Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1

Abstract

Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triple-helical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis.

Fig. 1.

Binding of MMP-1(E200A) to immobilized collagen I. (A) Binding of full-length MMP-1(E200A) and its individual Cat(E200A) and Hpx domains at 20 °C. (B) Binding of MMP-1(E200A) at different temperatures. (C) Binding of 1 μM MMP-1(E200A) in the presence of the active-site inhibitor GM6001 at different temperatures.

Fig. 2.

Screening of a triple-helical peptide library of collagen II with proMMP-1(E200A), MMP-1(E200A), Hpx domain, and Cat(E200A) domain. Error bars show SDs from three repeats. Critical Leu and Ile residues at P1′ and P10′ subsites are highlighted in the collagen II portion of the peptide sequences surrounding the collagenase cleavage site. ∼, bond cleaved by collagenases.

Fig. 3.

Collagen I footprint on MMP-1(E200A) determined by H/DXMS and mutagenesis. (A) Sites 1 and 3–6 are protected from deuterium incorporation, and site 2 shows enhanced deuterium incorporation upon collagen binding. The sites are mapped onto the crystal structure of MMP-1(E200A) (7). Residues potentially involved in collagenolysis that were mutated are indicated. Dashed line, predicted collagen binding direction. (B) Relative initial velocities of collagen I cleavage by MMP-1 and its variants normalized to WT.

Fig. 4.

Crystal structure of the MMP-1(E200A)–triple helical collagen peptide complex. (A) Sequence of the collagen peptide used for cocrystallization with MMP-1(E200A). Residues within 4 Å distance of the enzyme in the complex are colored. The subsite designation is indicated for the leading chain. (B) Stereoview of the MMP-1(E200A)–collagen peptide complex. The collagen chains are colored cyan (L), green (M), and red (T), and the enzyme is shown as a gray surface with areas within 4 Å distance of the L, M, and T chains colored correspondingly. Magenta sphere, active-site zinc ion. (C) Stereoview of the interactions between collagen chains (colored as in B) and the active site cleft of MMP-1(E200A). Dashed lines indicate hydrogen bonds. (D) Stereoview of the interactions of the collagen chains with the Hpx domain. Selected residues making enzyme-substrate contacts are labeled with respective colors and shown in stick representation (N, dark blue; O, red).

Fig. 5.

Modes of collagen binding to MMP-1. Shown is relationship between the unproductive and productive collagen binding modes (see text). In the sequence alignment on the left, the trailing chain is shown twice to emphasize the circular nature of the chain arrangement. The P1′ and P10′ residues are boxed, and residues interacting with MMP-1 are in bold. The structural models on the right show that the unproductive and productive binding modes are related by a simple rotation/translation of the collagen triple helix. Note that only the interactions with one collagen chain are changed (L in the unproductive mode, T in the productive mode); the interactions with the other two chains are the same in both modes.

Fig. 6.

Model of the first transition state of collagenolysis based on the productive complex. The upper rim of the catalytic site cleft anchors the M chain (green), and the Hpx domain anchors the L chain (cyan). These interactions position the T chain above the active site. Interdomain flexing of the enzyme bends the collagen triple helix and facilitates the insertion of Leu(P1′) of the T chain into the S1′ pocket.