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Structural analysis of inter-genus complexes of V-antigen and its regulator and their stabilization by divalent metal ions

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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.

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Abbreviations

Ysc-Yop:

Yersinia secretion component-Yersinia outer protein

T3SS:

Type III secretion system

SEC:

Size-exclusion chromatography

DLS:

Dynamic light scattering

CD:

Circular dichroism

SPR:

Surface plasmon resonance

MSA:

Multiple sequence alignment

MCS:

Multiple cloning site

LB:

Luria–Bertani

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

Ni-NTA:

Nickel-nitrilotriacetic acid

EGS:

Ethylene glycol bis[succinimidyl succinate]

Tm :

Melting temperature

PDI:

Polydispersity index

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Acknowledgments

The funding and fellowship for this research were provided by the Council of Scientific and Industrial Research (CSIR), CSIR-Network Project (UNSEEN) and Department of Science and Technology (DST), Government of India. The research work was carried out at CSIR – Indian Institute of Chemical Biology (IICB), Kolkata. We thank Dr. Supratim Dey and Dr. Urmisha Das for their kind assistance in protein designing. We acknowledge Dr. Samir Kumar Roy (Senior technical officer, IICB-Kolkata) for the SPR experiments and data analysis.

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    1. Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700032, West Bengal, India

      Abhishek Basu, Atanu Das, Abhisek Mondal & Saumen Datta

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    1. Abhishek Basu
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    A. Basu and A. Das contributed equally.

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    249_2015_1081_MOESM1_ESM.tif

    Online resource 1 Sequence similarity of V-antigen and its regulator belonging to Ysc family. (a) MSA of LcrV, LssV, AcrV and PcrV was done in clustal W2 and represented in Jalview (b) MSA of PcrG, LssG, LcrG, and AcrG was done in ClustalW2 and represented in Jalview (TIFF 46645 kb)

    Online resource 2 Intramolecular coiled-coil region in LcrG predicted by Multicoil server (TIFF 1479 kb)

    Online resource 3 Intramolecular coiled-coil region in LcrG predicted by Paracoil server (TIFF 1507 kb)

    Online resource 4 Intrinsically Disordered Region in LcrG predicted by PrDOS (TIFF 5103 kb)

    Online resource 5 Intrinsically Disordered Region in LcrG predicted by DisEMBL (TIFF 6132 kb)

    249_2015_1081_MOESM6_ESM.tif

    Online resource 6 Dynamic light scattering spectrum of a) LcrV-LcrG, b)PcrV-PcrG, c) LcrV-PcrG, and d)PcrV-LcrG, in presence and absence of calcium and magnesium ions (TIFF 251 kb)

    249_2015_1081_MOESM7_ESM.tif

    Online resource 7 SPR-based equilibrium-binding analysis plot of a) PcrG and LcrV interaction, b) LcrG and PcrV interaction (TIFF 308 kb)

    249_2015_1081_MOESM8_ESM.tif

    Online resource 8 SPR-based equilibrium-binding analysis plot of a) LcrG and LcrV interaction, b) PcrG and PcrV interaction, c) PcrG and LcrV interaction, d) LcrG and PcrV interaction, in presence of 20 mM calcium (TIFF 603 kb)

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    Basu, A., Das, A., Mondal, A. et al. Structural analysis of inter-genus complexes of V-antigen and its regulator and their stabilization by divalent metal ions. Eur Biophys J 45, 113–128 (2016). https://doi.org/10.1007/s00249-015-1081-2

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