Structural investigation into recombinant eye lens aquaporin (AQP0) and the effector proteins (BEPS) from "Bartonella henselae"

Abstract

Abstract Chapter IA: Co-axial association of recombinant eye lens aquaporin AQP0: Aquaporin-0 (AQP0) is the major membrane protein present in the vertebrate eye lenses. It has been proposed that AQP0 tetramers mediate contact between membranes of adjacent lens fiber cells, which would be consistent with the extraordinarily narrow intercellular space. Indeed, octamer formation with purified AQP0 in solution was observed by native gel electrophoresis and analytical ultracentrifugation methods. We obtained 3D crystals of AQP0 that diffract to 7.0 A resolution and molecular replacement was performed using the recently determined 3D structure of AQP0 from native source. The result shows that, within the cubic lattice, tetramers (point symmetry 42) are associated head-to-head. There are no direct octamer-octamer contacts and the crystal integrity is most probably maintained by detergent belts surrounding the membrane protein. Within the octamer, extracellular loops A and C interdigitate at the center and the perimeter of the octamer, respectively. The octamer formation has been compared with the AQP0 structure derived from 2D crystals using electron diffraction. Intriguingly, the mutual orientation of tetramers within the octamer is significantly different to that previously reported for 2D crystals. Clearly, the low resolution of the X-ray data permits only a comparison of the oligomeric arrangement. The interactions observed in the looselypacked 3D crystals presented here possibly represent the in vivo association mode between AQP0 tetramers from juxtaposed membranes in the eye lens. Abstract Chapter II: Structural studies on the effector proteins (Beps) of the VirB-Type IV secretion system in Bartonella henselae: Type IV transporters are one of the five major families of transporters that are capable of exporting virulence factors across the membranes of pathogenic bacteria. Members of the Type IV secretion system (T4SS) share structural homologies but they display diversity in the nature of the substrates that they transport. T4SS are multicomponent transporters of Gram-negative bacteria with functions as delivery of effector proteins into eukaryotic target cells in pathogenesis or DNA transfer in bacterial conjugation through the long pilus structure (VirB apparatus). Type IV transporters are produced by several bacterial pathogens. Bartonella henselae is one among the T4SSs which mediates the delivery of effector proteins into the eukaryotic cell via the process of translocation. Bartonella henselae is a Gram-negative, arthopod-borne zoonotic pathogen causing a broad range of clinical manifestations in incidentally infected humans. Transmission to humans occurs by cat scratch or bite of an infected cat flea causing vasoproliferative lesions, which result in the formation of tumours in the skin and/or inner organs. In vitro studies showed that the VirB T4SS of B. henselae mediates most of the virulence attributes associated with the interaction of B. henselae with human endothelial cells and they interfere with the signalling network of the host. We have characterized few of the Bartonella effector proteins (Beps) BepA, B, C and D and their domains of B. henselae for structural analysis. Crystals were obtained for the truncated version of BepA and it diffracted to 3.3 A resolution. Currently, we are in the process of investigating the data for structural elucidation of the BepA protein. Investigations on other proteins are under progress. In this study, we explain the preliminary results obtained from all the constructs and in detail for the BepA protein. Ultimately, obtaining structural information of these effector proteins will help us to investigate its functional importance in the eukaryotic host.