below Δμ−Cl ∼ 0 eV, the potential term −niΔμCl/AS in γ faster for the (1 × 1) 1ML configuration, and the (2 × 2) 1/2ML and (2 × 1) 1/2ML structures are now the most favourable. This is in agreement with previous LEED work that observed a 0.5 coverage c(2 × 2) Cl adsorption on Cu (110) exposed to HCl.26 However as we keep decreasing the electrochemical potential Δμ−Cl to the point beyond ∼−1.9 eV, the potential term becomes large enough to dominate over the adsorption energy and Δγ becomes positive for all the systems considered, hence no adsorption occurs. This is because the energy required to deposit an electron from the ion into the electrode becomes energetically too costly.Now that we have determined the energetically preferred Cl adsorption sites for the various configurations, we compute the free energy of adsorption Δ2+ and Cl− ions on a Cu (110) surface under different electrode potentials and ion concentrations. In these calculations, we consider Cl− adsorption configurations studied above but limit the coverage to the range 1/4 to 1, as lower coverages were found to be energetically unfavourable. Also, we employ the optimal Cl− adsorption sites found in the preceding section in our coadsorption study. This leads to the calculation geometries of (1 × 1) 1ML and (1 × 2) 1/2ML on the top site and (2 × 1) 1/2ML, (2 × 2) 1/2ML and (2 × 2) 1/4ML configurations on the short bridge site. For Cu2+ adsorption, we consider the same configurations as Cl− adsorption, i.e. (1 × 1) 1ML, (1 × 2) 1/2ML, (2 × 1) 1/2ML, (2 × 2) 1/2ML and (2 × 2) 1/4ML. However, we assume that adsorption occurs on the hollow site, as this is the position that leads to the formation of bulk Cu. The possible configurations for Cu2+ and Cl− adsorption are summarized in 2+ and Cl− configurations to generate 36 co-adsorption systems.In the next step, we look at the coadsorption of Cuγ for coadsorption, however we introduce between −0.4, −0.2, 0.0, 0.2, 0.4 electrons to the Cu (110) slabs under consideration to obtain Esurf,ads and Esurf,0 for different applied potentials USHE. The potential is evaluated by computing the work function W under different charging conditions and applying the relation USHE = W − 4.44V, giving rise to a potential variation on the order of 1V. The resulting total energies are fit to a quadratic polynomial in USHE so that the total energy can be evaluated at arbitrary USHE. In general, we setWe again apply a, b, c as well as a0, b0 and c0 are obtained from fits using total energies obtained from charged DFT calculations, and ECu and E2Cl are obtained from separate calculations on bulk fcc Cu and an isolated Cl2 molecule respectively. The coefficients a′, b′ and c′ are computed for each of the 36 co-adsorption configurations and the Δγ are compared at each USHE to identify the phase that minimizes Δγ. This procedure is repeated for the activity combinations , ,  and  and the results are plotted in The coefficients USHE between −2.0 to 2.0 V. Inspection of the plots shows that the nature of the stable configurations and shape of the Δγ plots are mostly independent of the activities aCl and aCu. This is because the term kBT ln(a) is much smaller than other energy terms in Δγ at room temperature kBT = 0.025 eV in the studied activity range. We conclude that the activity term only matters if the ion is present in very small concentrations. We list the a′, b′ and c′ values of the 5 stable phases in a′ ranges between −0.020 to 0.028 eV Å−2, that of b′ ranges between −0.276 to 0.436e Å−2, and c′ is between −0.326 and −0.001 eV Å−2. Comparing the possible values of a′ and b′, it can be concluded that the quadratic term is generally smaller than the linear term for the range of USHE considered and is therefore not consequential in determining the most stable phase. Near the extremities of the USHE range considered, the most stable phases Cu (1 × 1) 1ML and Cl (1 × 1) 1ML correspond to the systems with the largest magnitudes of b’ for the systems considered, and these large values originate from the large areal densities n/AS arising from the full coverage of adsorbates. Conversely for small values of |USHE|, both quadratic and linear terms fall away and Δγ is dominated by the constant c′ term. The contribution of this term is mostly the adsorption energy Eads plus the reduction potential term , with  and . This implies that stable configurations are the ones with low Eads along with the ones with large nCu due to the  term, which gives rise to the Cl− (2 × 2) 1/2ML configuration owing to its low Eads and Cu (1 × 1) 1ML due to its large nCu.As can be seen, there are five preferred phases over a range of Esurf,ads and Esurf,0 using neutral slabs for the 36 possible co-adsorption configurations, and generated a USHE independent Eads for computing Δγ. The results of these calculations are seen in γ curve remaining unchanged from the charged calculations. However, the boundaries separating each phase has shifted, and this is particularly pronounced for the boundary between Cu (1 × 1) 1ML and Cu (1 × 1) 1ML + Cl (2 × 2) 1/4ML. From this, it seems that charging the slabs has stabilized Cl− adsorption in the presence of Cu2+ relative to neutral calculations, however the main conclusions between charged and uncharged calculations remain unchanged and there is semi-quantitative agreement between results from these two sets of data.As a test of the charging formalism used in this study, we also compute the total energies − or Cu2+ ions vs. coadsorption of both Cu2+ and Cl− on Cu (110). To do this, we have calculated the Δγ for the most stable configurations in the presence of Cl− ions only, Cu2+ ions only and both Cl− and Cu2+ ions and plotted them in a−Cl and a2+Cu kept at 1.0.Finally, we compare the adsorption of only ClUSHE| is large, the most stable phases consist of adsorption of only a single type of ion. However, in the intermediate region of USHE between −1.4V and 0.2V, the coadsorption of both Cu and Cl ions is energetically preferable. In addition, we can determine if the presence of Cl− promotes the adsorption of Cu2+ on Cu (110). To do this we consider the following reactions Cl (2 × 2) 1/2ML + Cu2+ → Cu (1 × 1) 1ML + Cl (2 × 2) 1/2ML and Cu(110) + Cu2+ → Cu (1 × 1) 1ML. The first reaction is for Cu2+ adsorption on a Cl− covered Cu surface and the second is for Cu2+ on pristine Cu, and we expect these states to be stable in the potential range −0.68V < USHE < 0.15V. The free energy of adsorption for these reactions can be obtained by taking the difference between the black and blue curves of γ is almost identical for adsorption onto either the Cl adsorbed or pristine Cu (110). From this, we can see that the energetics of copper plating is fairly independent of the presence of Cl− ions on the Cu surface, and this strongly suggests that the presence of Cl− neither enhance nor deter the plating of Cu.For regions where |− as well as coadsorption of Cu2+ and Cl− on the Cu (110) surface using first-principles DFT calculations. The screening arising from the ionic solvent is modeled by solving the linearized Poisson–Boltzmann equation, and the electrode potential is tuned by adding or removing charge to the simulation system. By calculating the free energy of adsorption, we identify the preferred phases of Cl− on Cu(110) as the Cl (1 × 1) 1ML for positive Δμ−Cl and Cl (2 × 2) 1/2ML for negative Δμ−Cl, while for Δμ−Cl < −1.9 V, there are no stable adsorbed phases of Cl−. In addition, we find that for Cl− configurations at low coverage, the short bridge adsorption site is preferred as it maximizes binding between the Cl− adsorbate and Cu substrate, whereas for high coverage configurations where electrostatic repulsion between Cl− is dominant, the top configuration is more favorable as it minimizes charge transfer from Cu to Cl−. For our study of Cu2+ and Cl− coadsorption on Cu (110), we identify five different stable phases over a range of USHE, with the configurations at large |USHE| dominated by full coverage of either Cl− or Cu2+ adsorbates while that of low |USHE| being structures that have large adsorption energies Eads, such as Cl (2 × 2) 1/2ML. In addition, we found that calculating Δγ using neutral slab calculations gives semi-quantitative agreement with charged slab calculations that explicitly model the electrode potential, and that the presence of Cl− does not significantly enhance or deter the plating of Cu2+.In this work, we have studied the adsorption of ClThere are no conflicts to declare.


Transcription of endogenous retroviral elements are tightly regulated during development by members of the KRAB‐containing zinc finger proteins (KRAB‐ZFPs) and the co‐repressor Trim28 . KRAB‐ZFPs form the largest family of transcription regulators in mammals and initiate transcriptional silencing by tethering Trim28 to a target locus. Subsequently, Trim28 recruits chromatin modifying effectors resulting in the formation of heterochromatin. In the present study, we identify surface exposed residues on the central six turns of the Trim28 coiled‐coil region forming the binding interface for the KRAB domain. Using AlphaFold2 (AF2) we provide high confidence models of the interface between Trim28 and the KRAB domain and identified leucine 301 on each chain of the Trim28 monomer to act as a pin extending into a hydrophobic pocket on the KRAB domain surface. Site directed mutations in the Trim28‐KRAB binding interface abolished binding to the KRAB domain. Our work provides a detailed understanding of the specific interactions between the KRAB domain and the Trim28 coiled‐coil and how this interaction may be regulated during silencing events. KRAB‐ZFPsKRAB‐containing zinc finger proteins1The human genome contains endogenous retroviral elements (ERVs) as relics of historic retroviral infections that have been maintained in the germline and inherited by future generations. In human ancestral lineages it is estimated there were over 50 waves of retroviral endogenization events.The KRAB‐ZFPs contain an N‐terminal KRAB domain followed by an array of between three and 40 DNA binding C2H2 zinc finger domains.Trim28 (or KAP1 or TIF1β) belongs to the TRIM protein family of RING E3 ubiquitin ligases consisting of over 70 members in humans. Family member's share a conserved N‐terminal domain organization termed the RBCC, consisting of a RING finger domain, one or two B‐box domains, and a coiled‐coil region. One defining feature of TRIM proteins is their ability to oligomerize as homo‐dimers and higher order oligomers. The coiled‐coil regions of TRIM proteins forms an extended ~170 Å long α‐helix that exhibits a conserved heptad repeat forming a constitutive anti‐parallel homodimer.in vitro investigations on Trim28 binding to the KRAB domain of KRAB‐ZFPs independently determined that the binding stoichiometry is 1:2 KRAB‐ZFP: Trim28, indicating the KRAB binds at the two fold symmetry axis of the coiled‐coil domain.Two recent Protein structure prediction from the amino acid sequence has been a developing field for the past decades with the CASP14 experimentIn this study we demonstrate the surface exposed residues of the central six turns of the Trim28 coiled‐coil region form the binding interface for the KRAB domain. We provide high confidence models of the interface between Trim28 and the KRAB domain and identified leucine 301 on each chain of the Trim28 monomer to act as a pin extending into a hydrophobic pocket on the KRAB domain surface. We also validate the proposed model through site‐directed mutagenesis of interfacing residues on the Trim28 coiled‐coil that result in a reduction in binding affinity.22.1The coiled‐coil region of Trim28 is comprised of two 30‐turn amphipathic helices followed by a hairpin loop with the protein chain extending back toward the center of the helices and connecting to a small helix forming a four‐helix bundle.Using a series of pull‐down experiments we identified K297 in this central region as being critical for binding. To expand on this work we hypothesized that there are likely other mutations that, rather than abolishing binding completely, would result in a reduction in binding affinity that could be assessed using our surface plasmon resonance (SPR) binding assay.We undertook Biacore SPR on the Trim28 mutants to examine their binding to the immobilized KRAB domain from ZFP932. The KRAB domain was expressed with an N‐terminal Avitag resulting in a monobiotinylated construct that was then immobilized onto the surface of a biacore Sensor Chip CAP. Data were analyzed by either equilibrium or kinetic analysis.The interaction between Trim28 and ZFP932 has an affinity of 14.6 ± 1.1 nM consistent with our previous experiments. Analysis of the Turns 1–2 mutant binding to ZFP932 has an affinity of 2.7 ± 0.1 μM, a 185‐fold decrease in affinity. Additionally, K297 was located in the Top Face 6 mutant and was binding‐deficient suggesting that this is the key binding interface. This is supported by mutations on the opposing face of the coiled coil that showed minimal effect on binding with affinities of 34.5 ± 11.3 nM and 52.9 ± 7.8 μM for the bottom face 2/6 mutants, respectively. We were unable to use a kinetic analysis for Turns 3–4 and Turns 5–6 due to the fast on/off kinetics of binding. Equilibrium analysis of Turns 3–4 gave an affinity of 1.1 ± 0.1 μM and Turns 5–6 gave an affinity of 14.0 ± 0.7 μM showing mutations in these turns of the coiled‐coil can disrupt binding to ZFP932 as severe as the K297 mutations. The Turns 7–8 mutant displayed the same affinity as the wild‐type protein placing this area of the coiled‐coil outside the binding interface. Taken together, our binding data confirms that the KRAB domain binds the top face of the Trim28 coiled‐coil and spans the central region including residues between V293 and R311. Furthermore, it demonstrates that residues located on the top face of the Turns 1–6 are involved in the interaction expanding the binding interface compared to previous studies2.2Recent advances in protein folding algorithms have made it possible to model proteins with a high degree of accuracy. Alphafold has been shown to accurately predict the structure of proteins from sequence alone. Based on the conserved nature of the Trim28‐KRAB interaction, we hypothesized that sufficient co‐evolutionary information would be present for Alphafold2 to provide an accurate model of the interaction that could then be verified either by site‐directed mutagenesis or mutations present in existing literature.Models for individual protein subunits were accessed via the EBI AlphaFold 2 database. The AF2 predicted structure for Trim28 domains is consistent with published structures (UniProt Q13263) albeit as a model of the monomer rather than the dimer Figure . HoweverModels of the KRAB‐ZFP KRAB domain from multiple models in the AF2 database converge on a common fold consisting of four helices Figure . The N‐tTo understand the molecular determinants for binding of the KRAB domain with Trim28 we modeled Trim28 in complex with the KRAB domain from ZFP809 using AF2. This modeling is complicated by the 2:1 stoichiometry of the TRIM‐KRAB complex. To generate the model we initially used a sequence whereby the Trim28 RBCC (58–418) was duplicated with a 40‐glycine linker placed between monomers, the KRAB‐AB domain of ZFP809 (residues 1–74) was then appended to the sequence after another 40aa glycine linker to generate the appropriate 2:1 stoichiometry of the interaction.The model for the complex of Trim28 with the ZFP809 KRAB domain comprised of two similar chains of the Trim28 RBCC  and a single KRAB domain. AF2 recreated the expected anti‐parallel Trim28 dimer with the Bbox and RING domains placed in positions consistent with the published structures Figure . In our 2 from each molecule. The binding interface with Trim28 spans residues Lys290—Arg312 of chain A, and Val294—Asn308 for chain B  to be 445‐fold weaker than wildtype, and L301S (Kd = 30.8 ± 1.0 μM) to further diminished binding by 2,112‐fold. This mutagenesis confirms the importance of L301 with mutations to surrounding residues also resulting in a reduction in binding affinity but to a lesser degree.Our modeling of Trim28 in complex with KRAB domains produced high accuracy predictions consistent with known structures Trim28 and the KRAB domain in isolation.The asymmetric interface formed by the Trim28:KRAB complex is extremely tight in fitting with its biological role, the KRAB domain must recruit Trim28 to a single site within the genome. The two pseudo symmetric hydrophobic pockets on the KRAB domain allows it to bind in either orientation with essentially identical interactions. Comparison with the structure of the Trim28—SMARCAD1 Cue1 complex shows no overlap in the binding interface allowing both interactions to occur simultaneously. This is consistent with the Trim28 dimer functioning as a scaffold tethering multiple epigenetic regulators to KRAB‐ZFP binding sites.Consistent with the models presented, mutation of conserved amino acids within the KRAB‐A domain have previously been shown to abrogate Trim28‐mediated transcriptional repression.The KRAB‐B box has previously been shown to enhance the repression activity of the KRAB‐A box.Our study provides insights into the initial events during Trim28‐mediated transcription silencing. The affinity of Trim28 for the KRAB domain is low nanomolar ranging from 2 to 200 nM.44.1glutathione S‐transferase [GST] tag in pET49b with an maltose binding protein [MBP] tag) by a ligation independent cloning (LIC) method.DNA encoding murine Trim28 (1–834) and ZFP932_KRAB (5–76) was codon optimized and synthesized (GeneArt). The coding sequences for T28_RBCC (58–418) and ZFP932 (5–76) were amplified by PCR. These constructs were cloned into pET‐49b(+)‐MBP  cellsD‐1‐thiogalactopyranoside (IPTG) to log phase cultures prior to subsequent growth overnight at 18°C. Cells were lysed by cell disruption in 20 mM Tris–HCl (pH 8.0), 200 mM NaCl, 0.5 mM Tris(2‐carboxyethyl)phosphine (TCEP), 10% (v/v) glycerol, 0.1% (v/v) Triton X‐100. The MBP‐His‐tagged Trim28 constructs were purified using amylose affinity chromatography. The N‐terminal purification tags were removed by cleavage with 3C‐protease prior to further purification by size exclusion chromatography (SEC). The AviTagged ZFP932 KRAB domain was co‐expressed with pBirA in E. coli BL21 (DE3) LOBSTR grown in LB at 37°C to approximately 0.5 OD600. Cells were then induced with 1 mM IPTG and supplemented with 20 μM D‐biotin (Sigma) and grown overnight at 18°C. The MBP‐His‐tagged ZFP932_KRAB (5–76) was then purified as described above.All Trim28 constructs were expressed in 4.3Size‐exclusion chromatography coupled to multi‐angle laser light scattering (SEC‐MALLS) was used to determine the solution molecular weight and assess protein heterogeneity. Samples (100 μl) were applied to either a Superdex S200 Increase 10/300GL column equilibrated in 10 mM Tris/HCl pH 7.8, 150 mM NaCl, 0.1 mM TCEP mounted on a Dionex HPLC with a PSS SLD7000 7‐angle MALLS detector and Shodex RI‐101 differential refractive index detector. The weight average molecular weight was determined using PSS winGPC Unichrom software.4.4d was determined by fitting a two‐state reaction model using BiacoreX100 Evaluation Software version 2.0.1. For equilibrium analysis, a protein concentration series was prepared by serial diluting proteins with SPR buffer. Each concentration (from low to high) was injected over the reference and sample surfaces for 60 s, followed by a 600 s wash with SPR buffer. The data was fitted using BiacoreX100 Evaluation Software version 2.0.1.SPR was undertaken using a Sensor Chip CAP in a Biacore X100 instrument. CAPture reagent (GE) was loaded onto both the reference and sample surfaces at a flow rate of 5 μl/min for 120 s, achieving a resonance unit (RU) of ~2,500. Monobiotinylated KRAB domain was then loaded onto the sample surface at 10 μl/min achieving ~100 RU. Exactly, 1 μM of RBCC_R184D or RBCC coiled‐coil mutants were injected over the reference and sample surfaces for 60 s, followed by a 600 s wash with SPR buffer . K4.5Models were produced using AlphaFold2_advanced on Google Colab GPUs. The input were sequences of Trim28 RBCC (residues 58–418) and KRAB domains with the stoichiometry set to 2:1 Trim28:KRAB. No templates were included in the modeling and the models were relaxed using Amber‐Relax as part of the AF2 modeling process. Five models were produced for each complex and the rank1 model that has the highest overall confidence was chosen for analysis. No differences were observed in the Trim28:KRAB interface between the 5 models. Residues with pLDDT less than 50 and the B1 Bbox domains were removed prior to analysis.Jamie R. H. Taka: Conceptualization (supporting); investigation ; writing – original draft ; writing – review and editing . Yunyuan Sun: Investigation (supporting); writing – review and editing (supporting). David C. Goldstone: Conceptualization ; investigation ; supervision ; writing – original draft ; writing – review and editing .The authors declare no competing financial interests.Figure S1. Structural comparison between the modeled Trim28‐KRAB complexes indicates a uniform binding site. Predicted aligned error plots for the four models of the Trim28‐KRAB complexes with Trim28 consisting of chain A and B, and the KRAB domains consisting of chain C. The plots indicate a high confidence in the relative position of residues within the domains of the Trim28 RBCC. There is high confidence in the relative position of the KRAB domains to the coiled‐coil regions of each chain of Trim28.Figure S2. SEC‐MALLS analysis of the Trim28 RBCC coiled‐coil mutants. Protein elution profile is shown by differential refractive index traces (solid lines) and MW is represented by open circles. (a–c) Mutation of coiled‐coil residues to alanine and change of function mutations show a consistent elution profile and Mw as wildtype Trim28. (d) Mutation of K305E has weak concentration dependent self‐association.Figure S3. Mutation of conserved residues in the KRAB‐A box disrupts the interaction with Trim28 and transcriptional repression. The KRAB‐A box is highly conserved and responsible for the interaction with Trim28. Residues involved in the Trim28‐KRAB binding interface with from our AF2 models are marked by an asterisk. GST‐pulldown binding assay shows mutations of conserved KRAB‐A box residues disrupt the interaction with the Trim28 RBCC  and are congruent with a disruption of the binding interface identified from our structural models. GAL4‐KRAB‐A box mutations also disrupt the transcriptional repression activity of KZFPs . Exchanging the PRDM9 residues for amino acids occurring at the respective positions in ZFP10 confers repression activity, whereas wildtype PRDM9 is unable to repress transcription. (B) ZFP809 KRAB domain model showing location of conserved “D8V9,” “E16E17W18,” and “M33L34E35” motifs involved in the interaction with Trim28Click here for additional data file.