title: Structural analysis of inter-genus complexes of V-antigen and its regulator and their stabilization by divalent metal ions.
abstract: Gram-negative bacteria like Yersinia, Pseudomonas, and Aeromonas need type III secretion system (T3SS) for their pathogenicity. V-antigen and its regulator are essential for functioning of T3SS. There is significant functional conservation amongst V-antigen and its regulator belonging to the Ysc family. In this study, we have structurally characterized the inter-genus complexes of V-antigen and its regulator. ConSurf analysis demonstrates that V-antigens belonging to the Ysc family show high structural identity predominantly confined to the two long helical regions. The regulator of V-antigen shows high conservation in its first intramolecular coiled-coil domain, responsible for interaction with V-antigen. ∆LcrG(1-70) localizes within the groove formed by long helices of LcrV, as observed in PcrV-∆PcrG(13-72) interaction. Inter-genus complexes of LcrV-PcrG and PcrV-LcrG exhibited elongated conformation and 1:1 heterodimeric state like the native complex of PcrV-PcrG and LcrV-LcrG. Both native and inter-genus complexes showed rigid tertiary structure, solvent-exposed hydrophobic patches, and cooperative melting behavior with high melting temperature. LcrV-PcrG and PcrV-LcrG showed nanomolar affinity of interaction, identical to PcrV-PcrG interaction, but stronger than LcrV-LcrG interaction. Calcium (a secretion blocker of T3SS) propels all the complexes towards a highly monodisperse form. Calcium and magnesium increase the helicity of the native and inter-genus complexes, and causes helix-helix stabilization. Stabilization of helices leads to a slight increase in the melting temperature by 1.5-2.0 °C. However, calcium does not alter the affinity of interaction of V-antigen and its regulator, emphasizing the effect of divalent of cations at the structural level without any regulatory implications. Therefore, the structural conservation of these inter-genus complexes could be the basis for their functional complementation.
authors.affiliation: Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700032, West Bengal, India.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Basu
authors.affiliation: Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700032, West Bengal, India.
authors.forename: Atanu
authors.initials: A
authors.lastname: Das
authors.affiliation: Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700032, West Bengal, India.
authors.forename: Abhisek
authors.initials: A
authors.lastname: Mondal
authors.affiliation: Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700032, West Bengal, India. saumen_datta@iicb.res.in.
authors.forename: Saumen
authors.initials: S
authors.lastname: Datta
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000494:pharmacology; D008274:Magnesium / Q000737:chemistry* / Q000494:pharmacology; D008969:Molecular Sequence Data; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C088009:LcrG protein, Yersinia; C043908:LcrV protein, Yersinia; D052899:Pore Forming Cytotoxic Proteins; D008274:Magnesium; D002118:Calcium
keywords: Elongated conformation; Helix-helix stabilization; Inter-genus complex; Nanomolar binding affinity; Polydispersity index; Structural identity