Biomedical subjects

H Tordai

Publications and source records attributed to H Tordai.

6 recordsLinked to original sources

PubMed2001

Gelatin-binding region of human matrix metalloproteinase-2: solution structure, dynamics, and function of the COL-23 two-domain construct.

Human matrix metalloproteinase-2 (MMP-2) contains an array of three fibronectin type II (FII) modules postulated to interact with gelatin (denatured collagen). Here, we verify that the NMR solution structure of the third FII repeat (COL-3) is similar to that of the second FII repeat (COL-2); characterize its ligand-binding properties; and derive dynamics properties and relative orientation in solution for the two domains of the COL-23 fragment, a construct comprising COL-2 and COL-3 in tandem, with each domain possessing a putative collagen-binding site. Interaction of the synthetic gelatin-like octadecapeptide (Pro-Pro-Gly)(6) (PPG6) with COL-3 is weaker than with COL-2. We found that a synthetic peptide comprising segment 33-42 (peptide 33-42) from the MMP-2 prodomain interacts with COL-3 and, albeit with lower affinity, with COL-2 in a way that mimics PPG6 binding. COL-3 strongly prefers peptide 33-42 over PPG6, which suggests that intramolecular interactions with the prodomain could modulate binding of pro-MMP-2 to its gelatin substrate. In COL-23, the two modules retain their structural individuality and tumble independently. Overall, the NMR data indicate that the relative orientation of the modules in COL-23 is not fixed in solution, that the modules do not interact with one another, and that COL-23 is rather flexible. The binding sites face opposite each other, and their responses to, and normalized affinities for, the longer ligand PPG12 are virtually identical to those of the individual domains for PPG6, thus precluding co- operativity, although they may interact simultaneously with multiple sites of the extracellular matrix.

Amino Acid Sequence↗

PubMed1999

The PAN module: the N-terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins.

Based on homology search and structure prediction methods we show that (1) the N-terminal N domains of members of the plasminogen/hepatocyte growth factor family, (2) the apple domains of the plasma prekallikrein/coagulation factor XI family, and (3) domains of various nematode proteins belong to the same module superfamily, hereafter referred to as the PAN module. The patterns of conserved residues correspond to secondary structural elements of the known three-dimensional structure of hepatocyte growth factor N domain, therefore we predict a similar fold for all members of this superfamily. Based on available functional informations on apple domains and N domains, it is clear that PAN modules have significant functional versatility, they fulfill diverse biological functions by mediating protein-protein or protein-carbohydrate interactions.

Amino Acid Sequence↗

PubMed1999

The second type II module from human matrix metalloproteinase 2: structure, function and dynamics.

BACKGROUND: Matrix metalloproteinase 2 (MMP-2, gelatinase A, 72 kDa type IV collagenase) has an important role in extracellular matrix degradation during cell migration and tissue remodeling. It is involved in development, inflammation, wound healing, tumor invasion, metastasis and other physiological and pathological processes. The enzyme cleaves several types of collagen, elastin, fibronectin and laminin. Binding to collagen is mediated by three repeats homologous to fibronectin type II modules, which are inserted in the catalytic domain in proximity to the active site. RESULTS: We have determined the NMR solution structure of the second type II module from human MMP-2 (col-2). The module exhibits a typical type II fold with two short double-stranded antiparallel beta sheets and three large loops packed around a cluster of conserved aromatic residues. Backbone amide dynamics, derived from (15)N relaxation experiments, correlate well with solvent accessibility and intramolecular hydrogen bonding. A synthetic peptide with the collagen consensus sequence, (Pro-Pro-Gly)(6), is shown to interact with the module. CONCLUSIONS: Spectral perturbations induced by (Pro-Pro-Gly)(6) binding reveal the region involved in the interaction of col-2 with collagen. The binding surface comprises exposed aromatic residues Phe21, Tyr38, Trp40, Tyr47, Tyr53 and Phe55, and the neighboring Gly33-Gly37 segment.

Amino Acid Sequence↗

PubMed1999

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

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.

Amino Acid Sequence↗

PubMed1996

Structure and domain-domain interactions of the gelatin binding site of human 72-kilodalton type IV collagenase (gelatinase A, matrix metalloproteinase 2).

We have shown previously that all three fibronectin type II modules of gelatinase A contribute to its gelatin affinity. In the present investigation we have studied the structure and module-module interactions of this gelatin-binding domain by circular dichroism spectroscopy and differential scanning calorimetry. Comparison of the Tm values of the thermal transitions of isolated type II modules with those of bimodular or trimodular proteins has shown that the second type II module is significantly more stable in the trimodular protein coll 123 (Tm = 54 degrees C) than in the single-module protein coll 2 (Tm = 44 degrees C) or in the bimodular proteins coll 23 (Tm = 47 degrees C) and coll 12 (Tm = 48 degrees C). Analysis of the enthalpy changes associated with thermal unfolding of the second type II module suggests that it is stabilized by domain-domain interactions in coll 123. We propose that intimate contacts exist between the three tandem type 11 units and they form a single gelatin-binding site. Based on the three-dimensional structures of homologous metalloproteases and type II modules, a model is proposed in which the three type II units form an extension of the substrate binding cleft of gelatinase A.

Amino Acid Sequence↗

PubMed1994

The gelatin-binding site of human 72 kDa type IV collagenase (gelatinase A).

To identify structures critical for gelatin-binding of 72 kDa type IV collagenase (gelatinase A), fragments of this metalloproteinase have been expressed in Escherichia coli and assayed for their gelatin affinity. Each of the three fibronectin-related type II domains was found to have affinity for gelatin. Fragments containing all three tandem type II domains had significantly stronger affinity than any of the constituent units, indicating that they co-operate to form the high-affinity gelatin-binding site. Competition experiments have also shown that gelatinase A binds more tightly to gelatin than fibronectin and can displace the latter from denatured collagen.

Base Sequence↗