The catabolism of collagen (the most abundant protein in human body) is precisely regulated in normal physiology and a critical physiological process in the extracellular matrix. Abnormal collagen catabolism is key in the development of pathological conditions such as tumor growth and invasion, arthritis and fibrosis. However, the mechanism of collagenolisys still remains poorly understood. Thus, understanding the basic biology and pathology of collagenolysis can help in the development of novel therapeutic agents. Our preliminary results show that the conformational flexibility in MMP-1 is important and can exercises impact on its function. Long-term goal of the proposed research is to understand the molecular mechanism of collagenolysis and to apply this knowledge in developing novel therapeutics by integrated application of computational and experimental methods. The objective of the proposed research application will be accomplished by two specific aims:
Aim 1 Will define the conformational transition from initially-bound substrate to MMP-1 to the formation of catalytically productive MMP-1THP ES complex. The work hypothesis is that conformational flexibility plays crucial role in the formation of a productive ES complex and in particular the flexibility of the linker region is a driving force that triggers transition from initially bound MMP-1THP complex to catalytically productive ES complex. We suppose that the process continues with rotation of HPX domain and CAT domain and further unsplitting and insertion of the leading THP chain in the active site, where it is stabilized by interactions with the active site residues in the CAT domain. Experimental kinetic studies of the formation of the kinetically productive MMP-1THP complex using different clinically important mutations in THPs will be done and will be used for calibration and validation of the computational predictions.
Aim 2 Modeling the reaction mechanism of substrate's L-chain hydrolysis by MMP-1 and computer-aided design of selective mechanism-based inhibitors of MMP-1. The hypothesis that MMP-1collagen hydrolysis proceeds via reaction mechanism in four steps: first nucleophilic attack, then followed by hydrogen bond rearrangement, proton transfer and scissile bond C-N cleavage will be tested. In addition, we hypothesize that the conformational flexibility exercises sensitive effects on MMP-1 reaction path. These studies will help us reveal the intimate mechanism of collagen hydrolysis in MMP-1.
Abnormal collagenlysis leads to the development of pathological conditions, however the intimate mechanism of the process remains unknown. The proposed research will reveal structural and mechanistic features of the process of collagenolysis that can help for the development of novel therapeutic agents.
