Institute of Biomedical and Biomolecular Science (IBBS)
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Structure-Function Relationships in Matrix Metalloproteinases
Matrix metalloproteinases (MMPs) are a family of zinc-dependant endopeptidases that are important not only in normal physiological processes but also in conditions such as cancer, arthritis and cardiovascular disease. The MMPs are produced as inactive zymogens with an inhibitory pro-domain (PRO) blocking the active site of their catalytic (CAT) domains. The majority of MMPs also possess a hemopexin (HPX) domain which is multifunctional but in most cases allows the enzyme to discriminate between substrates (Figure 1). We are interested in answering two specific questions: (1) what is the series of events leading to MMP activation? and (2) what is the molecular mechanism of collagen breakdown (“collagenolysis”) by MMPs? The latter question is particularly intriguing as the active site cleft in the CAT domain is too narrow to accommodate the collagen triple helix. Thus, the enzyme must somehow unwind the helix prior to hydrolysing the polypeptide backbone.
Figure 1. Cartoon (left), space-filling (middle) and ribbon (right) representations of MMP-1 highlighting the CAT (green) and HPX (blue) domains. In the middle figure, the catalytic zinc ion (white) can be seen at the bottom of the active site cleft in the CAT domain.
We employ a variety of biophysical methods to perform multi-disciplinary research on the structure-function relationships in collagenolytic MMPs. This is illustrated in our recent article (Arnold et al., 2011) in which, using a combination of analytical ultracentrifugation (AUC), nuclear magnetic resonance (NMR) spectroscopy, surface plasmon resonance (SPR) and small-angle X-ray scattering (SAXS), we found that the interface between the CAT and HPX domains in MMP-1 conceals residues that are important for collagen recognition.
Biomolecular Structure Determination using Swarm Intelligence
The calculation of protein structures from NMR spectroscopy data is a laborious task. Typically, it involves the identification of hundreds to thousands of close-range interactions (nuclear Overhauser effects or NOEs) between pairs of hydrogen nuclei (protons) in the protein. These protons are identified by their chemical shifts and thus an NOE by a pair of chemical shifts. If multiple protons have the same (or very similar) chemical shifts then NOEs involving those protons are ambiguous, i.e. they cannot be assigned to a particular donor-acceptor proton pair without prior knowledge of the protein structure. However, the protein structure cannot be determined without assigning the ambiguous NOEs. This is known as “the NOE ambiguity problem” and solving it is usually the rate-limiting step in biomolecular structure determination from NMR data.
Figure 2. Swarm intelligence NMR. This schematic shows a swarm of seven biomolecular ants undergoing simultaneous restrained molecular dynamics and communicating via a set of communal inter-proton NOE restraints.
We have developed a novel method of solving the NOE ambiguity problem that is built upon the concept of swarm intelligence – the apparent cleverness that arises from the co-operation between simple beings such as social insects. In our swarm intelligence NMR method (Figure 2), a swarm of biomolecular ants explore their conformational space through molecular dynamics simulations, in an analogous fashion to spatial exploration by a real colony of ants. Any biomolecular ant that encounters a conformation which agrees well with the experimental data informs the other members of the swarm and encourages them to adopt that same structure. In so doing, the ants co-operate to find the solution structure of the protein without the need for the laborious explicit assignment of the ambiguous NOEs. This novel method has been patented and was used recently in the NMR structure determination of estrogen receptor ligands (Phillips et al., 2011).
- Dr. Andy Pickford, Senior Lecturer
- Dr. Chris Read, Senior Research Fellow (associate group member)
- Louise Butt, IBBS-funded PhD student
- Neha Karia, undergraduate project student
- Sahar Iftikhar, undergraduate project student
Former Lab Members
- Dr. Stephen Prior (University of Missouri, USA)
- Dr. Laurence Arnold (MRC National Institute for Medical Research, Mill Hill, London)
- Prof Gregg B Fields, Torrey Pines Institute for Molecular Studies, Florida
- Prof Iain D Campbell and Dr John Vakonakis, Dept of Biochemistry, University of Oxford
- Dr Uli Schwarz-Linek, University of St Andrews
- Dr Paul Cox, Pharmacy and Biomedical Sciences, University of Portsmouth
- Dr Markus Schäde, AstraZeneca (formely at Pfizer, Sandwich)
Four year PhD studentship opportunity on 'The molecular mechanism of matrix metalloproteinase-1 activation'
Informal enquires welcome to: firstname.lastname@example.org
Application deadline: 31st January 2012
Undergraduate Research Projects 2012-2013
Molecular mechanism of proMMP-1 activation.
Investigating collagen binding activity in stromelysin-1.
Optimising co-operation in swarm intelligence structure calculations.
Recombinant expression of collagen fragments from methylotrophic yeast.
Arnold LH, Butt LE, Prior SH, Read CM, Fields GB & Pickford AR (2011) The interface between catalytic and hemopexin domains in matrix metalloproteinase-1 (MMP-1) conceals a collagen binding exosite. (Manuscript submitted to J. Biol. Chem.)
Phillips C, Roberts LR, Schäde M, Bazin R, Bent A, Davies NL, Moore R, Pannifer AD, Pickford AR, Prior SH, Read CM, Scott A, Brown DG, Xu B & Irving SL (2011) Design and structure of stapled peptides binding to estrogen receptors. J. Am. Chem. Soc. 133, 9696-9.
Erat MC, Schwarz-Linek U, Pickford AR, Farndale RW, Campbell ID & Vakonakis I (2010) Implications for collagen binding from the crystallographic structure of fibronectin 6FnI1–2FnII7FnI. J. Biol. Chem. 285, 33764-70.
Millard CJ, Ellis IR, Pickford AR, Schor AM, Schor SL & Campbell ID (2007) The role of the fibronectin IGD motif in stimulating fibroblast migration. J. Biol. Chem. 282, 35530-5.
Wegener KL, Partridge AW, Han J, Pickford AR, Liddington RC, Ginsberg MH & Campbell ID (2006) Structural basis of integrin activation by talin. Cell 128, 171-82.
The Royal Society (2006-2007): "The Molecular Mechanism of Collagen Degradation by Matrix Metalloproteinase-1", £15,000
IBBS PhD Studentship (2006-2009): “Biophysical Characterisation of Collagen Binding by the Hemopexin Domain of Matrix Metalloproteinase -1 (MMP-1)” to Laurence Arnold.
Biotechnology and Biological Sciences Research Council (2008-2011): "Protein Structure Determination from Nuclear Magnetic Resonance (NMR) Spectroscopy using Swarm Intelligence", £312,209 (BB/F004532/1)
IBBS PhD Studentship (2009-2012): “The Role of Inter-Domain Interactions in Matrix Metalloproteinase Latency” to Louise Butt.