Stephen J. Perkins, Justin Hinshelwood, Yvonne J. K. Edwards and P. Vince. Jenkins, Department of Biochemistry and Molecular Biology, and of. Haematology.
Biochemical Society Transactions ( 1999) Volume 27, part 5
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The need for expression expertise in solid state NMR studies of membrane proteins and peptides - successes and wish lists
Biomembrane Structure Unit, Biochemistry Department, South Parks Road, Oxford, OX1 3 QU, UK Resolving molecular details for membrane-embedded peptides (toxins, ion channels) and proteins (receptors, transporters) is still high on the agenda of many structural biologists. Progress is being made, with crystallisation being only one hurdle and production, isolation, characterisation and reconstitution all presenting their own challenges. Solid state NMR is becoming a powerful addition to the arsenal of the structural biologist for membrane systems, not least because some of the problems associated with crystallography do not hamper the NMR approach. In particular, a functionally competent protein or peptide can be studied directly in association with lipids i n a heterogeneous biomembrane, or when reconstituted into bilayers. NMR-visible. non-perturbing isotopes ('"2, "N, I9F, *H) are being incorporated into specific residues or the backbone of a protein using expression or chemical means, permitting specific regions of the protein to be examined. Interatomic distances can be determined precisely (to better than 0.05nm) between NMR-visible atoms, and the orientation of residues and ligands determined to high precision (better than f 5"). Ligand structures (drugs, prosthetic groups, hormones) whilst at their site of action, and the residues involved, have been identified. Also functional intermediates (helix reorientation, ligand conformational changes) have been resolved, including mechanistic detail about electronic changes on binding. A progress report of some advances of the method will be presented here, with indications of how further progress is being planned and made, especially in combination with the power of expression technologies where appropriate, for sugar transporters, GPCRs ATPases, a ligandgated ion channel and membrane peptides. (For general references for the use of solid state NMR i n biology see www.bioch.ox.ac.uk/-oubsu/www/ssnmrb/ssnmrb.html and for the membrane work from the group. see www.bioch,ox.ac.uk/-awattsl).
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Integrin a2p1 is a collagen receptor that plays a n essential role in t h e adhesion of platelets duri ng pri mary haemostasis. a2pl binding to collagen is magnesium dependant a n d is mediated through t h e 200 residue inserted 'I' d o m a i n of t h e alpha su b u n i t (a2-I). T h e triple-helical st ruct ure of collagen is required for aZ-I domai n recognition a n d several groups have reported similar binding affinities in t h e range 50-500 nanomolar. W e have previously reported t h e crystal structure of t h e u2-I d o mai n determined to high resolution. T h e a2-I d o m a i n st r u c t ure adopt s t he dinucleotide binding fold, a nd contains a metal i o n dependent adhesion site ( M I D A S ) motif wi th b o u n d Mg2+ a t t h e t o p of the beta-sheet. This structure revealed a n e w helix (the C-helix) prot rudi ng f r o m the MIDAS site face w h i c h creates a groove centred o n t he magnesium ion. Recently t h e specific collagen sequences recognised by t h e 1x2-I d o m a i n have been identified as a 6 residue segment GFPGER using overlapping synthetic peptides derived f r o m a bovine collagen t y p e I fragment. T h e ER residues of this segment have been s h o w n to play a n essential role i n binding. A combination of molecular modelling, site directed mutagenesis a n d crystallo g r a p h y provides insight i nt o t he interaction between the GFPGER binding site on collagen a n d t he MIDAS site o n the integrin I d o mai n.
0 1999 Biochemical Society
STRUCTURE AND FUNCTION OF VWF-A DOMAINS IN COMPLEMENT AND COAGULATION
Stephen J. Perkins, Justin Hinshelwood, Yvonne J. K. Edwards and P. Vince Jenkins, Department of Biochemistry and Molecular Biology, and of Haematology. Royal Free Campus, Royal Free and Univerhity College Medical School, Rowland Hill Street, London NW3 2PF, U.K. The von Willebrand factor type A (vWF-A)domain occurs in the complement system ot immune defence in factor 9, C2 and complement receptor type 3 (CR3) and in blood coagulation as one of the four domain types in von Willebrand factor. A combination of ( I ) analogy and (2) homology modelling approaches together with (3) structural studies of a recombinant vWF-A domain from Factor B has revealed many functional propertieb of this protein superfamily. ( I ) In 1995, sequence, spectroscopic and structural data showed that the vWF-A domain was predicted to be similar i n its overall fold to the rasp21 nucleotide binding protein. The subsequent publication of the vWF-A crystal structure from CR3 verified the prediction. A post-mortem analysis 01 the prediction showed that most of the details of the structure had been correctly identified, with an accuracy of 77% for the secondary structure, with the only minor difference heing that a P-hairpin structure found at one end of the structure was reversed in its orientation. (2) Our understanding of the sequence and structure of vWF-Adomains was next used to interpret genetic mutations in the vWF-AI and -A2 domains that cause Type 2B and 2M von Willebrand's Disease, hased on homology modelling of the vWF-A domain. The analysis o l 2X vWF-AI sequences from mammalian species identified a conserved set 0 1 basic residues that were not conserved in Over 70 other vWF-A sequenceb, and this led to the prediction of a heparin-binding site on one side of the structure. Single amino acid mutations in von Willebrand factor that result in Type 2 8 disease were localised to this heparin-binding region of the vWF-AI domain and involved the upregulation of vWF activity. Mutations that resulted in downregulation of vWF activity result in Type 2M disease, and these were located to the other side of the vWF-AI structure in a region associated with GPlb receptor binding. (3) Since it could he concluded that the vWF-AI domain displayed allosteric properties, the question of whether allosteric properties also existed in the vWF-A domain of facror B was examined. NMR and CD spectroscopy studies indicated that conformational changes occurred i n thc vWF-A domain as a function of the ligand present, and these will he discussed.
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A1 Structural studies of t he integrln a2-I domai n L o n a s Ernsley and Robert Liddington Department of Biochemistry, Leicester University, Leicester, L E I 7RH
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Functional Role of A-Domains in Type VI Collagen. A. Shuttleworth, Ball,S., Baldock, C., FakhouryHajeer,H. and Kielty,C.M. University of Manchester. 2.205 Stopford Oxford Rd, Manchester M13 9PT Collagen VI is one of the most ubiquitous and interactive extracellular macromolecules. It has a short triple helix, its non-collagenous sequence is dominated by numerous Adomains, and it has a unique biphasic intracellular and extracellular mode of assembly. Whilst the hndamental principles of triple helix formation undoudtedly apply to collagen V1, the molecular recognition signals for trimerisation are unique and its higher order intracellular oligomerisation is probably driven by its multiple A-domains. We have produced a variety of constructs comprising the collagenous domains plus the cysteine-rich carboxy-terminal flanking sequences with or without the carboxy and aminoterminal domains of all three chains and used these in an in vitro translation system t o investigate chain association. Triple-helix formation was monitored by proteolytic resistance and oligomerisation by SDS-PAGE. Differences in the ability of the three chains t o associate was evident and this association was shown t o be influenced by the presence of specific A-domains at the carboxy-terminal. In addition w e have produced recombinant peptides of Nterminal A-domains of the a3(VIII) chain and are currently examining structure differences between the various Adomains of type VI collagen. These studies are allowing us to define the regions of the molecule that direct chain association and control molecular interactions This work is supported by the BBSRC and MRC.
