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Abstract

Membrane proteins are poor targets for structural biology because of several factors including their relatively larger apparent molecular size and slow molecular motion when isolated in detergents, and their poor propensity to form well-ordered crystals. ese inherent characteristics render them difficult to be studied with well-established struc- ture determining techniques such as X-ray crystallography and solution NMR spectroscopy. Solid-state NMR, on the other hand, does not rely on availability of high quality crystals or molecular tumbling motion, hence serves as a promising tool for structure determination of membrane proteins, amyloid fibrils etc. YadA is a membrane protein from Yersinia enterocolitica and is a distinctive member of a family of non-fimbrial, non-pillus adhesins, named trimeric autotransporter adhesins. Its transmembrane anchor domain forms a highly stable trimeric β-barrel to let the sticky head pass through to adhere to the host cell surface in the extracellular environment. is adherence is the first step in causing several enteric foodborne diseases i.e., enterocolitis, diahrrea etc. Any information about structure and dynamics of domains involved in autotransport would significantly help understanding this mechanism. A de novo structure of the beta barrel membrane protein was obtained on the basis of MAS solid-state NMR data [1,2]. Various homo- and heteronuclear multidimensional NMR spectra were used to gather secondary and tertiary structure information. Several functional aspects of YadA-M were revealed from the structure. It was found that a quartet of helical residues (named the “ASSA” region) displays random-coil-like chemical shis, low order- parameters, reduced helix propensity, and a drop in signal intensity pointing towards a relatively increased flexi- bility. Based on combined structural and evolutionary studies, our work is in strong favour of the ‘hairpin model’ as mechanism of the autotransport. In addition, an uncommon, non-covalent S...O interaction was reported which was observed between the side-chain oxygen atoms of S38-S39 and the sulphur atom of M96. Based upon these findings we carried out point mutations in the ASSA region. Replacing the first Alanine in the ASSA region with a Proline residue shows marked structural changes. e mutant protein seems to have been stalled during autotrans- port. A high-resolution structure of the trapped conformation would give valuable functional information on the autotransport activity of TAAs.