Purification and characterization of the human stromelysin catalytic domain expressed in Escherichia coli

Ye, Qi Zhuang; Johnson, Linda L.; Hupe, Donald; Baragi, Vijaykumar M.

doi:10.1021/bi00160a038

Cited by 79 publications

(65 citation statements)

References 25 publications

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“…It was decided to study stromelysin at pH 7, in the presence of 20-fold excess of CaCI, to avoid the [local] unfolding. Stromelysin activity is just a factor of two lower than optimum at pH 7 (Ye et al, 1992). We speculate that the unfolding at pH 6.0 occurs in the Ca2+ binding site regions because CaZ+ affinity is known to decrease 10-fold with a pH change from 7 to 5.5 (Wilhelm et at., 1993).…”

Section: Role Of Metalmentioning

confidence: 77%

“…As stromelysin catalytic domain is most active at pH 6.0 (Ye et al, 1992), a logical choice would be to study the molecule at or near that pH, similar to the work reported by Gooley et at. (1994).…”

Section: Role Of Metalmentioning

confidence: 99%

“…The stromelysin catalytic domain was expressed in Escherichia coli and purified from inclusion bodies as described previously by Ye et al (1992). Preparation of the labeled samples, typically about 1 mM, and of the buffers (containing I O rnM Tris-dll-HCI, pH 7.0, 20 mM CaCI,, 15% acetonitrile43 and 8% D,O) was as described by .…”

Section: N M R Data Collection and Handlingmentioning

confidence: 99%

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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 Metalmentioning

confidence: 77%

Section: Role Of Metalmentioning

confidence: 99%

Section: N M R Data Collection and Handlingmentioning

confidence: 99%

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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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“…Matrilysin (PUMP; MMP-7) naturally lacks the C-terminal hemopexinlike domain and is an active enzyme [24]. Stromelysin and 72-kDa type IV collagenase are active with this domain removed [25][26][27]. On the other hand, fibroblast and neutrophil collagenases lacking the C-terminal domain lose the degradative activity against collagens [28,29].…”

Section: Expression and Characterization Of Rat 92-kda Type IV Collagmentioning

confidence: 99%

Cloning of rat 92‐kDa type IV collagenase and expression of an active recombinant catalytic domain

Xia¹,

García²,

Chen³

et al. 1996

FEBS Letters

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A full-length cDNA for rat 92-kDa type IV collagenase was isolated and sequenced. RNase protection assay revealed tissue specific differential expression of the 92-kDa type IV collagenase in the rat during development. Natural and modified forms of the 92-kDa type IV collagenase were expressed. One active protein, 92-CD, contained only the putative catalytic domain. Large quantities of the 92-CD were expressed in Escherichia coli, extracted from inclusion bodies, purified, and refolded to an active form. This recombinant protein was able to cleave denatured and native collagen and was inactivated by known MMP inhibitors.

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“…Truncated forms of the proenzymes lacking the C-terminal domain have been constructed by using DNA technology and these are the forms that were used to determine the published structures. Some kinetic studies have been performed on these domain deletion mutants and they have been shown to have catalytic and specificity characteristics similar to those of their full-length counterparts [12,13]. As part of a study designed to characterise more fully the full-length and short form enzymes, we have compared the zinc content of full-length prostromelysin-1 and progelatinase A with that of the truncated mutants, N-prostromelysin-1 and N-progelatinase A.…”

Section: Introductionmentioning

confidence: 99%

The second zinc atom in the matrix metalloproteinase catalytic domain is absent in the full‐length enzymes: a possible role for the C‐terminal domain

Willenbrock

Murphy²,

Phillips

et al. 1995

FEBS Letters

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Domain deletion mutants of the matrix metaHoproteinases consisting of the catalytic domain only contain two zinc atoms per molecule. One is essential for catalysis, while the other may fulfil a structural role. We have analysed the zinc contents of both the full-length and the truncated mutants of prostromelysin-1 and progelatinase A and report that the second zinc atom is not present in the full-length form of the proenzymes. Thus it seems likely that the role proposed for this zinc atom in maintaining the structure of the enzyme catalytic domain is performed by the C-terminal domain in the full-length enzyme.

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Purification and characterization of the human stromelysin catalytic domain expressed in Escherichia coli

Cited by 79 publications

References 25 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

Cloning of rat 92‐kDa type IV collagenase and expression of an active recombinant catalytic domain

The second zinc atom in the matrix metalloproteinase catalytic domain is absent in the full‐length enzymes: a possible role for the C‐terminal domain

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