| This thesis concerns three peptide ligands. Chapter 2 focuses on the structural/functional characterization of the first ligand, the 55 residue C-terminal segment of the alpha2-plasmin inhibitor (A2PI). A2PI is the major regulator of the blood-clot removing serine protease plasmin (Plm). Plasminogen (Pgn), the zymogen of Plm is a multidomain protein, consisting of an N-domain, five kringle repeats and a trypsin-like serine protease. Upon activation and subsequent autocatalytic hydrolysis of the N-domain, Plm undergoes a conformational change from a closed, compact configuration to an open form which binds to exposed lysines on the fibrin clot via the kringles, positioning the protease to lyse the clot. The aforementioned C-terminal peptide (A2PIC) disrupts fibrinolysis through competitive interactions with the kringles, the nature of which are explored in chapter 2. Chapter 3 concerns the folding of the second peptide ligand, the 77 residue Pgn N-domain. The N-domain of Pgn is thought to function as a switch that drives Pgn from its closed to open form. Chapter 3 argues that flexibility or plasticity may be an inherent feature of the N-domain. In order to serve as a switch, the N-domain may have to shuttle from a modestly beta-sheet structure to a loosely folded state. Chapter 4 introduces another protein, Matrix Metalloproteinase 2 (MMP-2). MMP-2 is modularly organized, consisting of a pro-domain, a zinc protease catalytic unit, a hemopexin-like domain and an array of three fibronectin type II (FN2) domains. MMP-2 and Pgn share many features. Both are extracellular proteins thought to be involved in a wide variety of processes such as cell migration, tissue remodeling, tumor invasion and metastasis. Both are multimodular proteins, consisting of regulatory, binding and protease motifs. Moreover, in both MMP-2 and Pgn, there are multiple copies binding domains, three FN2 repeats in MMP-2, and five kringle motifs in Pgn. Chapter 4 explores the affinity of each of the three FN2 repeats, both as individual domains and as components of mulit-FN2 arrays toward a third ligand, the triple helical peptide, Hel3, a mimic of collagen. Each FN2 binds Hel3, albeit with various affinities, implying a processive interaction between MMP-2 and collagen. |
