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

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 Å 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 Å 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.