Structure function and mechanics of the Moraxella catarrhalis adhesin UspA1

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

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.

Technical Summary

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.

Publications

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Description 1. We have determined crystal structures for 3 segments of UspA1[ 527-566 (CEACAM1 binding region), 42-345 and 42-366 (head domain and fibronectin binding region)]. In total these account for about 60% of the overall structure. The remaining parts of the structure were readily amenable to homology modeling (strong coiled-coil propensity and very highly conserved transmembrane region) permitting assembly of an overall composite model for UspA1. 2. Binding studies and site-directed mutagenesis have been used to identify the binding sites on UspA1 for two of its receptors, CEACAM1 and fibronectin. Small-angle X-ray scattering (SAXS) has been used to determine molecular envelopes for both the UspA1:CEACAM1 and UspA1:fibronectin complexes, models for which have been assembled from the individual crystal structures of each component. These structures suggest receptor binding is associated with UspA1 bending. 3. A specialised form of atomic force microscopy, the lateral molecular force microscope which as a vertical rather than horizontal cantilever, has been developed and used to measure the adherance and properties of the UspA1 layer at the Moraxella surface. These data correlate well with the studies of isolated UspA1 fragments, and show that UspA1 molecules appear to undergo significant bending motions at the cell surface when receptors are bound. This is one of the very first demonstrations of such changes in situ at the cell surface.
Exploitation Route As above Fundamental studies that could help direct the design and implementation of novel anti-bacterials
Sectors Pharmaceuticals and Medical Biotechnology

 
Description An important technological development from this project is the Lateral Molecular Force Microscope (LMFM) that has been developed with Dr Antognozzi's laboratory for these studies. This is a specialised atomic force microscope in which the cantilever is placed vertical rather than horizontal to the specimen, allowing more accurate measurements in the x-y plane. Combined with TIRF visualisation of the sample, this device has enabled us to measure behaviour of the UspA1 adhesins directly in situ at the cell surface - one of the primary aims of this project. The LMFM is now being used and further developed for a range of other biological investigations, including similar studies of other adhesins at bacterial cell surfaces. This is significant as it has not previously been possible to observe molecular changes directly on membranes, with most studies of this kind instead using isolated proteins. These studies have also resulted in the production of a large number of plasmids for bacterial expression of a range of recombinant proteins. These include 6 constructs for segments of Moraxella UspA1 (overlapping fragments covering most of the extracellular domain), 8 constructs for segments of human fibronectin (variously containing FNIII domains 6-15), human CEACAM1, and a range of single-point mutations of both CEACAM1 and the UspA1 stalk. We have also recombinantly expressed a range of segments from the homologous UspA2, also from Moraxella. The peptide studies have also resulted in a series of synthetic peptides with a range of assembly properties. These include designs with the CEACAM1 binding site of UspA1, in addition to seqences with tetrameric and hexameric assembly properties.
First Year Of Impact 2010
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic