Structural basis of the action of thermolysin and related zinc peptidases

Matthews, Brian W.

doi:10.1021/ar00153a003

Cited by 727 publications

(615 citation statements)

References 76 publications

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“…This was verified for collagenases by crystallography and for stromelysin by diagnostic imidazole chemical shifts (Gooley et al, 1993). The fourth ligand in uninhibited matrix metalloproteinases is expected to be a water molecule able to carry out nucleophilic attack on the scissile carbonyl once polarized by a conserved glutamate in helix B acting as a general base, like a mechanism proposed for thermolysin by Matthews (1988). Hydroxamate-substituted inhibitors like the one used in this study must displace this water in providing the fourth and fifth ligands.…”

Section: Role Of Metalsupporting

confidence: 52%

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor

Doren

Kurochkin

et al. 1995

Protein Science

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Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed 0-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The &sheet and a-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemisfry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal Si subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic Si surface subsite, and a third hydrophobic side chain (Pi) lies at the surface.Keywords: catalytic domain; hydroxamate; matrix metalloproteinase 3; multidimensional NMR; protein structure; stromelysinAs a member of the zinc-and calcium-dependent family of matrix metalloproteinases (MMPs), which hydrolyze the extracellular matrix, stromelysin participates in the tissue remodeling of health and disease. Regarding the shared domain structure of the MMPs, their subfamilies, specificity, transcriptional regulation, activation, and posttranslational regulation, see reviews of Woessner (1991), Matrisian (1992), Birkedal-Hansen et al. (1993), andNagase (1995). The involvement of MMPs in tissue remodeling includes that of development, tissue resorption, reproduction, angiogenesis, and wound healing, and that of sev-

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Section: Role Of Metalsupporting

confidence: 52%

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor

Doren

Kurochkin

et al. 1995

Protein Science

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“…Elimination of one of these interactions upon substitution of Ala at P3 with Pro (compounds 3, 4 versus compounds 1, 2) could possibly account for the loss of activity by the serine proteases, although steric restrictions resulting from this substitution may also contribute [24]. More recent X-ray crystallographic investigations of the complexes formed between thermolysin and its inhibitors also led to a proposal as to the mode of binding of extended peptide substrates to thermolysin [27,28]. Again, the substrate is bound to the enzyme as in an antiparallel fl-pleated sheet, with two hydrogen bonds contributed by the backbone of the substrate residue at position P2 and one hydrogen bond by the NH group of the residue at position P].…”

Section: Resultsmentioning

confidence: 99%

“…Again, the substrate is bound to the enzyme as in an antiparallel fl-pleated sheet, with two hydrogen bonds contributed by the backbone of the substrate residue at position P2 and one hydrogen bond by the NH group of the residue at position P]. However, additional anchorage points of the substrate to the enzyme are assigned to the NH and CO groups of the residue at position P~, with the phenolic oxygen of Tyr-157 and the active site zinc, respectively [28]. Apparently, the release of one of these interactions upon substitution of the N-terminal Ala with Pro (compounds 3, 4 versus compounds 1, 2) is of a much smaller effect in thermolysin compared with its effect in the serine proteases (Table 4).…”

Section: Resultsmentioning

confidence: 99%

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

et al. 1996

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Tripeptide derivatives like 3-carboxypropanoylalanyl-alanyMeucine 4-nitroanilide or 3-carboxypropanoylalanyl-alanyl-phenylalanine 4-nitroanilide are very sensitive substrates for neprilysin (kcat > 102 s-Z; kcatlKm >-106 s -~ "M-~) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (kcat ~ I s-~-34 s-~). By substituting the N-terminal alanine of the substrates with proline, the catalytic efficiency of the enzymic reaction, by the serine proteases, is diminished by 2-3 orders of magnitude, whereas that by neprilysin and thermolysin decreases only slightly. These effects demonstrate that structural alterations in peptide substrates that impair secondary sub-site interactions with one class of peptidases may enhance the selectivity of the substrates towards another class of peptidases.

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“…In TLN, the position of Glu143, which acts as a general base in catalysis, overlaps its CPD counterpart, Glu270 (60). The positively charged arginine cluster in CPD is replaced by His231, which is also thought to stabilize the oxyanion transition state (61). Consequentially, the 10-fold lower potency of D-Cys toward TLN as compared to CPD may be due to replacement of the positively charged arginine residues with a neutral histidine residue, which can only form a single hydrogen bond rather than the two that the guanidinium group can form.…”

Section: Possible Implications For Cpa Interaction With D-penmentioning

confidence: 99%

Crystal Structure of Carboxypeptidase A Complexed with d-Cysteine at 1.75 Å − Inhibitor-Induced Conformational Changes^,

2000

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D-Cysteine differs from the antiarthritis drug D-penicillamine by only two methyl groups on the -carbon yet inhibits carboxypeptidase A (CPD) by a distinct mechanism: D-cysteine binds tightly to the active site zinc, while D-penicillamine catalyzes metal removal. To investigate the structural basis for this difference, we solved the crystal structure of carboxypeptidase A complexed with D-cysteine (D-Cys) at 1.75-Å resolution. D-Cys binds the active site zinc with a sulfur ligand and forms additional interactions with surrounding side chains of the enzyme. The structure explains the difference in potency between D-Cys and L-Cys and provides insight into the mechanism of D-penicillamine inhibition. D-Cys binding induces a concerted motion of the side chains around the zinc ion, similar to that found in other carboxypeptidase-inhibitor crystal structures and along a limited path. Analysis of concerted motions of CPD and CPD-inhibitor crystal structures reveals a clustering of these structures into distinct groups. Using the restricted conformational flexibility of a drug target in this type of analysis could greatly enhance efficiency in drug design.

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Structural basis of the action of thermolysin and related zinc peptidases

Cited by 727 publications

References 76 publications

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

Crystal Structure of Carboxypeptidase A Complexed with d-Cysteine at 1.75 Å − Inhibitor-Induced Conformational Changes^,

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Structural basis of the action of thermolysin and related zinc peptidases

Cited by 727 publications

References 76 publications

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

Crystal Structure of Carboxypeptidase A Complexed with d-Cysteine at 1.75 Å − Inhibitor-Induced Conformational Changes,

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Crystal Structure of Carboxypeptidase A Complexed with d-Cysteine at 1.75 Å − Inhibitor-Induced Conformational Changes^,