Solution structure of inhibitor-free human metalloelastase (MMP-12) indicates an internal conformational adjustment

Abstract

Macrophage metalloelastase or matrix metalloproteinase-12 (MMP-12) appears to exacerbate atherosclerosis, emphysema, aortic aneurysm, rheumatoid arthritis, and inflammatory bowel disease. An inactivating E219A mutation, validated by crystallography and NMR spectra, prevents autolysis of MMP-12 and allows us to determine its NMR structure without an inhibitor. The structural ensemble of the catalytic domain without an inhibitor is based on 2813 nuclear Overhauser effects (NOEs) and has an average RMSD to the mean structure of 0.25 A for the backbone and 0.61 A for all heavy atoms for residues Trp109-Gly263. Compared to crystal structures of MMP-12, helix B (hB) at the active site is unexpectedly more deeply recessed under the beta-sheet. This opens a pocket between hB and beta-strand IV in the active-site cleft. Both hB and an internal cavity are shifted toward beta-strand I, beta-strand III, and helix A on the back side of the protease. About 25 internal NOE contacts distinguish the inhibitor-free solution structure and indicate hB's greater depth and proximity to the sheet and helix A. Line broadening and multiplicity of amide proton NMR peaks from hB are consistent with hB undergoing a slow conformational exchange among subtly different environments. Inhibitor-binding-induced perturbations of the NMR spectra of MMP-1 and MMP-3 map to similar locations across MMP-12 and encompass the internal conformational adjustments. Evolutionary trace analysis suggests a functionally important network of residues that encompasses most of the locations adjusting in conformation, including 18 residues with NOE contacts unique to inhibitor-free MMP-12. The conformational change, sequence analysis, and inhibitor perturbations of NMR spectra agree on the network they identify between structural scaffold and the active site of MMPs.

Figure 1

(a) NMR chemical shift perturbations of MMP-12 by inhibitors or E219A mutation at the active site. The radial chemical shift changes are defined as: Δω_HN = [(Δω_H)2 + (Δω_N/5)²]^1/2 relative to the peak positions in inhibitor-free MMP-12(E219A) . Red circles mark differences from MMP-12(F171D) bound to NNGH . Blue triangles mark differences from MMP-12 bound to Wyeth’s proprietary inhibitor . Black squares mark differences of the short-lived, wild-type spectrum from that of E219A-substituted MMP-12. Helices are marked by cylinders and β-strands by arrows. (b) The backbone of the ensemble of 20 accepted structures (PDB code 2POJ) is plotted in stereo. It is colored gold where inhibitor causes Δω_HN > 1.0 ppm, orange-red where 1.0 ppm > Δω_HN > 0.3 ppm, and violet where 0.3 ppm > Δω_HN > 0.1 ppm. Calcium ions are colored orange and zinc gray.

Figure 2

Comparison of coordinates of MMP-12 X-ray and NMR structures having the E219A mutation (1JK3 and 2POJ, respectively) with X-ray structures lacking this inactivating point mutation. Black squares indicate the average Cα RMSD of the crystal structure of MMP-12(E219A) with batimastat bound (1JK3) to crystal structures with wild-type (1JIZ , 1ROS 17) or F171D mutant sequence (1RMZ , 2OXU , 2HU6 80). The inhibitor-free solution structure with the E219A mutation (2POJ) is compared to crystal structures of active MMP-12 with F171D mutation (blue triangles, 2OXU), NNGH-inhibited F171D variant (red triangles, 1RMZ), and batimastat-inhibited E219A variant (green squares, 1JK3).

Figure 3

(a) NMR structure of MMP-12 free of inhibitor (blue; 2POJ) is superposed with crystal structures of MMP-12 with inhibitor washed away (red; 2OXU) or a hydroxamate inhibitor bound (either E219A-substituted 1JK3 in green or 1JIZ in gold), as shown in stereo. The inhibitor CGS27023A from 1JIZ is drawn with ball and sticks. Lines point out closer approaches of hB with sI, sIII or hA in the solution structure without inhibitor. For clarity, the S-shaped III-IV loop and N-terminal six or seven residues are clipped from view. Zinc and calcium ions in the solution structure are gray and orange, respectively. (b) Dotted blue lines indicate 25 NOEs that distinguish the ligand-free solution structure from X-ray structures using crystallization with inhibitors. Side chain and backbone groups involved in the contacts unique to this NMR structure are plotted with sticks.

Figure 4

Comparison of MMP-12’s active site surfaces and internal pocket in (a) the inhibitor-free solution structure (model 1 of 2POJ), (b) structure from crystals formed with acetohydroxamate inhibitor subsequently washed out to allow activity (2OXU), and (c) crystal structure with batimistat bound but not shown (1JK3). A viewing slab 12 Å thick clips off near and far surfaces to reveal internal cavities. Surface-accessible hydrogen atoms are colored light gray, carbon green, nitrogen blue, and oxygen red. A sphere marks the zinc ion in the active site. Its histidine ligands are drawn with sticks. The arrow at right in each panel points to the S₁’ specificity pocket. The arrow at left in each panel points to the buried cavity. The middle arrow in (a) points to a pocket unique to the solution structure of inhibitor-free MMP-12(E219A).

Figure 5

Residues were ranked in importance to structure and function of MMP-12 and other MMPs using the real-valued Evolutionary trace method ; , and marked on the solution structure of MMP-12. The first, second, and third most important deciles are colored green, yellow, and red, respectively. Panel (a) plots the backbone. Panel (b) also plots the side chain heavy atoms with sticks and dots for the top three deciles of residues. Zinc and calcium ions are gray and orange, respectively.