The gelatin-binding site of the second type-II domain of gelatinase A/MMP-2

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

We have shown previously that all three fibronectin type-II modules of gelatinase A contribute to its gelatin affinity. In the present work the second type-II module was subjected to site-directed mutagenesis in order to localize its gelatin-binding site. The functional integrity of mutant proteins was assessed by their affinity for gelatin using gelatin-Sepharose affinity chromatography. The structural integrity of the mutant proteins, i.e. their resistance to thermal and chaotropic agent-induced denaturation, was characterized by CD spectroscopy. Our studies show that, in the case of mutants R19L, R38L, K50G, K50R and R19L/R38L, the mutations had no significant effect on the structure and gelatin affinity of the type-II module, excluding the direct involvement of these residues in ligand binding. In the case of mutants Y25A, Y46A, D49A and Y52A, the mutations yielded proteins that were devoid of gelatin affinity. Structural characterization of these proteins, however, indicated that they had also lost their ability to fold into the native structure characteristic of the wild-type domain. In the case of mutant Y37A, the structure and stability of the mutant protein is similar to the wild-type module. However, its gelatin affinity was severely impaired compared with the wild-type protein. The fact that the Y37A mutation impairs ligand binding without detectable distortion of the module's architecture suggests that Y37 is directly involved in ligand binding. Homology modeling based on the three-dimensional structure of the second type-II module of PDC-109 places Y37 on the right-hand rim of a hydrophobic pocket that includes residues F20, W39, Y46, Y52 and F54, and thus provides proof for the involvement of this pocket in ligand binding.

Original languageEnglish
Pages (from-to)513-518
Number of pages6
JournalEuropean Journal of Biochemistry
Volume259
Issue number1-2
DOIs
Publication statusPublished - Jan 15 1999

Fingerprint

Matrix Metalloproteinase 2
Gelatin
Matrix Metalloproteinases
Binding Sites
Mutant Proteins
Ligands
Mutation
Affinity chromatography
Agarose Chromatography
Mutagenesis
Proteins
Denaturation
Structural integrity
Site-Directed Mutagenesis
Affinity Chromatography
Fibronectins
Sepharose
Spectrum Analysis
Hand
Hot Temperature

Keywords

  • Angiogenesis
  • Gelatinase A
  • Metastasis

ASJC Scopus subject areas

  • Biochemistry

Cite this

The gelatin-binding site of the second type-II domain of gelatinase A/MMP-2. / Tordai, H.; Patthy, L.

In: European Journal of Biochemistry, Vol. 259, No. 1-2, 15.01.1999, p. 513-518.

Research output: Contribution to journalArticle

@article{9569570eaf614db0b0553dbd75618c1d,
title = "The gelatin-binding site of the second type-II domain of gelatinase A/MMP-2",
abstract = "We have shown previously that all three fibronectin type-II modules of gelatinase A contribute to its gelatin affinity. In the present work the second type-II module was subjected to site-directed mutagenesis in order to localize its gelatin-binding site. The functional integrity of mutant proteins was assessed by their affinity for gelatin using gelatin-Sepharose affinity chromatography. The structural integrity of the mutant proteins, i.e. their resistance to thermal and chaotropic agent-induced denaturation, was characterized by CD spectroscopy. Our studies show that, in the case of mutants R19L, R38L, K50G, K50R and R19L/R38L, the mutations had no significant effect on the structure and gelatin affinity of the type-II module, excluding the direct involvement of these residues in ligand binding. In the case of mutants Y25A, Y46A, D49A and Y52A, the mutations yielded proteins that were devoid of gelatin affinity. Structural characterization of these proteins, however, indicated that they had also lost their ability to fold into the native structure characteristic of the wild-type domain. In the case of mutant Y37A, the structure and stability of the mutant protein is similar to the wild-type module. However, its gelatin affinity was severely impaired compared with the wild-type protein. The fact that the Y37A mutation impairs ligand binding without detectable distortion of the module's architecture suggests that Y37 is directly involved in ligand binding. Homology modeling based on the three-dimensional structure of the second type-II module of PDC-109 places Y37 on the right-hand rim of a hydrophobic pocket that includes residues F20, W39, Y46, Y52 and F54, and thus provides proof for the involvement of this pocket in ligand binding.",
keywords = "Angiogenesis, Gelatinase A, Metastasis",
author = "H. Tordai and L. Patthy",
year = "1999",
month = "1",
day = "15",
doi = "10.1046/j.1432-1327.1999.00070.x",
language = "English",
volume = "259",
pages = "513--518",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "1-2",

}

TY - JOUR

T1 - The gelatin-binding site of the second type-II domain of gelatinase A/MMP-2

AU - Tordai, H.

AU - Patthy, L.

PY - 1999/1/15

Y1 - 1999/1/15

N2 - We have shown previously that all three fibronectin type-II modules of gelatinase A contribute to its gelatin affinity. In the present work the second type-II module was subjected to site-directed mutagenesis in order to localize its gelatin-binding site. The functional integrity of mutant proteins was assessed by their affinity for gelatin using gelatin-Sepharose affinity chromatography. The structural integrity of the mutant proteins, i.e. their resistance to thermal and chaotropic agent-induced denaturation, was characterized by CD spectroscopy. Our studies show that, in the case of mutants R19L, R38L, K50G, K50R and R19L/R38L, the mutations had no significant effect on the structure and gelatin affinity of the type-II module, excluding the direct involvement of these residues in ligand binding. In the case of mutants Y25A, Y46A, D49A and Y52A, the mutations yielded proteins that were devoid of gelatin affinity. Structural characterization of these proteins, however, indicated that they had also lost their ability to fold into the native structure characteristic of the wild-type domain. In the case of mutant Y37A, the structure and stability of the mutant protein is similar to the wild-type module. However, its gelatin affinity was severely impaired compared with the wild-type protein. The fact that the Y37A mutation impairs ligand binding without detectable distortion of the module's architecture suggests that Y37 is directly involved in ligand binding. Homology modeling based on the three-dimensional structure of the second type-II module of PDC-109 places Y37 on the right-hand rim of a hydrophobic pocket that includes residues F20, W39, Y46, Y52 and F54, and thus provides proof for the involvement of this pocket in ligand binding.

AB - We have shown previously that all three fibronectin type-II modules of gelatinase A contribute to its gelatin affinity. In the present work the second type-II module was subjected to site-directed mutagenesis in order to localize its gelatin-binding site. The functional integrity of mutant proteins was assessed by their affinity for gelatin using gelatin-Sepharose affinity chromatography. The structural integrity of the mutant proteins, i.e. their resistance to thermal and chaotropic agent-induced denaturation, was characterized by CD spectroscopy. Our studies show that, in the case of mutants R19L, R38L, K50G, K50R and R19L/R38L, the mutations had no significant effect on the structure and gelatin affinity of the type-II module, excluding the direct involvement of these residues in ligand binding. In the case of mutants Y25A, Y46A, D49A and Y52A, the mutations yielded proteins that were devoid of gelatin affinity. Structural characterization of these proteins, however, indicated that they had also lost their ability to fold into the native structure characteristic of the wild-type domain. In the case of mutant Y37A, the structure and stability of the mutant protein is similar to the wild-type module. However, its gelatin affinity was severely impaired compared with the wild-type protein. The fact that the Y37A mutation impairs ligand binding without detectable distortion of the module's architecture suggests that Y37 is directly involved in ligand binding. Homology modeling based on the three-dimensional structure of the second type-II module of PDC-109 places Y37 on the right-hand rim of a hydrophobic pocket that includes residues F20, W39, Y46, Y52 and F54, and thus provides proof for the involvement of this pocket in ligand binding.

KW - Angiogenesis

KW - Gelatinase A

KW - Metastasis

UR - http://www.scopus.com/inward/record.url?scp=0033555301&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033555301&partnerID=8YFLogxK

U2 - 10.1046/j.1432-1327.1999.00070.x

DO - 10.1046/j.1432-1327.1999.00070.x

M3 - Article

C2 - 9914534

AN - SCOPUS:0033555301

VL - 259

SP - 513

EP - 518

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 1-2

ER -