Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein–protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell.  Salmonella enterica is a facultative intracellular pathogen capable of causing disease in humans and a broad range of animal hosts. The medically relevant Salmonella enterica serovars are among the leading causes of gastroenteritis and bacteremia worldwide1. The pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium) is largely mediated by two distinct type III secretion systems encoded on Salmonella pathogenicity islands 1 and 2 2. These type III secretion systems transport bacterial effector proteins into the cytoplasm of the host cell where they specifically target host processes to promote both invasion and intracellular survival of S. Typhimurium3. Effectors secreted by the T3SS1 largely function to promote invasion into epithelial cells, whereas effectors secreted by the T3SS2 promote intracellular survival and replication within both epithelial and phagocytic cells4.S. Typhimurium as an intracellular pathogen is establishing a unique intracellular replicative niche. A subset of secreted effectors contribute to formation of the Salmonella containing vacuole (SCV) to protect intracellular S. Typhimurium from host defenses4. At 4–6 h post invasion, S. Typhimurium induces formation of filamentous structures known as Salmonella-induced filaments (SIFs) that radiate outwards from the SCV along microtubules9. The two interrelated processes of SIF biogenesis and SCV membrane maintenance are in large part resultant from controlled interactions with the host endomembrane system mediated by the T3SS2-secreted effectors SifA, SopD2, SteA, PipB2, SseJ, SseF, and SseG10.Critical to the success of 11. Host binding partners for SifA, SopD2, SteA, PipB2, SseJ, SseF, and SseG have previously been identified largely through single-effector transfection studies (reviewed in Ref.10). Such studies express a single effector out of the context of a native infection, and therefore would not be expected to demonstrate a phenotype if multiple T3SS2 effectors cooperate to form SIFs or maintain the SCV membrane. The overlapping and redundant nature of many T3SS2 effectors further hampers efforts to discover novel effector functions. Characterization of the protein–protein interactions mediating these critical infection stages is key to understanding the significance of T3SS2-secreted effectors during S. Typhimurium infection.We have previously shown that effector phenotypes during infection are nuanced and complex in ways that are not captured by single effector studies as it is likely that effectors work in concert to mediate multiple aspects of SIF biogenesis or SCV membrane maintenance12. This methodology permits identification and quantification of effector binding partners, whether that be a host protein or a secreted bacterial effector. Our lab previously used the SILAC method to identify protein–protein interactions during S. Typhimurium infection by incorporating stable isotope-labelled amino acids into the entire proteome of host cells, followed by effector transfection, immunoprecipitation of effector complexes, and analysis by mass spectrometry13.We employed SILAC (stable isotope labelling with amino acids in cell culture) to identify cognate binding partners of the effectors associated with SIF biogenesis, SCV membrane maintenance, and intracellular positioning of the SCV during infection. SILAC is a quantitative mass spectrometry-based technique that relies on non-radioactive isotopic labelling of particular amino acids to detect differences in protein abundances in different samples allowing identification of specific and non-specific protein–protein interactionsS. Typhimurium strains secreting increased abundance of HA-tagged T3SS2-effectors through overexpression of the T3SS2 transcriptional regulator SsrB. Using this approach, we demonstrate the utility of examining the role of multiple effectors during infection and find a common host binding partner for two T3SS2 effectors.We hypothesize that the nuanced phenotypes resulting from effector deletion studies arise from effectors targeting the same host protein or forming an effector-effector complex. To investigate this, we examined a series of T3SS2-secreted effector protein–protein interactions during infection by performing a series of immunoprecipitation experiments in SILAC-labelled host cells infected with E. coli MFDpir strain, media were supplemented with dl-2,6-Diaminopimelic acid (DAP) at a final concentration of 0.3 mM when appropriate. Antibiotics were used at the following concentration when required: streptomycin 50 µg/mL, chloramphenicol 30 µg/mL.Bacterial strains used in this work are described in Table 21 as were plasmids pRE112∆sifA, pRE112∆sopD2, pRE112∆pipB2, pRE112∆steA, pRE112∆sseJ, and pRE112∆sseF11. Complementation vectors and gene deletion/insertion vectors were routinely maintained in E. coli DH10B and MC1061λpir, respectively. Primers are listed in Table Plasmids constructed and used in this study are listed in Table ssrB was constructed using primers KK_001 and KK_002 to amplify linear pRE112 from its KpnI to SacI unique restriction sites. For PCR, Phusion High-Fidelity DNA Polymerase (NEB) was used. Linearized pRE112 plasmid backbone was subsequently digested with DpnI (NEB) to remove any remaining circular template DNA. To generate an unmarked gene insertion, the region upstream of the ssrB insertion site was amplified using primers KK_003 and KK_004 and the region downstream of the insertion site was amplified using primers KK_007 and KK_008. The ssrB coding region and native promoter were amplified using primers KK_005 and KK_006. Linearized pRE112, upstream insertion site fragment, ssrB fragment, and downstream insertion site fragment were assembled to form pRE112-ssrB using Gibson Assembly22.The plasmid pRE112-pipB2, steA, sseJ, and sseF were amplified from SL1344 genomic DNA using the indicated primers: pipB2: KK_009 and KK_010, sseJ: KK_011 and KK_012, steA: KK_013 and KK_014. The pSseF-2HA plasmid was constructed by amplifying the sseABCDEFG operon promoter the using primers KK_015 and KK_016. Expression and secretion of SseF is dependent on the chaperone protein SscB23. The coding regions of sscB and sseF were amplified using primers KK_017 and KK_018. The sseABCDEFG operon promoter was ligated to upstream end of sscB using Gibson Assembly. Each coding region plus native promoters was SalI/XhoI digested and ligated into SalI/XhoI digested pSopD2 with T4 DNA ligase (Invitrogen).Protein coding and regulatory regions of 21. Briefly, pSopD2-2HA was SalI/XhoI digested. Protein coding regions plus 300–500 bp upstream of the coding region containing each gene’s native promoter were amplified from SL1344 genomic DNA, SalI/XhoI digested, and ligated into SalI/XhoI digested pSopD2-2HA with T4 DNA ligase (Invitrogen) resulting in a double hemagglutinin (HA)-tag at the C-terminal end of each effector all under the control of each effector’s native promoter. All cloned constructs were verified by DNA sequencing (Genewiz).Plasmids pPipB2-2HA, pSteA-2HA, pSseJ-2HA, and pSseF-2HA were constructed from the pSopD2-2HA plasmidpipB2, and the codons for the first 5 amino acids of PipB2. The amplified linearized plasmid was XhoI (NEB) digested and ligated back together, resulting in a re-circularized plasmid with truncated pipB2 fused to the double HA tag under the control of the pipB2 native promoter (pACYC-2HA). The resulting plasmid was transformed into E. coli DH10B for plasmid propagation and sequencing. Plasmid was sequenced (Genewiz) prior to transformation into the T3SS2+ssrB overexpression strain (see below) resulting in the T3SS2+pACYC-2HA strain.To generate the plasmid pACYC-2HA, the plasmid pPipB2-2HA was simultaneously amplified and linearized using primers KK_019 and KK_020. The entire plasmid except for amino acids 6–350 of PipB2 were amplified including: the entire backbone of pACYC184, the native promoter for ssrB resulting in the T3SS2+ hypersecretion mutant was created by conjugating MFDpir transformed with the plasmid pRE112-ssrB with wild type SL1344 to make an unmarked chromosomal duplication of ssrB in the SL1344 chromosome between the genes dppA and SL1344_3597, specifically at the 3,837,749 base pair position according to the genome assembly NCBI_accession:NC_016810. Pro-conjugation single crossover mutants between pRE112-ssrB and the SL1344 chromosome were selected on LB agar plates containing chloramphenicol. Sucrose counter-selection was performed as described previously24 to select for the second crossover event, thus inserting ssrB into the chromosome and creating the T3SS2+ mutant. Unmarked complete deletion mutants in the T3SS2+ background were generated as described elsewhere11.The chromosomal duplication of ® CCL-2™) were routinely cultured in normal growth media: Dulbecco’s Modified Essential Medium (DMEM) (Hyclone) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco), 1% (v/v) Glutamax (Gibco), and 1% (v/v) nonessential amino acids (Gibco). Cells were maintained at 37 °C in a 5% CO2 atmosphere.HeLa cells  (Hyclone), diluted in DMEM, and added to the HeLa cells at a multiplicity of infection (MOI) of 100:1. The infection proceeded for 15 min at 37 °C in 5% CO2 before non-internalized bacteria were removed by three washes in DPBS and cells incubated in growth media containing 100 µg/mL gentamicin until 2 h post-infection, followed by growth media containing 10 µg/mL gentamicin for the remainder of the experiment. HeLa cells were infected for a total of 8 h.HeLa cells were infected as previously describedSalmonella antibody CSA-1 (Kirkegaard and Perry Laboratories) was used at a dilution of 1:300. The mouse anti-LAMP1 antibody H4A3c developed by J.T. August and J.E.K. Hildreth, obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa , was used at a dilution of 1:300. The rabbit anti-Annexin A2 polyclonal antibody  was used at a dilution of 1:300. Alexa 647-labelled phalloidin and secondary antibodies were obtained from Thermo Fisher Scientific and used at a dilution of 1:500: Alexa plus 405-conjugated donkey anti-goat, Alexa 488-conjugated donkey anti-mouse, Alexa 568-conjugated donkey anti-goat, and Alexa 568-conjugated donkey anti-rabbit.The primary goat polyclonal anti-26. In brief, cell monolayers seeded on glass coverslips were fixed with 4% (v/v) paraformaldehyde in DPBS at room temperature for 10 min and washed three times in DPBS. Excess paraformaldehyde was quenched with 50 mM ammonium chloride for 10 min at room temperature followed by two washes in DPBS. Cells were permeabilized in ice-cold acetone for 5 min at − 20 °C and then blocked in 1% bovine serum albumin  (Sigma) in DPBS for 30 min at room temperature. Cells on coverslips were then incubated with primary antibodies diluted in 1% BSA in DPBS at room temperature for 1 h followed by three washes in DPBS. Secondary antibodies diluted in 1% BSA in DPBS were added to the coverslips and incubated at room temperature for 1 h and then washed once with DPBS. Cells were then incubated for 10 min at room temperature with DAPI (Invitrogen) diluted in DPBS followed by two DPBS washes. Cells were then washed in deionized water prior to mounting with ProLong Gold Antifade Mountant (Life Technologies) on glass slides. Microscopy was performed using Olympus IX81 (100× objective) and Zeiss Axio Imager M2 (100× objective) microscopes using SlideBook 4.1.0 and Zeiss Zen Pro softwares, respectively. Images were further analyzed with ImageJ version 2.1.0/1.53c and Adobe Photoshop version 21.0.1. Confocal microscopy was performed using a Leica SP8 confocal microscope (100× objective) using Leica Application Suite X software and Adobe Photoshop version 21.0.1.Cells were fixed, permeabilized, and stained essentially as previously described+-tubules, we imaged at least one infected cell per field of view, then quantified the number of infected and uninfected cells per field of view. Infected cells were then scored for the presence or absence of LAMP1+-tubules radiating outwards from labelled Salmonella. At least 60 infected cells per strain were scored blind in each experiment, and each experiment was repeated at least three times.To quantify the number of infected cells with LAMP1Salmonella and the nuclei of infected cells, immunofluorescent images were analyzed by a custom CellProfiler software (version 4.2.1)27 pipeline. Host nuclei (stained blue) and Salmonella (red) were identified as primary objects and the minimum distance (edge to edge) between the two was measured (in µM) and enumerated. At least 200 Salmonella-to-nucleus distances were measured, with three replicates.To quantify the distance between S. Typhimurium, processed for immunofluorescence microscopy, and stained for Salmonella, LAMP1, and DNA/nucleus as described above, as well as actin with Alexa 647-labelled phalloidin.Human AnxA2 expression was silenced via RNA interference (RNAi) using commercially available AnxA2 small hairpin RNA (shRNA) lentiviral particles . Scrambled control shRNA lentivirus particles were used as a negative control . Briefly, HeLa cells in twelve-well tissue culture plates at approximately 50% confluency were treated with DMEM containing 10 µg/mL of Polybrene  and infected by adding shRNA lentiviral particles at a MOI of 1 for 24 h, following the manufacturer’s instructions. To select stable cells expressing the shRNA, HeLa cells were treated with puromycin dihydrochloride  at a final concentration of 10 µg/mL. Knockdown efficiency was assessed by western blotting of whole-cell lysates using rabbit α-Annexin A2 polyclonal antibodies , with Beta Tubulin used as a loading control and probed with mouse α-Beta Tubulin antibodies (Sigma). These AnxA2 knockdown and control cells were then infected with 2H4-lysine and 0.1 mM 13C6-arginine (Cambridge Isotope Laboratories) for heavy cells or normal isotopic abundance of l-lysine and l-arginine (Sigma) for light cells. Cells were maintained in labelling media for at least 5 cell divisions to ensure complete labelling as described elsewhere28.HeLa cells were split from normal growth media into arginine and lysine-free DMEM (Caisson Laboratories Inc.) supplemented with 10% heat inactivated dialyzed FBS (Gibco) and either 1 mM 6 cells per 15 cm culture plate 16–24 h prior to infection. For all HeLa cell infections: overnight bacterial cultures were subcultured 1:33 in LB without antibiotics and incubated for 3 h at 37 °C with shaking. 1 mL of bacterial cultures was pelleted and resuspended in Dulbecco’s Phosphate-Buffered Saline (DPBS) (Hyclone), diluted in the appropriate isotope-supplemented DMEM, and added to the HeLa cells at a multiplicity of infection (MOI) of ≈ 100:1. Infections proceeded for 15 min at 37 °C in 5% CO2, after which non-internalized bacteria were removed by three washes in DPBS and cells incubated in growth media containing 100 µg/mL gentamicin until 2 h post-infection, followed by growth media containing 10 µg/mL gentamicin for the remainder of the experiment. HeLa cells were infected for a total of 8 h.Light and heavy labelled HeLa cells were seeded at a density of approximately 4.5 × 10+∆sifA-pSifA-2HA, T3SS2+∆sopD2-pSopD2-2HA, T3SS2+∆pipB2-pPipB2-2HA, T3SS2+∆steA-pSteA-2HA, T3SS2+∆sseJ-pSseJ-2HA, T3SS2+∆sseF-pSseF-2HA, or T3SS2+pACYC-2HA (vector control)  Table , while sg for 10 min in a centrifuge set to 4 °C. The supernatant was removed, and cell pellets were resuspended in RIPA buffer ) and incubated on ice for 10 min. Cell lysates were centrifuged for 20 min in a 4 °C microcentrifuge at maximal speed to pellet cell debris. Protein concentrations were determined using a Bicinchoninic Acid Assay (BCA) (Sigma). Lysates from cells infected with each of the seven different T3SS2+ strains were then mixed at a 1:1 ratio with lysate from the opposite SILAC labelled HeLa cells infected with the vector control T3SS2+-pACYC-2HA. e.g., lysate from light labelled HeLa cells infected with T3SS2+∆sifA-pSifA-2HA was combined at a 1:1 ratio with lysate from heavy labelled HeLa cells infected with T3SS2+-pACYC-2HA. 25 µL of anti-HA magnetic beads (Thermo Fisher Scientific) was added to the mixed lysates and incubated with end-over-end rotation overnight at 4 °C. Immunoprecipitated proteins were eluted from the magnetic beads using a basic elution protocol according to manufacturer’s specifications. Eluted proteins were separated by SDS-PAGE, bands excised, and subjected to in-gel tryptic digestion and proteomic analyses as previously described29.Infected light and heavy labelled cells were washed three times in ice cold DPBS and manually detached from the culture dish containing 5 mL ice-cold DPBS and transferred into 15 mL tubes. Cells were spun at 300×27. Peptides were searched on MaxQuant version 1.6.7.030 against UniProt sequence database (uniport.org) for Homo sapiens , S. Typhimurium , and software provided common contaminants (246 entries). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE28 partner repository with the dataset identifier PXD025582.The resulting peptides of the immunoprecipitated proteins after in-gel tryptic digestion were analyzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS) using a Bruker Impact II Qtof as described elsewhere32. Overnight cultures of S. Typhimurium strains in LB-broth were subcultured 1:100 in fresh LB broth containing no antibiotics. Cultures were grown at 37 °C for 6 h with shaking after which the optical density at 600 nm was measured. 3 mL of culture were centrifuged at 9500×g for 10 min at 4 °C. Culture supernatant was passed through a 0.22 µm filter, precipitated with trichloroacetic acid  at a final concentration of 10% (v/v), and incubated on ice at 4 °C overnight.Effectors secreted by SPI-1 encoded type III secretion system (T3SS1) were analyzed by way of a SPI-1 secretion assay as described elsewhere33. 1 mL of overnight S. Typhimurium cultures was washed twice in a low phosphate and low magnesium-containing medium (LPM), then inoculated at a 1:50 dilution in 30 mL of LPM at a pH of 5.8. LPM composition was 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 80 mM MES, 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 24 µM MgCl2, 337 µM PO43−. Cultures were grown at 37 °C with shaking for 9 h after which the optical density at 600 nm was measured. Bacteria were collected by centrifugation for 30 min at 3000×g in a 4 °C centrifuge. Culture supernatant was passed through a 0.22 µm filter, precipitated with TCA at a final concentration of 10% (v/v), and incubated on ice at 4 °C overnight.Effectors secreted by T3SS2 were analyzed by way of a SPI-2 secretion assay as previously describedg at 4 °C. Precipitate pellets were then washed in ice cold acetone and centrifuged at maximal speed in a 4 °C microfuge. Pellets were air-dried before resuspension in a volume of 2× SDS-PAGE sample buffer  normalized to A600 of the original culture and boiled for 10 min. Samples were separated by SDS-PAGE using 12% polyacrylamide gels and stained with Coomassie Brilliant Blue R-250.For T3SS1-secreted proteins: The TCA insoluble fraction was collected by centrifugation for 45 min at 13,000×g at 4 °C. Pellets were solubilized with a volume of 2× SDS-PAGE sample buffer adjusted according to the A600 of the original culture.For T3SS2-secreted proteins: The TCA insoluble fraction was collected by centrifugation for 45 min at 14,000×600, were separated on SDS-12% polyacrylamide gels, transferred to Pure Nitrocellulose membranes (Bio-Rad) using a wet transfer cell. Membranes were blocked in blocking buffer  Tween 20 (TBS-T) and 5% (w/v) non-fat milk) overnight at 4 °C. Blots were incubated with the following primary antibodies in blocking buffer for one hour at room temperature: rabbit affinity-purified antibodies raised against recombinant SseB and SseD (1:1500), rat anti-HA monoclonal antibody , or mouse anti-beta-tubulin . Three 5-min wash steps were performed using TBS-T followed by room temperature incubation of membranes with secondary antibodies. Secondary antibodies conjugated to horseradish peroxidase  were diluted to 1:5000 in blocking buffer. Antibody complexes were detected using ClairtyTM Western ECL Substrate  prior to detection in a BioRad Gel Imaging System.Proteins from equivalent numbers of bacterial cells, as determined by the A+∆sopD2-pSopD2-2HA, T3SS2+∆pipB2-pPipB2-2HA, and T3SS2+-pACYC-2HA were grown in LB for 9 h at 37 °C with shaking. SPI-2 expression was induced by washing cell pellets from cultures twice in LPM 2SO4, 0.5 mM K2SO4, 80 mM MES, 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 24 µM MgCl2, 337 µM K2HPO4/KH2PO4 (pH 7.4)33) prior to inoculation at a 1:50 dilution in 400 mL of LPM at a pH of 5.8. Cells were pelleted by centrifugation and culture supernatant was filter sterilized by passage through a 0.22 µm filter. Sterilized culture supernatant was concentrated by passage through a 10 kDa molecular weight cut-off centrifugal filter unit (Millipore Sigma). Protein concentration of the concentrated culture supernatant was determined by BCA assay. Approximately 20 µg of concentrated culture supernatant was mixed with 5 µg of recombinant human His6-tagged annexin A2  and incubated with end-over-end rotation at 4 °C overnight. Samples were incubated with Ni-Sepharose High Performance Beads (Sigma) for two hours at room temperature. Beads were washed four times with TBS-T , resuspended in 160 µL SDS-PAGE loading buffer, and boiled for 10 min prior to Western blot analysis.Strains T3SS2Samples were separated on 12% polyacrylamide gels and transferred to Pure Nitrocellulose (Bio-Rad) using a wet transfer cell. Membranes were blocked with blocking buffer ) overnight at 4 °C. Membranes were incubated with the following primary antibodies diluted in blocking buffer for 1 h at room temperature: α-HA  diluted 1:2000, mouse α-beta tubulin (Abcam) diluted at 1:1500, and α-Annexin A2 Clone 5  diluted 1:2000. Membranes were washed three times for five minutes each with TBS-T prior to incubation with the following secondary antibodies diluted in blocking buffer for one hour at room temperature: HRP-conjugated goat α-rat or goat α-mouse (Sigma) diluted 1:5000. Membranes were washed as described above and treated with ClarityTM Western ECL Substrate (Bio-Rad) prior to chemiluminescent developing.https://string-db.org/). Graphs were made using Prism9 (GraphPad).Mass spectrometry data were analyzed in Perseus version 1.6.7.0. STRING analysis was performed using the STRING database online tool version 11.0 . Analysis was performed using a Mann–Whitney test to compare two groups and two-way ANOVA for more than one group with Tukey’s multiple comparison test as appropriate. Aggregate results represent the mean ± standard error on the mean.S. Typhimurium SL1344 that specifically secretes increased amounts of T3SS2-secreted effectors, but a similar amount T3SS1-secreted effectors relative to wild type SL1344.A barrier to identifying specific and significant T3SS2-secreted effector interactions is that the quantity of T3SS2-secreted effectors pales in comparison to the abundance of both T3SS1-secreted effectors and host proteins. This hampers identification of T3SS2-effector binding partners as their low abundance confounds separation of specific binding partners from the background noise of mass spectrometry. It is therefore inadequate to rely on native expression of T3SS2 effectors to identify binding partners. To mitigate this limitation, we created an isogenic strain of 37. Overexpression of plasmid-encoded SsrB has previously led to discovery of additional T3SS2-secreted effectors within an in vitro T3SS2-secretome analysis38 but ssrB overexpression has to our knowledge never been used to identify effector-host binding partners. ssrB was overexpressed by duplicating ssrB including its native promoter within the chromosome of SL1344 in an intergenic region distant from SPI-2 and between the genes dppA and SL1344_3597 (a putative xanthine permease). The ssrB overexpression strain will hereafter be referred to as the T3SS2+ strain.The two-component regulatory system SsrA/SsrB encoded within SPI-2 is a major regulator of virulence associated events linked to SPI-2. The response regulator SsrB binds to all promoters within SPI-2 and is indispensable for expression of the T3SS2 secretion apparatus and its associated effectors encoded within SPI-2, as well as effectors encoded outside SPI-2ssrB in the T3SS2+ strain neither increased nor decreased T3SS1-secretion relative to wild type  provide a convenient means to assess T3SS2 secretionseD Fig. b and SseseD Fig. c relativseD Fig. e. The ΔsssrB overexpression does not appear to have an effect on T3SS1 secretion we hypothesized that increased T3SS2-secretion in the T3SS2+ strain would affect primarily T3SS2-, but not T3SS1-, mediated processes. It follows then that increased T3SS2-secretion should increase T3SS2-mediated events such as increased SIF biogenesis frequency during infection. The T3SS2+ strain does not infect a significantly higher proportion of HeLa cells relative to wild type and the T3SS2-defective ΔssaR strain  relative to wild type infected cells (24.94 ± 2.61% of infected cells with SIFs)  were generated in the T3SS2+ΔsopD2, T3SS2+ΔsseJ, and T3SS2+ΔsseF compared to their respective uncomplemented counterpart strains  and not part of the background noise associated with mass spectrometry. Thus, these proteins demonstrate a relative difference in quantity between control and infection, thus indicating enrichment of that protein and suggestive of an interaction with the tagged effector. 273 proteins passed this threshold which were then used for subsequent analysis.We set out to systematically investigate effector-effector and effector-host protein interactions during 47 on these proteins for each of the six effector-IPs to identify trends or common interaction partners or networks amongst the effector-IPs. STRING is a database of known protein–protein interactions including both direct and indirect associations. Direct associations are protein–protein binding events and indirect associations are proteins interacting together on a similar pathway, but not directly binding to each other. STRING analysis of the proteins with SILAC ratios greater than 1.2 from the SopD2-IPs and PipB2-IPs revealed several identified proteins in common that may interact either directly or indirectly  suggesting that vimentin and alpha-actinin-4 may non-specifically bind to the anti-HA coated magnetic beads.Vimentin was found with a median 1.579-fold and 1.530-fold increase in SopD2 and PipB2 IPs relative to control, respectively. AnxA2 was found with a median 1.213-fold and 1.313-fold increase in SopD2 and PipB2 IPs relative to control, respectively. Alpha-actinin-4 was found with a median 1.225-fold and 1.268-fold increase in SopD2 and PipB2 IPs relative to control, respectively. Finally, plectin was found with a median 1.629-fold and 1.752-fold increase in SopD2 and PipB2 IPs relative to control, respectively. It is important to note that vimentin and alpha-actinin-4 were also found with SILAC ratios around 1 in ControlS. Typhimurium invasion sites in a T3SS1-dependent manner and is required for efficient invasion48. We decided to validate the interaction between AnxA2 and both SopD2 and PipB2 given that AnxA2 plays an important role in multiple steps of host cell membrane trafficking49. S. Typhimurium alters host endosome maturation in a T3SS2-dependent manner, and AnxA2 immunoprecipitated along with the T3SS2 effectors SopD2 and PipB2. To do so, we performed a reciprocal pull-down, using purified His6-tagged AnxA2 to enrich for SopD2-2HA and PipB2-2HA. Concentrated culture supernatant from T3SS2+∆sopD2-pSopD2-2HA, T3SS2+∆pipB2-pPipB2-2HA, and T3SS2+ -pACYC-2HA grown under SPI-2 inducing conditions was mixed with His6-tagged AnxA2, AnxA2 complexes were pulled-down against the His-tag, and analyzed by Western blotting. Both SopD2-2HA and PipB2-2HA were successfully pulled-down along with His6-AnxA2 , to the Ni-Sepharose beads makes it difficult to conclude with certainty that PipB2 specifically binds to AnxA2, and alternative methods and future experiments might be needed to validate this interaction. However, in three independent experiments increased amounts of PipB2 were pulled down in the presence of AnxA2 as compared to in the absence of AnxA2  and other host proteins known to be involved in SIF biogenesis and SCV membrane maintenance identified in previous studies  and immunoprecipitation coupled with mass spectrometry (IP-MS) to investigate T3SS2-secreted effectors known to manipulate host intracellular trafficking 60. This is the common approach owing to the relatively low abundance of T3SS2 effectors present within the host cell during infection. We demonstrated in a previous study that S. Typhimurium requires multiple effectors to establish its replicative niche within host cells, highlighting the need to study effectors as an ensemble rather than individually11. We hypothesize that multiple effectors may bind a single host target, either cooperating simultaneously or sequentially, to exert their function. Consequently, the absence of any single effector, whether through deletion or transfection of select effectors, may drastically impact identification of host binding partners. Thus, we created an isogenic S. Typhimurium strain that translocates an increased abundance of T3SS2 effectors into host cells during infection. To our knowledge, no one has attempted to identify multiple T3SS2-effector binding partners during infection while not heavily relying on transfection.A popular approach to studying T3SS2-effectors—namely SifA, SopD2, PipB2, SteA, SseJ, SseF, and SseG—is to transfect individual effectors into host cells and identify cognate binding partnersSalmonella-infected cells  and fungal (such as Cryptococcus neoformans) infections, but may also be exploited for adhesion, invasion and proliferation by certain bacterial  and viral (such as human papillomavirus) pathogens61. AnxA2 has previously been shown to be involved in T3SS1-mediated Salmonella invasion48, has only recently been shown to interact with SseI and SseL63 in macrophages, but has not been shown to be involved in T3SS2-mediated processes in epithelial cells. We hypothesize that AnxA2 could be involved in the T3SS2-mediated linkage between the SCV and the actin nest surrounding the SCV.Here, we demonstrated that the T3SS2-secreted effector SopD2, and likely PipB2, target the host protein AnxA2. We first showed that SopD2-2HA and PipB2-2HA bind to AnxA2 by using SILAC-based IP-MS analysis of proteins from lls Fig. , and thells Fig. , althoug∆sopD2 and ∆pipB2 have reduced replication within macrophages and also have altered subcellular localization relative to wild type infected cells65. These altered phenotypes in the ∆sopD2 and ∆pipB2 single mutants could be explained by altered interactions with the S. Typhimurium-induced vacuole-associated actin polymerization (VAP). The VAP plays an important role in SCV membrane integrity and may be involved in regulating the subcellular localization of the SCV during infection67. AnxA2 functions at sites of actin association with membranes enriched in cholesterol, such as the membrane of the SCV68. More specifically, AnxA2 is enriched at the junction between vesicles and actin, possibly functioning as a barbed-end capping protein49. Given that SopD2 and PipB2 bind AnxA2, we hypothesize that SopD2 and PipB2 recruit AnxA2 to the interface between the SCV and VAP to stabilize the F-actin filaments and link the SCV to the VAP. Deletion of SopD2 would lessen contact, or even completely detach the SCV from VAP, resulting in the observed scattered distribution of SCVs within the host cell cytoplasm as well as the decreased replication in macrophages for the ∆sopD2 mutant. Indeed, we show that RNAi knockdown of AnxA2 in HeLa cells alters intracellular positioning of SCV of wild type S. Typhimurium . As previously mentioned, past studies relied heavily on transfection of single effectors into host cells to identify interacting proteins, discounting the possibility that two effectors are required for binding to the same host protein. Here, we infected host cells with S. Typhimurium with its entire T3SS2-secreted effector arsenal at its disposal, a strategy with its own advantages and disadvantages over transfection.We were unable to identify any known interactors from previous studies in this work. However, it is important to note that our method differed in key ways from previous studies (reviewed in Ref.+ssrB overexpression strain which increases expression and secretion of T3SS2-secreted effectors. Nevertheless, the amount of tagged effector within the host cell is likely orders of magnitude less than that of a transfected effector. Protein complex formation is dependent on the stoichiometry of the interacting proteins. High protein concentrations resultant from effector transfection will shift the formation/dissociation equilibrium towards strong binding with one protein present at a greater molar ratio than the effector e.g., the known interactors70. Given the low concentration of tagged effectors in our study, the formation/dissociation equilibrium is shifted toward what would be natively expected during normal cell infection, allowing for identification of different binding partners. This may explain why we did not identify known interactors.The biggest disadvantage to our technique is that the concentration of each effector within the host cell is low as compared to effectors introduced by transfection. Low effector concentration within the host cell makes it difficult to separate specific effector interactions from the background noise during mass spectrometry. We attempted to mitigate this limitation by creating and using the T3SS2The advantage of our native infection-based mass spectrometry study is that all effectors are present, permitting both effector-effector interactions and maintaining all effectors at biologically relevant levels, enabling identification of new effector binding partners. Effector transfection may introduce artificially high signal from the more highly expressed host proteins and drown out true effector binding partners. The absence of known binding partners in our study does not indicate a failed experiment, but rather highlights the differences in techniques used to identify host binding partners for T3SS2-secreted effectors.S. Typhimurium native infection in SILAC labelled cells as a valuable strategy to identify T3SS2-secreted effector binding partners. Using this technique, we demonstrate that SopD2 and PipB2 both bind to AnxA2 which may play a role in SCV intracellular positioning during infection. Further work is required to ascertain the precise role of AnxA2 during infection and the impact of AnxA2 binding to SopD2 and PipB2. Successful identification of a host protein associated with S. Typhimurium infection, and two T3SS2-effectors that bind that host protein, potentially explain the nuanced phenotypes observed in the single effector deletion mutants. Our work highlights that a global view of T3SS2-secreted effectors during infection is necessary to unravel the complexities of the S. Typhimurium intracellular replicative niche.Collectively, this study highlights the advantages of Supplementary Information.


PDCD4 stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the PABP, which also stimulates peptide release, PDCD4 activity in translation termination increases. PDCD4 regulates translation termination by facilitating the binding of release factors to the ribosome, increasing the GTPase activity of eRF3, and dissociating eRF3 from the posttermination complex. Using a toe-printing assay, we determined the first stage at which PDCD4 functions—binding of release factors to the A-site of the ribosome. However, preventing binding of eRF3 with PABP, PDCD4 suppresses subsequent rounds of translation termination. Based on these data, we assumed that human PDCD4 controls protein synthesis during translation termination. The described mechanism of the activity of PDCD4 in translation termination provides a new insight into its functioning during suppression of protein biosynthesis.Programmed cell death 4 protein (PDCD4) regulates many vital cell processes, although is classified as a tumor suppressor because it inhibits neoplastic transformation and tumor growth. For example, PCDC4 has been implicated in the regulation of transcription and mRNA translation. PDCD4 is known to inhibit translation initiation by binding to eukaryotic initiation factor 4A and elongation of oncogenic c- and A-myb mRNAs. Additionally, PDCD4 has been shown to interact with poly(A)-binding protein (PABP), which affects translation termination, although the significance of this interaction is not fully understood. Considering the interaction between PABP and PDCD4, we hypothesized that PDCD4 may also be involved in translation termination. Using  Programmed cell death 4 protein (PDCD4) is a broad regulator of vital processes occurring in cells, such as cell cycle progression , transcrThe PDCD4 protein can migrate between the nucleus and cytoplasm as it contains two nuclear export signals. Under normal growth conditions, PDCD4 is mainly located in the nucleus; however, PDCD4 is predominantly cytoplasmic upon serum withdrawal . The locA). The MA3 domain belongs to the families of HEAT  repeat domains. HEAT is a structural motif consisting of repeats of two α-helices connected by a short loop (A) to suppress its RNA helicase activity, thus suppressing the translation of mRNA with structured 5′UTRs (in vitro binding experiments (A), and the major site of this interaction is with the RNA recognition motif 1 to 2 (RRM) domains of PABP.The mechanism of PDCD4 functioning in the cytoplasm appears to be closely related to the regulation of protein translation. PDCD4 contains the N-terminal RNA-binding domain and two MA3 domains located in the central and C-terminal regions of the protein A. The MAort loop . The MA3ort loop . The MA3ort loop A to supperiments . The N-teriments . PDCD4 ieriments . Additioeriments , 26. Onleriments A, and thvia its N domain -tagged PDCD4 or PDCD4 without the tag  was used in the experiments.Previously, we showed that PABP facilitates translation termination th eRF3а . Since Pet al. (A) was fixed in the P-site by the mutant eRF1(AGQ), which is unable to hydrolyze peptidyl-tRNA to accomplish the process. These complexes were purified from the rabbit reticulocyte lysate (RRL) by sucrose density centrifugation at the high ionic strength, which resulted in eRF1(AGQ) dissociation from the ribosome. The addition of release factors to purified preTC(NL-polyA) triggered peptidyl-tRNA hydrolysis and the release of NLuc from the ribosome. This, in turn, can be detected by luminescence. To address the possible effect of PDCD4 in the complex with PABP on the translation termination process, eRF1, eRF3a, PDCD4, and PABP were added to the purified preTC(NL-polyA) simultaneously or in different combinations, and the luminescence was measured as a function of time.The final step of translation termination is the hydrolysis of peptidyl-tRNA and the release of the nascent peptide from the ribosome. Analysis of peptide release efficiency was performed using the highly sensitive Termi-Luc approach developed recently by Susorov et al. . PreTCs et al. A was fixB). Yet, the proteins act synergistically, as the joint addition of PDCD4 and PABP increased peptide release more strongly than the separate addition of proteins. To ensure that the effect of PDCD4 is not associated with the stabilization of release factors or the nonspecific prevention of their sorption in test tubes because of very low eRF concentrations, we added GST to the reaction in the presence of release factors as a negative control. This addition revealed no effect on peptide release.Clearly, PDCD4 activates translation termination in the presence of PABP and, quite unexpectedly, in its absence B. Yet, tA). Thus, the stimulation of peptide release from the terminating ribosome by PDCD4 indicates its involvement in the process of human translation termination.Importantly, we used a low concentration of the release factors, to detect the maximum stimulation of PDCD4. The titration curves of the release factors demonstrated that the efficiency of PDCD4 stimulation of peptide release depended on the concentration of release factors A. Thus, in vivo levels of PDCD4 and release factors were determined by the analysis of published mass spectrometry data . As a result, a preTC(MVHL) containing a stop codon at the A site of the ribosome and peptidyl-tRNA at the P site was formed. After purification of the preTC(MVHL) from the unbound translation factors by sucrose gradient centrifugation, it was possible to study the influence of individual proteins on the subsequent translation termination stages. During translation termination, eRF1, in the complex with eRF3, recognized the stop codon and formed a termination complex 1 (TC1); then, eRF3 hydrolyzed GTP and positioned eRF1 in the peptidyl transferase center of the ribosome, forming TC2 (A). This triggered peptidyl-tRNA hydrolysis, and the complex passed into the posttermination stages (postTC1 and postTC2), followed by ribosome recycling. Addition of the non-hydrolysable GTP analogue, GDPCP, to the translation termination inhibited GTP hydrolysis and thus transition to the TC2 stage (A). The G183A mutant of eRF1 (eRF1(AGQ)), which was unable to hydrolyze peptidyl-tRNA, inhibited the transition to the postTC and freezed the termination process at the TC2 stage (A). Therefore, it is possible to investigate events occurring during TC formation in a great detail , indicating the involvement of PDCD4 in all stages of translation termination. It should be noted that we observed stimulation of TC1 formation in the presence of GDPCP by PDCD4, but it was weaker than in the presence of GTP.To study the effect of PDCD4 on the formation of termination complexes, PDCD4 along with eRF1 or eRF1(AGQ) and eRF3a was incubated with preTC(MVHL) in the presence of GTP or GDPCP, and the efficiency of TC1, TC2, and postTC1 formation was analyzed using a fluorescent toe-print assay .To study the possible effect of PDCD4 in the complex with PABP on TC2 formation, PDCD4, eRF1(AGQ), eRF3a, and PABP were incubated with preTC(MVHL), and the efficiency of TC2 formation was analyzed. We showed that PABP did not interfere with PDCD4 in the stimulation of TC2 formation and even increased the efficiency of TC2 formation in the presence of a poly(A) tail B and S3ATherefore, we confirmed PDCD4 activity in translation termination observed in peptide release experiments and demonstrated that this protein is involved in different termination steps.32P]-labeled GTP, we detected an increase in GTPase activity of the full-length eRF3a or its truncated form eRF3c, in the presence of PDCD4 (B). Interestingly, PDCD4 stimulated the GTPase activity of eRF3a more strongly than eRF3c. This finding correlates with the stimulation of translation termination in previously described experiments. To demonstrate the key role of PDCD4 in the GTPase activity of eRF3, we performed peptide release experiments with the non-hydrolyzable GTP analogues GDPCP, GMPPNP, and GTP-γ-S (C). When the GTP analogues were added to peptide release, no stimulation of translation termination by PDCD4 was detected. Thus, stimulation of peptide release by PDCD4 is performed via activation of the GTPase reaction on the ribosome.One of the intermediate stages of translation termination is GTP hydrolysis performed by eRF3, which occurs after eRF1-eRF3 binding to the stop codon in the ribosome. Using the GTPase test with  vector (Promega), and 50 nt poly(A) tail.The NLuc mRNA was transcribed 2, 0.2 mM ATP and GTP, 0.04 mM 20 amino acids (Promega), 2 mM DTT, and 8 mM creatine phosphate to a final volume of 1 ml. The mixture was preincubated with 1 μM eRF1(AGQ) at 30 °C for 10 min, followed by the addition of NLuc mRNA to a final concentration of 8 μg/ml, resulting in the formation of preTCs. Next, KOAc concentration was adjusted to 300 mM, and the mixture was layered on a 10 to 35% linear sucrose gradient in buffer containing 50 mM HEPES-KOH, pH 7.5, 7.5 mM Mg(OAc)2, 300 mM KOAc, and 2 mM DTT. The gradients were centrifuged in a SW-41 Ti (Beckman Coulter) rotor at 18,000 rpm for 14 h. Fractions enriched with preTC NL were collected. PreTC NL was aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C. Peptide release was performed in a solution containing 7.5 nM preTC, 50 mM Hepes-KOH, pH 7.5, 0.25 mM spermidine, 2 mM DTT, 0.2 mM GTP, and 1% NLuc substrate  in the presence of release factors (8 nM eRF1(AGQ) alone or with 8 nM eRF3a/eRF3c) and proteins of interest . Luminescence was measured at 30 °C using a Tecan Infinite 200Pro (Tecan). The peptide release kinetic curves were generated, and the standard deviation was calculated using Microsoft Excel.A reaction mixture containing 50% RRL (Green Hectares) was supplemented with 30 mM Hepes-KOH (pH 7.5), 50 mM KOAc, 1 mM Mg(OAc)E. coli strain BL21 with subsequent protein purification on Ni-NTA agarose and ion-exchange chromatography  from the linear fragment of the pET28-MVHL-UAA plasmid containing the T7 promoter, four CAA repeats, β-globin 5′-UTR, and ORF (encoding for the peptide MVHL), followed by the stop codon UAA and 3′-UTR, comprising the remaining natural β-globin coding sequence and, optionally, a 50 nt poly(A) tail. PreTCs on MVHL mRNA with and without poly(A) were assembled and purified as previously described  supplemented with 400 U RiboLock RNase inhibitor (Thermo Fisher Scientific), 1 mM ATP, 0.25 mM spermidine, 0.2 mM GTP, 75 μg calf liver total tRNA , 75 pmol human 40S and 60S purified ribosomal subunits, 200 pmol eIF2, 60 pmol eIF3, 400 pmol eIF4A, 150 pmol eIF4B, and 125 pmol each of human eIF2, ΔeIF4G, eIF1, eIF1A, eIF5, and ΔeIF5B for the formation of the 80S initiation complex. PABP (37 pmol) was added to the reaction to obtain the MVHL-poly(A)-PABP complex. After 15 min, 200 pmol of human eEF1H and 38 pmol of eEF2 were added to form the elongation complex. The ribosomal complexes were then purified via centrifugation in a Beckman SW55 rotor for 95 min at 4 °C and 48,000 rpm in a 10 to 30% (w/w) linear SDG prepared in buffer A with 5 mM MgCl2. Fractions corresponding to the ribosomal complexes were diluted threefold with buffer A containing 1.25 mM MgCl2  and used in experiments.The 40S and 60S ribosomal subunits, as well as eukaryotic translation factors eIF2, eIF3, eEF1H, and eEF2, were purified from RRL or HeLa lysate, as previously described . The humtography . Human etography . mRNAs . All data were normalized to the ribosomal shift efficiency calculated in the control experiments.First, 0.03 pmol preTCs incubated with release factors (0.35 pmol eRF1(AGQ) alone or 0.02 pmol eRF1(AGQ)/eRF1 with 0.06 pmol eRF3a/eRF3c) and proteins of interest  at 37 °C for 10 min in the presence of 0.2 mM GTP or GDPCP were supplemented with equimolar amounts of MgCleviously . Using s4Cl, 30 mM KCl, 6 mM MgCl2) containing 2 μM [γ-32P] GTP with a specific activity of 2.16 μCi/pmol was added. Next, 4 pmol of PDCD4, and its domains MA3m, MA3c, MA3m-c, or eIF4A were added to the reaction. The reaction was initiated by adding 1 pmol of eRF3a or eRF3c, and the final reaction volume was 10 μl. After incubation for 10 min at 37 °C, the reaction was stopped by adding 500 μl of a solution containing 5% charcoal in 50 mM NaH2PO4 to bind free GTP. Finally, the charcoal was precipitated via centrifugation at 12,000g, and 380 μl of the supernatant was subjected to liquid scintillation counting to quantify the released [32P] phosphate.The ribosomal 40S and 60S subunits (1 pmol each) were associated at 37 °C for 10 min, and then, a GTPase buffer  containing 0.2 mM GTP equilibrated with MgCl2 at a final volume of 20 μl. The reaction mixture was incubated for 1 h at 4 °C, followed by the addition of 20 μl of 50% glutathione-sepharose 4B , equilibrated with the binding buffer for 5 min at 37 °C with shaking. After centrifugation , the supernatant was removed, and the resin was washed four times with 500 μl of binding buffer. Then, 1 μg of TEV protease diluted in 16 μl of binding buffer without GTP was added to the resin and incubated for 10 min at 37 °C. The linker between GST and PDCD4 in the GST-PDCD4 molecule was cleaved, releasing the complex of PDCD4 and its interacting proteins into the solution. The supernatant was collected through a mini-spin column , leaving the resin on the filter. Then, the supernatant was diluted to 30 μl with sample buffer. Ten microliters of the final sample was used for Western blot analysis using the following antibodies: anti-PDCD4  anti-eRF3 , anti-eRF1 , and anti-PABP . As positive control for antibodies, inputted proteins were added in the following dilutions relative to the experimental samples: ×1/5 for GST-PDCD4, ×1/600 for eRF1, eRF3, and PABP.To study protein–protein interactions, ications . GST-PDC2, 2 mM DTT, 0.1 mM spermidine with 0.2 mM GTP equilibrated with 0.2 mM MgCl2 at 37 °C for 10 min. The reaction volume was 100 μl. Subsequently, TCs were incubated with 1% formaldehyde at 4 °C for 1 h. Glycine was added up to 0.1 M to stop the cross-linking reaction. The preTC NL was centrifuged into a 10 to 30% (w/w) linear SDG, as described above. The whole gradient was fractionated into 13 equal fractions (385 μl), followed by precipitation with 10% trichloroacetic acid. The protein pellets were dried and analyzed using western blotting analysis with corresponding antibodies raised against PDCD4 , eRF1 , eRF3 , RLP9 (Abcam 182556), and RPS15 .1.35 pmol purified preTCs NL or 10 pmol of 40S and 80S ribosome were incubated with 10 pmol eRF3a, 10 pmol eRF1(AGQ), and 10 pmol PDCD4 either separately or together in buffer containing 20 mM Tris-HCl pH 7.0, 10 mM MgClt test was used to compare the mean values between the groups. For multiple comparisons, the Bonferroni correction was used.All experiments were performed with at least two replicates. A two-tailed alkalaeva@eimb.ru) upon reasonable request.The data that support the findings of this study are contained within the article and the supporting information. All source data generated for this study are available from the corresponding author (Dr Elena Alkalaeva; This article contains The authors declare that they have no conflicts of interest regarding the contents of this article.


Knowledge of a structure–function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca2+ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca2+, R342W and Q426R were stabilized at higher Ca2+ concentrations than the wild-type form, and R402P was completely insensitive to Ca2+. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca Here, we focus on the missense mutations, which result in a substitution of a single amino acid. Clinically important mutations also involve frameshifts, which have more pronounced effects, insertion–deletion polymorphisms (indels), which result in excision or insertion of a DNA fragment, and mutations involving non-coding sequences, for example introns . Pu. Pug forst 2021) .3+ ions to hOtolC1q and its mutants was assessed using steady-state fluorescence. Terbium (III) chloride was dissolved in MilliQ water to a final concentration of approximately 0.5 M. Exact concentration of TbCl3 was determined by titration of diluted stock solution with EDTA in the presence of xylenol orange. Aliquots of diluted TbCl3 were added to 2 mL 3.7 µM protein solution in a 10 × 10 mm quartz SUPRASIL® cuvette  and incubated for 15 min at room temperature. Subsequently, fluorescence emission at 520-580 nm was recorded using an excitation wavelength of 280 nm using a Fluorolog-SPEX fluorimeter  equipped with a Peltier heating accessory set at 20 °C. The bandwidth was set at 5 nm for both excitation and emission monochromators. A cut-off filter absorbing below 350 nm was installed in the emission path. Obtained fluorescence intensities were processed and fitted to a model based on work by Gonzalez et al. , c—protein concentration [g/L] and l—optical pathlength of a cuvette [cm]. The secondary structure content was estimated using CDPro [Circular dichroism of 0.2 mg/mL proteins in H10Na500G5 with 1 mM EDTA, 0.1 mM CaClequation :(3)θmrw=ng CDPro .® centerpieces and quartz windows, or sapphire windows for samples containing DTT. The proteins were analyzed at concentrations of 0.1, 0.25 and 0.5 mg/mL in H10Na500G5 with 1 mM EDTA or 10 mM CaCl2. Additional measurements were made for 0.25 mg/mL protein with EDTA and CaCl2 supplemented with 1 mM DTT. Effect of Tb3+ was analyzed by centrifuging 0.25 mg/mL protein with 7-fold molar excess of TbCl3. Assembled cells with the samples were preincubated in the ultracentrifuge for 3 h at 20 °C and then centrifuged at 50,000 rpm  overnight. The absorbance scans at 280 nm were collected continuously with 0.003 cm resolution. The scans were time-corrected [https://sedfitsedphat.nibib.nih.gov/, accessed 16 August 2021) using a continuous c(s) distribution model [http://www.jphilo.mailway.com/download.htm, accessed 16 August 2021). Maximum entropy regularization with p = 0.95 was used. Simplex and Marquardt–Levenberg algorithms were alternately used until the RMSD converged. Among the results of the calculations were sedimentation coefficients (s), sedimentation coefficients corrected for water at 20 °C , weight-averaged sedimentation coefficients (MWapp) and frictional ratios (f/f0). c(s) distributions were visualized using GUSSI  [Sedimentation velocity analytical ultracentrifugation (SV AUC) was conducted in a Beckman Coulter ProteomeLab XLI analytical ultracentrifuge  with an An60Ti rotor and assembled cells with two-channel 12 mm charcoal filled Eponorrected  and analon model  with at st 2021)  and Orig2, 1 mM CaCl2, 10 mM CaCl2, 100 mM CaCl2, and 7-fold molar excess of TbCl3. Final sample volume was 20 µL. The samples and the non-protein controls (Tm) were determined from the derivative of fluorescence with increasing temperature (dF/dT).Thermal shift assay (TSA) was conducted as described . Five µMcontrols  were ali


X-ray and neutron scattering showed that the Guinier RG values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients  A), which potentially gives the Fab and Fc regions in IgG3 a greater rotational freedom than in IgG1, IgG2, and IgG4, which have shorter hinges of lengths 15, 12, and 12 residues, respectively . A complex-type biantennary glycan with a Man3GlcNAc2 core and two NeuNAc.Gal.GlcNAc antennae is attached at Asn297 (EU numbering) on each CH2 domain   is availFig. 12 domain  Fig. 2)B). A com2 domain  and infl2 domain . The str2 domain .Figure 2The issues of the unknown IgG3 molecular structure and a role for its glycans were here addressed by a joint application of AUC, SAXS, small-angle neutron scattering (SANS), and atomistic modeling to myeloma human IgG3. This powerful solution structural strategy has recently provided molecular structures for glycosylated and deglycosylated monoclonal IgG1, and also human myeloma IgG2 , 17. AUC(i)A). Both forms eluted as a main symmetrical peak at 16.43 and 16.38 ml for glycosylated and deglycosylated IgG3, respectively (A). Trace amounts of a possible dimer just before 15 ml were discarded. This process ensured that the IgG3 sample was monodisperse with no aggregates present immediately before analytical ultracentrifugation or scattering experiments.Deglycosylation of IgG3 was completed after 10 h. The elution of the IgG3 PNGase F-treated product from a gel filtration column slightly preceded that for native glycosylated IgG3 A. Both fectively A. Trace (ii)B). Under reducing conditions, two bands were present corresponding to the heavy chain (with an apparent mass of ∼50–70 kDa) and the light chain (with an apparent molecular mass of ∼25 kDa) (B). These apparent molecular masses were as expected from the known sequence  B. These sequence .(iii)C). These peaks were unaffected by longer PNGase F digestion times. The existence of five peaks is attributed to up to as many as six weakly occupied O-linked glycans of sizes 1.9 to 2.8 kDa in the IgG3 hinge (2Man3GlcNAc4 that gives a mass of 1622 Da.Liquid chromatography mass spectrometry measurements showed a major peak and four minor peaks for glycosylated (in black) and deglycosylated (in magenta) IgG3 in a mass range of 160.2 to 169.5 kDa for glycosylated IgG3 and 156.6 to 166.0 kDa for deglycosylated IgG3 C. These G3 hinge . The impHuman IgG3 was purified in high yields from human myeloma serum . In ordeA). From the c(s) analyses, the molecular masses of the IgG3 monomer peak were 108 (glycosylated) and 122 kDa (deglycosylated) in light water. In heavy water the molecular masses of the IgG3 monomer peak were 139 (glycosylated) and 156 kDa for deglycosylated. These values were comparable with the composition-calculated masses of 158 and 154 kDa for the glycosylated and deglycosylated IgG3 monomers, respectively. These also agree well with the values from mass spectrometry of 165 and 161 kDa for glycosylated and deglycosylated IgG3, respectively (C). In the resulting size distribution analyses c(s), a clear monomer peak that monitored the overall IgG3 solution structure was observed at average s20,w values of 5.82 ± 0.06 S for glycosylated IgG3 and 6.29 ± 0.05 S for deglycosylated IgG3 in light water  in the IgG3 mass was not seen because of noisy data at low concentrations. These s20,w values were consistent with previous AUC studies of glycosylated IgG3 that reported s20,w values of 6.11 ± 0.03 S  is unchanged after deglycosylation. Variable minor peaks at 3 to 4 S were seen adjacent to the main monomer peak (M) ; they were attributable to minor fragmentation of IgG3 and/or artefacts caused by small sample–buffer optical mismatches. In conclusion, given that no concentration dependences were seen in Figure 4B, these datasets showed that IgG3 was monomeric in solution, as required for scattering studies.Sedimentation velocity experiments characterized the masses and solution structures of glycosylated and deglycosylated IgG3. The SEDFIT analyses of the boundaries involved fits of as many as 600 scans, and the good agreement between the experimental boundary scans and fitted lines is clear left, A. From tectively C. In theFig. 4A; . BecauseThe solution structures of glycosylated and deglycosylated IgG3 samples were characterized by X-ray and neutron scattering. The two methods provided different perspectives of the same solution structure. X-rays in light water buffers detect the hydration shell surrounding the protein structure, whereas neutrons in heavy water buffers see a much reduced effect of this hydration shell because of the different solute–solvent contrast in use , 19, 20.RG and RXS-1/RXS-2 values monitor the elongation of the overall IgG3 structure and its approximate cross-sectional structures, respectively. Guinier analyses resulted in high-quality linear plots for all samples and revealed three distinctive regions of the I(Q) curves, which is seen in the scattering curves for antibodies . The mean X-ray RG values that monitor the overall structure for glycosylated and deglycosylated IgG3 samples were similar, being 6.95 ± 0.06 and 7.02 ± 0.05 nm, respectively  is an approximate monitor of the cross-sectional Fab and Fc arrangement in glycosylated IgG3 and deglycosylated IgG3. The mean X-ray RXS-1 values for glycosylated and deglycosylated IgG3 were both typically 1.41 ± 0.2 nm, showing that the spatial arrangement of the Fab and Fc regions was unchanged following glycan removal. These values were notably reduced from RXS-1 values of 2.5 (±0.1) nm measured for monoclonal IgG1 before and after deglycosylation  nm. This indicates that the averaged cross-sections of the two Fab and one Fc region were unchanged before and after deglycosylation. Similar RXS-2 values of 1.4 (±0.1) nm were recently reported for monoclonal IgG1 before and after deglycosylation . A concentration dependence was not observed for IgG3, this being seen from the I(0)/c values that remained unchanged within error (B). This difference between the neutron and X-ray datasets is attributable to the fewer data points obtained with neutrons, leading to reduced precision in the datasets. The mean neutron RG values for glycosylated and deglycosylated IgG3 were similar at 6.23 ± 0.04 and 6.29 ± 0.04 nm, respectively  is derived from Fourier transformation of the scattering curve I(Q) and provides structural information in real space on glycosylated and deglycosylated IgG3. The X-ray and neutron P(r) analyses gave RG values that were similar to those from the X-ray Guinier analyses, showing that the two analyses were self-consistent  curve intersects zero on the r axis and were generally 28 nm (A). The neutron maximum lengths L of glycosylated and deglycosylated IgG3 were 25 to 28 nm. These were slightly smaller when compared with the X-ray value of 28 nm (B). These reductions in the neutron RG and L values compared with the X-ray values have been previously seen in our earlier joint SAXS and SANS studies of antibodies  curves corresponded to the most frequently occurring distances between scattering elements within the IgG3 structures. For the IgG3 samples, three peaks, M1, M2, and M3, were seen (A and B). Two peaks, M1 and M2, are characteristic for IgG1, IgG2, and IgG4 antibodies . For the X-ray data, the M1 peak was observed at 3.53 ± 0.06 nm for glycosylated IgG3 and 3.58 ± 0.09 nm for deglycosylated IgG3. The M2 peak was observed at 8.97 ± 0.47 nm for glycosylated IgG3 and 9.11 ± 0.04 nm for deglycosylated IgG3. Although not well resolved by X-rays, the positions of the M3 peak varied between 16 and 19 nm. The M3 peak was observed at 17.0 ± 1.5 nm for glycosylated IgG3 and 17.2 ± 1.9 nm for deglycosylated IgG3. The neutron P(r) analyses reflected the same trends as the X-ray data (D), although the M3 peak was better resolved in the neutron P(r) curves (B). The neutron M1 peak was seen at 3.8 ± 0.5 nm for glycosylated IgG3 and 3.7 ± 0.2 nm for deglycosylated IgG3, the M2 peak was seen at 9.7 ± 0.2 nm for glycosylated IgG3 and 9.7 ± 0.1 nm for deglycosylated IgG3, and the M3 peak was seen at 14.2 ± 0.1 nm for glycosylated IgG3 and 14.4 ± 0.6 nm for deglycosylated IgG3. Both the X-ray and neutron analyses were thus consistent with each other and support the premise that the two Fab regions were well separated from the Fc region in solution.The maxima in the ere seen , A and Btibodies , 22, 23.eviously . In IgG3 regions  and 7. N regions C. For thray data D, althou) curves B. The neA–G. The Fab and Fc regions were joined by a 62-residue hinge peptide , built using PyMOL. This hinge was energy minimized using NAMD with the CHARMM36 forcefield to generate a relaxed structure, then the latter was simulated in triplicate using all-atom molecular dynamics in NAMD for 100 ns (A). From this, a principal component analysis (PCA) (B–E). Five conformations that corresponded to the mid-point of each cluster were selected for attachment to the Fab and Fc regions to give five deglycosylated full-length IgG3 models. The corresponding five glycosylated IgG3 models were created by adding complex-type biantennary glycans to the two Asn-297 side chains in the Fc region (B). The five glycosylated IgG3 starting structures were each energy minimized.Despite the observation by scattering of small but detectable changes in the scattering curves of monoclonal IgG1 following glycan removal , no such peptide ; Fig. 2Er 100 ns A. From tis (PCA)  on the cis (PCA) , B–E. Fic region B. The fi(a)R-factor versus RG distribution with clear minima in all eight cases (A and B). The minima agreed with the experimental RG values (A). The minima showed that enough trial X-ray models had been generated to result in good fits in each case. Filtering of the models to select these with the lowest R-factors gave the 100 best-fit models for each concentration R-factor versus RG neutron distributions (C and D). The minima agreed with the experimental RG values (B). The range of R-factors was now higher than the X-ray values because of the reduced precision of the neutron scattering curves.For neutrons, the same outcome was found, thus confirming the reproducibility of the curve fits. Comparisons of the same 135,135 and 73,905 theoretical curves with the neutron scattering curves at 0.65 to 1.85 mg/ml showed again that 100 best-fit structures could be identified at clear minima in each of the ibutions , C and DG values B. The raPhysically realistic IgG3 models without steric overlaps or clashes were created for comparison with the experimental X-ray curves. By varying the torsion angles at three flexible regions at the start and end of the two IgG3 hinges  Fig. 1)Fig. 1), (a)A–D; I(Q) and P(r) X-ray curve fits confirmed the validity of the modeling fits (A and B).For the X-ray fits, the PCA indicated a difference between the glycosylated and deglycosylated IgG3 models E–H; I(Q) curve fits were obtained; however, the fit of these curves was inferior to the fit obtained in the X-ray fits at high Q (C and D). An incoherent scattering flat background correction of 2.0% of I(0) in the neutron fits accounted for a mismatch between the sample and the buffer background in heavy water (P(r) curves were well replicated in the modeled best-fit curves.For the neutron fits, the PCA also indicated differences between the glycosylated and deglycosylated IgG3 models 2·I(Q)/I(0) versus Q·RG for the experimental scattering curves at the highest concentrations in use and the scattering curves from the modeled best fit structures. These plots indicate whether the macromolecule in question is globular in its structure or possesses intrinsically disordered regions (A), the Q·RG values for the experimental glycosylated IgG3 peak was 2.24, in good accord with the modeled values of 2.25 before energy minimization and 2.23 after energy minimization. The Q·RG values for the experimental deglycosylated peaks of 2.26 were also in good accord with the modeled deglycosylated peaks of 2.26 before energy minimization and 2.33 after energy minimization. For the neutron Kratky curves (B), the Q·RG value for the experimental glycosylated IgG3 peak was shifted to 4.79, which were similar to the modeled peaks at 4.80 before energy minimization and 4.80 after energy minimization. For deglycosylated IgG3, the Q·RG value for the experimental peak was 4.66, which was larger than the modeled values of 3.88 before and after energy minimization. The existence of a single and not double Kratky peak for IgG3 was most likely due to the large separation between the Fab and Fc regions in IgG3, meaning that these three regions showed independent scattering properties . For the curve fits, five such hinges were simulated using energy minimization and molecular dynamics, and all showed extended but variable structures (C). The maximum length of the hinge between Glu216 and Pro285 (E) ranged between 7.67 and 10.80 nm, in good agreement with the increased length of IgG3 compared with IgG1, IgG2, and IgG4. The simulations do not predict a regular polyproline helix structure with no flexibility, and this outcome concurs with the scattering curves.The most prominent feature of the IgG3 models is the polyproline disulfide-linked hinge region A. For thructures C. The mad Pro285 E ranged RG values derived from Guinier analyses were significantly larger at 6.97 nm by X-rays and 6.26 nm . The maximum length L of IgG3 was significantly larger at 25 to 28 nm  curve for IgG3 showed three maxima M1, M2, and M3, whereas the other IgG subclasses only showed two maxima M1 and M2. The three maxima in the P(r) curves for IgG3 were well replicated by the atomistic modeling , P(r), and Kratky curves were obtained with a conformationally variable but essentially static set of IgG3 molecular models , IgG2 (7.2 S), and IgG4 (6.5 S) demonstrated that IgG3 has a more elongated structure than IgG1, IgG2, and IgG4 . The longer hinge may enable the Fc glycans to play a more local role in stabilizing the CH2 domain pair, and we note the loss of FcγR receptor binding in aglycosylated IgG3 Recently it has been reported that human IgG1 associates at cell surfaces to form Fc hexamers, with one Fab region bound to the antigenic surface and the other Fab region being unbound and located at the height of the Fc regions; this hexameric arrangement confers superior C1q complement activation , 33, 34.(ii)H domains; Fig. 2C). This outcome indicated that our best fit IgG3 solution structures showed a similar range of Fab separations to those seen for these antigenic separations on a surface.The bivalent binding of antibody to precise patterns of hapten-coated antigen surfaces was studied for the four human IgG subclasses . Studies(iii)e.g., targeting structures around the Node of Ranvier in the central nervous system, where these autoantibodies are of the IgG3 isotype. In acute autoimmune neuropathy, IgG3 was detected as the predominant autoantibody in patients with Guillain–Barré syndrome  was prepared from human myeloma serum stored at −20 °C. The serum was thawed at 37 °C, and spun at 3000 rpm for 15 min A volume of 12.5 ml of clear serum was dialyzed into 1 L of 0.01 M phosphate buffer at pH 7.5, repeated three times. IgG was isolated from other serum components using a DE 52 Whatman (DEAE) column (12 cm × 12 cm), in 0.01 M phosphate buffer pH 7.7, varying the salt concentration sequentially . The most IgG3 was found from the salt concentration eluate of 0.01 M. IgG3 was further purified using a 1 ml prepacked Staphylococcus Protein A coupled to a Sepharose column (Cytiva). Staphylococcus Protein A binds IgG1, IgG2 and IgG4, but not IgG3, which flowed through the column. The purity of IgG3 following purification from serum was confirmed using SDS-PAGE.To remove the N-glycans from IgG3, enzymatic deglycosylation was achieved using peptide:N-glycosidase F (PNGase F) , selected owing to its ability to remove glycans completely from the glycosylated Asn residues . Thus 205W38)   . The hinD: 1BBJ)  in orderD: 1BBJ) . This muD: 1BBJ) . The N-lantennae . From thwas 12.8 . The X-rwas 12.8 . The bufm/z range 1000 to 3200 in profile mode. The raw data were converted to zero charge mass spectra using the maximum entropy deconvolution algorithm in the MassHunter software version B.07.00 (Agilent). The single glycan mass was found by subtracting the mass of fully glycosylated IgG3 from that for deglycosylated IgG3 and halving this mass.The completeness of deglycosylation was verified by Superose 6 gel filtration, SDS-PAGE, and mass spectrometry. In the Mass Spectrometry Facility at the Chemistry Department University College London, the antibodies were analyzed on an Agilent 6510 Quadrupole time-of-flight liquid chromatography mass spectrometry system (Agilent). A volume of 10 μL of each sample was injected onto a PLRP-S, 1000A, 8 μM, 150 mm × 2.1 mm column, which was maintained at 60 °C at a flow of 0.3 ml/min. The separation was achieved using mobile phases A (water with 0.1% formic acid) and B  using a gradient elution. The column effluent was continuously electrosprayed into the capillary electrospray ionization source of the Agilent 6510 QTOF mass spectrometer, and electrospray ionization mass spectra were acquired in positive electrospray ionization mode using the c(s), for which the algorithm assumes that all species have the same frictional ratio f/f0. The final SEDFIT analyses used a fixed resolution of 200 and optimized the c(s) fits by floating f/f0 and the baseline until the overall root mean square deviations and visual appearance of the fits were satisfactory. The percentage of oligomers in the total loading concentration was derived using the c(s) integration function. The buffer viscosity and density were the experimental values. The observed s values were corrected to s20,w by:s is the sedimentation coefficient, the subscripts T,B refer to the temperature of the buffer and 20,w refers to water at 20 °C, ρ is the solvent density, η is the solvent viscosity, and Analytical ultracentrifugation data for glycosylated and deglycosylated IgG3 were obtained on two Beckman XL-I instruments equipped with AnTi50 rotors. Data were collected at 20 °C, at a rotor speed of 30,000 rpm in two-sector cells with column heights of 12 mm for approximately 6 h. Sedimentation analyses were performed using direct boundary Lamm fits of up to 600 scans using SEDFIT (version 15.01b) , 46. SEDQ range from 0.04 to 4 nm−1 . The glycosylated (0.50–2.46 mg/ml) and deglycosylated (0.50–1.00 mg/ml) IgG3 samples in light water buffer were loaded onto a 96-well plate and placed into an EMBL Arinax sample holder (X-ray scattering data were obtained during one beam session (July 2019) on Instrument B21 at the Diamond Light Source at the Rutherford Appleton Laboratory, operating with a ring energy of 3 Gev and a beamline operational energy of 12.4 keV . A PILATe holder , 49. Thie holder . An autoQ range from 0.05 to 4 nm−1. The data were acquired using a two-dimensional 3He detector with 512 × 512 pixels of 7.5 × 7.5 mm2 size. Samples of volume 1 ml were measured in 2-mm path length circular banjo cells for 1 to 7 h in a thermostatted sample rack at 20 °C. Data were reduced using MANTID software  and deglycosylated (0.65–1.21 mg/ml) IgG3 samples in heavy water buffer were obtained in one session (October 2020) on instrument SANS2D at the ISIS pulsed neutron source at the Rutherford Appleton Laboratory . This cosoftware . The MANch pulse . Using SRG, the cross-sectional radius of gyration RXS, and the molecular mass. The scattering curve I(Q) intensities at low Q are defined by the RG value, which is the averaged distance of each scattering point from the center of scattering. In a given solute–solvent contrast, the radius of gyration RG is a measure of structural elongation if the internal inhomogeneity of scattering densities within the protein has no effect. Guinier analyses at low Q gave the RG value and the forward scattering at zero angle I(0)  :lnI(Q)=lQ·RG range up to 1.5 and was used in our previous studies Q]Q0 is obtained from QRXS-1 and RXS-2, respectively. The RXS-1 parameter represents the averaged overall spatial separation of the Fab and Fc regions, this being one shorter Fab-Fab separation and two longer Fab–Fc separations (A), whereas the RXS-2 parameter represents the averaged spatial cross-section of each of the two Fab and one Fc regions. The RG and RXS analyses were performed using SCT  in reciprocal space into real space to give the distance distribution function P(r) was carried out using GNOM (version 5.0) . Th. ThRXS-1and IgG4 , 52. Indion 5.0) , 57.P(r)P(r) corresponds to the distribution of distances r between the volume elements in the macromolecule. This yields the maximum dimension of the macromolecule L and its most commonly occurring distance vector M in real space. For this P(r) analysis, the X-ray I(Q) curve utilized up to 788 data points in the Q range between 0.09 and 1.50 nm−1 for both glycosylated and deglycosylated IgG3. The neutron P(r) curve utilized up to 134 I(Q) data points in the Q range between 0.165 and 1.60 nm−1 for both glycosylated and deglycosylated IgG3.216ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3APELLGGP285 sequence.Starting structures were created for each of glycosylated and deglycosylated IgG3 based on the sequence of http://www.charmm-gui.org/). Protonable residues were identified using the PDB2PQR website (http://apbs-rest-test.westus2.cloudapp.azure.com/pdb2pqr) and their protonated states adjusted for pH 6 in CHARMM-GUI. The TIP3P model was used to simulate explicit water molecules. The octahedral solvation box was used as this allowed for at least 1.0 nm from the protein in each axis, and 0.138 M NaCl was added. The CHARMM36 force field was used (−1 and a Nosé-Hoover Langevin-piston (This 62-residue hinge structure was subjected to energy minimization and molecular dynamics (MD) independently of the Fab and the Fc regions in order to identify hinge conformations. Hinge structures were prepared for all-atom molecular dynamics simulations using Glycan Reader and Modeler , 60, 61 was used , and allwas used . A time was used . To assuwas used . For then-piston , 68 was n-piston .239SVFLFPPK246 (EU numbering) in the IgG3-Fc crystal structure that was kept unchanged in the MD simulations while allowing the hinge to move. This 239SVFLFPPK246 peptide was the anchor site for superimposing the hinge to the Fc region. The two N-linked Asn297 glycans on the Fc CH2 domains were approximated as complex-type biantennary oligosaccharides with a Man3GlcNAc2 core and two NeuNAc.Gal.GlcNAc antennae (https://github.com/dww100), which was energy minimized using NAMD  in order to create “CONECT” records for these glycosidic bonds. The CHARMM force field parameters and protein structure file, including those for the disulfide bridges and glycans, were generated using the CHARMM-Gui GlycanReader tool  and that of Pro285 (the first residue in the hinge region). To attach the Fc region to the hinge, which was not straightforward, MD simulations of the hinge included a small segment antennae . The glying NAMD  for 1 nsder tool , 71, 72 der tool , 75, 76.217LKTP220 and 285PS286 on one hinge of IgG3, and 217LKTPLG222 on the other hinge (B and 2E). For deglycosylated IgG3 these peptides were 217LKT219 and 283 GG284 on one hinge, and 217LKTP220 on the other hinge of IgG3. These six peptides corresponded to surface-accessible structures outside the structurally defined Fab and Fc regions and the disulfide-linked hinge core. These peptides were structurally varied in TAMC to create the required IgG3 conformers for testing against the scattering curve, while the rest of the IgG3 model was held rigid. Allowing 217LKTP220, 217LKTPLG222, or 217LKT219 to be variable on both IgG3 hinges enabled both Fab regions to be conformationally mobile. Allowing 285PS286 or 283 GG284 to be variable enabled the Fc region to be mobile too. For each of these linker residues, the backbone phi (ϕ) and psi (ψ) torsion angles were varied in 15o steps. During the Monte Carlo simulations, many attempted steps were physically unrealistic and were therefore discarded by TAMC. Overall, for glycosylated IgG3, 50,000 moves were attempted for each of the five starting hinge structures. Of these, 31% to 61% were rejected because of inappropriate steric clashes to leave a total of 135,135 of 250,000 starting models that were accepted. For deglycosylated IgG3, in which the glycan chains were omitted, 250,000 moves were attempted, of which 57% to 85% of the models were rejected to leave 73,905 models that were accepted.For the Monte Carlo simulations to generate trial structures, the residues in the five starting IgG3 structures were renumbered and their naming nomenclature was adjusted to match the required format for the Torsion Angle Monte Carlo (TAMC) module on SASSIE-web for this to work . The IgGer hinge B and 2E.I(Q) using an all-atom expression for the scattering intensity in which the orientations of the Q vectors are taken from a quasi-uniform spherical grid generated by the golden ratio (I(Q) and would require much computational effort (IModel(Qi) and the interpolated experimental curves IExpt(Qi), this function being analogous to that used in protein crystallography:Qi is the Q value of the ith data point, IExpt(Qi) is the experimental scattering intensity, IModel(Qi) is the theoretical modeled scattering intensity, and I(0)  curve to zero Q. The resulting 825 and 146 I(Q) values in the Q range of 0.0 to 1.5 nm−1 were utilized for the SAXS and SANS curve fits, respectively, and defined the Q spacing for use in SasCalc and the R-factors. The use of χ2 analyses to evaluate the fits was not possible because this requires the experimental data points to have errors associated with them, which were not available when interpolating the curve. For the SANS curve fits, a correction of 2.0% of I(0) was required to allow for a flat incoherent background that was attributed to the proton content of IgG3 and the heavy water dialysis buffer  . Lower R I(0)  with reference codes SASDLZ2 and SASDL23.All data are contained within this article. The two single best-fit models for glycosylated and deglycosylated IgG3 from X-ray searches at 1.00 mg/ml  are avaiThis article contains The authors declare that they have no conflicts of interest with the contents of this article.