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Title: Interaction between triple-helical collagens and human collagenases
Author: Manka, Szymon W.
ISNI:       0000 0004 2696 4123
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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Collagens are the major structural proteins in animal tissues. Their degradation is essential in embryogenesis and development, while unbalanced collagen breakdown is seen in diseases such as arthritis, atherosclerosis and cancer. Fibril-­forming collagens I, II and III consist of three polypeptide chains forming a triple helix, which is resistant to cleavage by most proteases. Collagenases of the matrix metalloproteinase family (MMP-­1, MMP-­8, and MMP-­13) degrade fibrillar collagens by locally unwinding the helix, followed by cleavage into ¼ and ¾ fragments. They comprise two domains, the catalytic (Cat) domain and the hemopexin (Hpx) domain, which are connected via a flexible linker. Both domains are essential for collagenolysis, but the exact sites of collagenase-­collagen interactions and how they unwind collagen remain elusive. This thesis addresses the roles of individual collagenase domains, and the sites in both the enzyme and the substrate that are involved in collagen binding and unwinding, focusing on the fibril-­forming collagens and human MMP-­1 as a prototype. MMP-­1 bound to immobilised collagen I with markedly higher affinity than its Hpx domain alone. The Cat domain alone failed to bind to collagen, but in the full-­length enzyme it participated in collagen binding. Above 25°C the two-­ domain binding involved the catalytic site cleft. Triple-­helical peptide (THP) Toolkits of collagens II and III were screened for MMP-­1 binding, and the collagenase binding motif has been established. It contains two hydrophobic residues within a 9 residue distance. Finally, hydrogen/deuterium exchange mass spectrometry (H/DXMS) experiments indicated two potential collagen binding sites: 285-­316 and 349-365 in the Hpx domain, and suggested a possibility of a dynamic interaction of the collagenase N-­terminus with collagen. These results imply that the two domains of collagenase bind to collagen in a cooperative manner. Based on the THP binding and H/DXMS data a 3D model of collagenase-­collagen interaction has been proposed. It assumes that collagenase utilises hydrophobic interactions to unwind the collagen helix via perturbation of the hydrogen-­bond network which stabilises the helix.
Supervisor: Nagase, Hideaki ; Visse, Robert ; Wait, Robin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral