Collagenase unwinds triple-helical collagen prior to peptide bond hydrolysis

Abstract

Breakdown of triple-helical interstitial collagens is essential in embryonic development, organ morphogenesis and tissue remodelling and repair. Aberrant collagenolysis may result in diseases such as arthritis, cancer, atherosclerosis, aneurysm and fibrosis. In vertebrates, it is initiated by collagenases belonging to the matrix metalloproteinase (MMP) family. The three-dimensional structure of a prototypic collagenase, MMP-1, indicates that the substrate-binding site of the enzyme is too narrow to accommodate triple-helical collagen. Here we report that collagenases bind and locally unwind the triple-helical structure before hydrolyzing the peptide bonds. Mutation of the catalytically essential residue Glu200 of MMP-1 to Ala resulted in a catalytically inactive enzyme, but in its presence noncollagenolytic proteinases digested collagen into typical 3/4 and 1/4 fragments, indicating that the MMP-1(E200A) mutant unwinds the triple-helical collagen. The study also shows that MMP-1 preferentially interacts with the alpha2(I) chain of type I collagen and cleaves the three alpha chains in succession. Our results throw light on the basic mechanisms that control a wide range of biological and pathological processes associated with tissue remodelling.

Figure 1

Alignment of the triple-helical peptide with the active site of MMP-1. (A) Collagen triple-helical peptides described by Kramer et al (2001) were manually aligned into the active site of the catalytic domain of porcine MMP-1 determined by Li et al (1995) using Insight II/Discover and the image was produced with Swiss PDB view (Guex and Peitsch, 1997). (B) The alignment model of MMP-1 and the triple-helical peptides in (A) were rotated 90° to the left. The location of the catalytic Zn²⁺ is indicated by an arrow. The active site shown as a cleft is unoccupied by the triple-helical peptide substrate. Pink, catalytic domain; blue, Hpx; purple, zinc ion.

Figure 2

Digestion of collagen I and gelatin I by MMP-1. Collagen I (30 μg) and gelatin I (30 μg) were incubated with 6 nM (A, B) or 40 nM (C, D) active MMP-1 at various temperatures for up to 2 h. The reaction was terminated by addition of 20 mM EDTA and subjected to SDS–PAGE under reducing conditions. TC^A and TC^B are 3/4 fragments and 1/4 fragments of α1(1) and α2(1) chains, respectively.

Figure 3

Digestion of collagen I in the presence of MMP-1(E200A) by MMP(ΔC). (A) Collagen I (30 μg) was incubated with 6 μM MMP-1(E200A) or (B) 0.7 μM MMP-1(ΔC) at 25°C for the indicated time. (C) Gelatin I (30 μg) was incubated with 0.7 μM MMP-1(ΔC). (D) Collagen I (30 μg) was made to react with an increasing amount of MMP-1(ΔC) in the presence of 6 μM MMP-1(E200A). The reaction products were analyzed as in Figure 2.

Figure 4

Digestion of collagen I by MMP-3(ΔC) or HLE in the presence of MMP-1(E200A). Collagen I (30 μg) was incubated with (A) 7.6 μM MMP-3(ΔC) or (B) 0.4 μM HLE in the presence of 6 μM MMP-1(E200A) at 25°C for up to 48 h. The reactions were terminated with 20 mM EDTA for MMP-3(ΔC) and 1 mM phenylmethylsulfonyl fluoride for HLE and the products were analyzed as in Figure 2.

Figure 5

Cleavage sites in collagen I by MMP-1(ΔC), MMP-3(ΔC) and HLE detected in the presence of MMP-1(E200A). The N-terminal amino-acid sequence of the TC^B fragments of α1(I) and α2(I) chain was determined as described in Materials and methods. In the case of HLE, only the α1(I) chain fragment was determined. The residues in the α2(I) chain indicated by brackets are the predicted cleavage sites of HLE based on enzyme specificity.

Figure 6

MMP-1(E200A) bound to GM6001X is unable to unwind collagen I. Collagen I (30 μg) was incubated with 2.5 μM MMP-1(E200A) and 0.4 μM HLE in the absence or presence of 10 μM GM6001X at 25°C for up to 48 h. The reaction of MMP-1(ΔC) and HLE was terminated by 1 mM phenylmethylsulfonyl fluoride and the products were analyzed as in Figure 4.

Figure 7

Thermal transition curve for guinea-pig type I collagen with or without MMP-1(E200A). Pepsin-treated guinea-pig type I collagen (3 μM) was incubated with or without an equimolar concentration of MMP-1(E200A) or MMP-3(E202A), and molar ellipticities were recorded at 222 nm while the temperature increased from 5 to 70°C at a rate of 35°C/h.

Figure 8

Unwinding of collagen by MMP-1 occurs only locally. Collagen I (30 μg) was incubated with 0.05 μM MMP-1 with or without chymotrypsin (CT) (20 μg/ml) at 25°C for up to 6 h. CT alone was incubated with collagen I and gelatin I as controls.

Figure 9

Collagenolytic activity expressed by reassociation of the catalytic domain and the Hpx domain. Collagen I (30 μg) was made to react with (A) 2μM MMP-1(E200A) and 0.8 μM MMP-1(ΔC), and (B) 4 μM Hpx_MMP-1 and 0.8 μM MMP-1(ΔC) at 25°C for the indicated period of time. The reactions were terminated with 20 mM EDTA and the products were analyzed as in Figure 2.

Figure 10

Detection of intermediate products during collagenolysis. Nonpepsin-treated collagen I was made to react with (A) 0.1 μM full-length collagenase or (B) 0.8 μM MMP-1(ΔC) and 6 μM MMP-1(E200A). The reactions were stopped with 20 mM EDTA and the products were analyzed by SDS–PAGE with 5% total acrylamide under reducing conditions. γ^A and β^A are the 3/4 fragments of γ and β chains generated by MMP-1. I₁, I₂, I₃ and I₄ indicate intermediate products.

Figure 11

Steps involved in collagenolysis. (A) Collagenase binds to and locally unwinds collagen before it cleaves the triple-helical interstitial collagen. (B) MMP-1(E200A) binds preferentially to the α2(I) chain and unwinds the triple-helical collagen, but is unable to cleave collagen. The unwound collagen becomes susceptible to noncollagenolytic proteinases indicated as ‘P' (e.g., MMP-1(ΔC), MMP-3(ΔC) and HLE), and the α1(I) chains are initially cleaved. This reaction requires the trimolecular complex formation of the unwinder (MMP-1(E200A)), a cutter proteinase and collagen.