Award details

Structure function and mechanics of the Moraxella catarrhalis adhesin UspA1

Reference	BB/F007256/1
Principal Investigator / Supervisor	Professor R Brady
Co-Investigators / Co-Supervisors	Dr Massimo Antognozzi, Professor Emeritus Mumtaz Virji, Professor Dek Woolfson
Institution	University of Bristol
Department	Biochemistry
Funding type	Research
Value (£)	490,930
Status	Completed
Type	Research Grant
Start date	01/01/2008
End date	31/03/2011
Duration	39 months

Abstract

UspA1 is a high-molecular weight (3x~90kDa) transmembrane surface adhesin protein from Moraxella catarrhalis that is believed to form an extended (600-800 A) rod-like structure. In order to progress our understanding of both bacterial adhesins and extended coiled-coil structures, in this proposal we aim to explore the overall structure and dynamics of UspA1. Existing and novel recombinant truncated forms of UspA1 will be expressed in E.coli, in each case with cleavable tags to facilitate their purification. The purified recombinant proteins will then be subjected to crystallisation trials (Objective 1). Individual protein components and their complexes with receptor fragments (Objective 2) will be included in the crystallisation screens. When crystals are obtained, their structures will be determined by X-ray diffraction methods by either molecular replacement or anomalous dispersion phasing techniques. SAXS and EM studies will be used to generate molecular envelopes from larger segments of UspA1, allowing an overall description of the UspA1 structure and its complexes (Objectives 1 & 2). Intact and appropriately truncated forms of UspA1 will be examined using a specialised TDFM form of AFM in order to ascertain the response of the adhesin to forces, in particular to identify the range and type of bending motions that result (Objective 3). These studies will be performed on both membrane-embedded and isolated UspA1 forms. Finally, analysis of the coiled-coil stem of UspA1 will lead to the design and synthesis of mutant forms of UspA1 (Objective 4) that could assist and extend the studies in Objectives 1-3. These stabilised, truncated forms could also be incorporated in functional assays, and may form the basis for further studies aimed at developing novel therapeutics.

Summary

Infectious bacteria use finger-like extensions from their surfaces to recognise, bind to and invade their human host cells. This proposal aims to study one such molecule, called UspA1, that forms such extensions on the bacterium Moraxella catarrhalis - a common cause of many human respiratory tract infections. In the first part of this proposal, in order to understand how UspA1 works, initially we plan to work out what it looks like. We will do this by applying a range of techniques such as X-ray crystallography, small-angle X-ray scattering and electron microscopy to determine the molecular shape of UspA1. As UspA1 is a very large molecule, we plan to approach this problem in stages - determining the structures of fragments of UspA1 initially, and then assembling these to understand the overall structure. We will also study the shapes of UspA1 bound to some of the proteins found on the human cells in order to extend our knowledge of how UspA1 works. Secondly, as UspA1 forms an extended lollipop-like structure at the bacterium surface, logic dictates that to enable the bacterium to approach sufficiently close to the host cell to invade it, UspA1 must bend or change shape in some way. Therefore, we plan to use a specialised form of atomic force microscopy to study the strength and direction of forces required to induce such bends. We aim to do this using both isolated molecules of UspA1 and also with UspA1 embedded on the bacterium surface. We want to know if there are particular regions of UspA1 that are more prone to bending, whether such bending is gradual or forms kinks in the structure, and whether bending is induced or encouraged in the presence of the receptor molecules from the human host cells. These studies are especially important because UspA1 belongs to a large family of proteins with similar structures, and little is known about the dynamic flexibility of these common proteins. Finally, as we learn more about this common molecular structure, it should become feasible to make changes in its composition to alter its properties. Therefore, we aim to design mutated forms of the protein that retain its overall important structural features, but which are smaller in size. These will be useful tools for us in exploring the overall structure of UspA1, and may also form the basis for future therapeutics based on the UspA1 molecule.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Industrial Biotechnology, Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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