Functional and structural characterization of PII-like protein CutA does not support involvement in heavy metal tolerance and hints at a small-molecule carrying/signaling role.
The PII-like protein CutA is annotated as being involved in Cu2+ tolerance, based on analysis of Escherichia coli mutants. However, the precise cellular function of CutA remains unclear. Our bioinformatic analysis reveals that CutA proteins are universally distributed across all domains of life. Based on sequence-based clustering, we chose representative cyanobacterial CutA proteins for physiological, biochemical, and structural characterization and examined their involvement in heavy metal tolerance, by generating CutA mutants in filamentous Nostoc sp. and in unicellular Synechococcus elongatus. However, we were unable to find any involvement of cyanobacterial CutA in metal tolerance under various conditions. This prompted us to re-examine experimentally the role of CutA in protecting E. coli from Cu2+ . Since we found no effect on copper tolerance, we conclude that CutA plays a different role that is not involved in metal protection. We resolved high-resolution CutA structures from Nostoc and S. elongatus. Similarly to their counterpart from E. coli and to canonical PII proteins, cyanobacterial CutA proteins are trimeric in solution and in crystal structure; however, no binding affinity for small signaling molecules or for Cu2+ could be detected. The clefts between the CutA subunits, corresponding to the binding pockets of PII proteins, are formed by conserved aromatic and charged residues, suggesting a conserved binding/signaling function for CutA. In fact, we find binding of organic Bis-Tris/MES molecules in CutA crystal structures, revealing a strong tendency of these pockets to accommodate cargo. This highlights the need to search for the potential physiological ligands and for their signaling functions upon binding to CutA. DATABASES: Structural data are available in Protein Data Bank (PDB) under the accession numbers 6GDU, 6GDV, 6GDW, 6GDX, 6T76, and 6T7E.
['Selim|Khaled A|KA|0000-0002-2974-9483', 'Tremiño|Lorena|L|', 'Marco-Marín|Clara|C|', 'Alva|Vikram|V|', 'Espinosa|Javier|J|', 'Contreras|Asunción|A|0000-0001-6591-9294', 'Hartmann|Marcus D|MD|0000-0001-6937-5677', 'Forchhammer|Karl|K|0000-0003-3199-8101', 'Rubio|Vicente|V|0000-0001-8124-1196']
D000222:Adaptation, Physiological D000595:Amino Acid Sequence D001426:Bacterial Proteins D002151:Calorimetry D003300:Copper D018360:Crystallography, X-Ray D019216:Metals, Heavy D008958:Models, Molecular D009154:Mutation D046937:Nostoc D011487:Protein Conformation D055503:Protein Multimerization D017386:Sequence Homology, Amino Acid D015398:Signal Transduction D046940:Synechococcus
PII-like protein CutA heavy metal tolerance Nostoc sp. PCC 7120 PII superfamily Synechococcus elongatus PCC 7942 cyanobacteria signal transduction