PDBsum entry 2poj

Hydrolase

PDB id: 2poj

Contents

Protein chain

164 a.a. *

Metals

_CA ×3

_ZN ×2

* Residue conservation analysis

PDB id:

2poj

Name:

Hydrolase

Title:

Nmr solution structure of the inhibitor-free state of macrophage metalloelastase (mmp-12)

Structure:

Macrophage metalloelastase. Chain: a. Fragment: catalytic domain. Synonym: hme, matrix metalloproteinase-12, mmp-12, macrophage elastase, me. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: mmp12, hme. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

20 models

Authors:

R.Bhaskaran,S.R.Van Doren

Key ref:

R.Bhaskaran et al. (2007). Solution structure of inhibitor-free human metalloelastase (MMP-12) indicates an internal conformational adjustment. J Mol Biol, 374, 1333-1344. PubMed id: 17997411 DOI: 10.1016/j.jmb.2007.10.028

Date:

26-Apr-07 Release date: 04-Dec-07

Protein chain

P39900  (MMP12_HUMAN) -  Macrophage metalloelastase from Homo sapiens

Seq: 470 a.a.

Struc: 164 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.24.65 - macrophage elastase.
• Reaction: Hydrolysis of soluble and insoluble elastin. Specific cleavages are also produced at 14-Ala-|-Leu-15 and 16-Tyr-|-Leu-17 in the B chain of insulin.
• Cofactor: Ca(2+); Zn(2+)

DOI no: 10.1016/j.jmb.2007.10.028

J Mol Biol 374:1333-1344 (2007)

PubMed id: 17997411

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

R.Bhaskaran, M.O.Palmier, N.A.Bagegni, X.Liang, S.R.Van Doren.

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.

Selected figure(s)

Figure 2.
Fig. 2. Comparison of the 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)^31 to crystal structures with wild-type (1JIZ^32 and 1ROS^17) or F171D mutant sequence (1RMZ,^35 2OXU,^40 and 2HU6^48). 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.
Fig. 3. (a) The NMR structure of MMP-12 free from inhibitor (blue; 2POJ) superposed with the crystal structures of MMP-12 with the inhibitor washed away (red; 2OXU),^40 or bound to hydroxamate inhibitor (either E219A-substituted 1JK3^31 in green or 1JIZ^32 in gold), as shown in stereo. The inhibitor CGS-27023A 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 an inhibitor. For clarity, the S-shaped III–IV loop and the N-terminal six or seven residues are clipped from view. Zinc and calcium ions in the solution structure are in 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 contacts unique to this NMR structure are plotted with sticks.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 374, 1333-1344) copyright 2007.

Figures were selected by an automated process.