Keith Brew

Tissue inhibitors of metalloproteinases: evolution, structure and function.

The matrix metalloproteinases (MMPs) play a key role in the normal physiology of connective tissue during development, morphogenesis and wound healing, but their unregulated activity has been implicated in numerous disease processes including arthritis, tumor cell metastasis and atherosclerosis. An important mechanism for the regulation of the activity of MMPs is via binding to a family of homologous proteins referred to as the tissue inhibitors of metalloproteinases (TIMP-1 to TIMP-4). The two-domain TIMPs are of relatively small size, yet have been found to exhibit several biochemical and physiological/biological functions, including inhibition of active MMPs, proMMP activation, cell growth promotion, matrix binding, inhibition of angiogenesis and the induction of apoptosis. Mutations in TIMP-3 are the cause of Sorsbys fundus dystrophy in humans, a disease that results in early onset macular degeneration. This review highlights the evolution of TIMPs, the recently elucidated high-resolution structures of TIMPs and their complexes with metalloproteinases, and the results of mutational and other studies of structure-function relationships that have enhanced our understanding of the mechanism and specificity of the inhibition of MMPs by TIMPs. Several intriguing questions, such as the basis of the multiple biological functions of TIMPs, the kinetics of TIMP-MMP interactions and the differences in binding in some TIMP-metalloproteinase pairs are discussed which, though not fully resolved, serve to illustrate the kind of issues that are important for a full understanding of the interactions between families of molecules.

TIMP-3 Binds to Sulfated Glycosaminoglycans of the Extracellular Matrix*

Of the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B β-strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases.

Crystal structures of apo- and holo-bovine alpha-lactalbumin at 2. 2-A resolution reveal an effect of calcium on inter-lobe interactions.

High affinity binding of Ca2+to α-lactalbumin (LA) stabilizes the native structure and is required for the efficient generation of native protein with correct disulfide bonds from the reduced denatured state. A progressive increase in affinity of LA conformers for Ca2+ as they develop increasingly native structures can account for the tendency of the apo form to assume a molten globule state and the large acceleration of folding by Ca2+. To investigate the effect of calcium on structure of bovine LA, x-ray structures have been determined for crystals of the apo and holo forms at 2.2-Å resolution. In both crystal forms, which were grown at high ionic strength, the protein is in a similar global native conformation consisting of α-helical and β-subdomains separated by a cleft. Even though alternative cations and Ca2+ liganding solvent molecules are absent, removal of Ca2+ has only minor effects on the structure of the metal-binding site and a structural change was observed in the cleft on the opposite face of the molecule adjoining Tyr103 of the helical lobe and Gln54 of the β-lobe. Changes include increased separation of the lobes, loss of a buried solvent molecule near the Ca2+-binding site, and the replacement of inter- and intra-lobe H-bonds of Tyr103 by interactions with new immobilized water molecules. The more open cleft structure in the apo protein appears to be an effect of calcium binding transmitted via a change in orientation of helix H3 relative to the β-lobe to the inter-lobe interface. Calcium is well known to promote the folding of LA. The results from the comparison of apo and holo structures of LA provide high resolution structural evidence that the acceleration of folding by Ca2+ is mediated by an effect on interactions between the two subdomains.

Crystal structures of guinea-pig, goat and bovine alpha-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase.

BACKGROUND The regulation of milk lactose biosynthesis is highly dependent on the action of a specifier protein, alpha-lactalbumin (LA). Together with a glycosyltransferase, LA forms the enzyme complex lactose synthase. LA promotes the binding of glucose to the complex and facilitates the biosynthesis of lactose. To gain further insight into the molecular basis of LA function in lactose synthase we have determined the structures of three species variants of LA. RESULTS The crystal structures of guinea-pig, goat and a recombinant from of bovine LA have been determined using molecular replacement techniques. Overall, the structures are very similar and reflect their high degree of amino acid sequence identity (66-94%). Nonetheless, the structures show that a portion of the molecule (residues 105-110), known to be important for function, exhibits a variety of distinct conformers. This region lies adjacent to two residues (Phe31 and His32) that have been implicated in monosaccharide binding by lactose synthase and its conformation has significant effects on the environments of these functional groups. The crystal structures also demonstrate that residues currently implicated in LAs modulatory properties are located in a region of the structure that has relatively high thermal parameters and is therefore probably flexible in vivo. CONCLUSIONS LAs proposed interaction site for the catalytic component of the lactose synthase complex is primarily located in the flexible C-terminal portion of the molecule. This general observation implies that conformational adjustments may be important for the formation and function of lactose synthase.

Cloning and mapping of a testis-specific gene with sequence similarity to a sperm-coating glycoprotein gene ☆

A testis-specific gene Tpx-1, located between Pgk-2 and Mep-1 on mouse chromosome 17, was isolated from a cosmid clone, and its cDNA sequences were determined. The predicted coding sequence of Tpx-1 isolated from BALB/c mice showed 64.2% nucleotide and 55.1% amino acid sequence similarity with that of a rat sperm-coating glycoprotein gene, the protein product of which is secreted by the epididymis. To examine the evolutionary relationship between Tpx-1 and a sperm-coating glycoprotein gene, the cDNA sequence of TPX1, the human counterpart of Tpx-1, was determined. The comparison of the predicted coding sequences of Tpx-1 and TPX1 showed 77.8% nucleotide and 70% amino acid sequence similarity. Since Tpx-1 (from mouse) is more similar to TPX1 (from man) than it is to a rat sperm-coating glycoprotein gene, we conclude that Tpx-1 (TPX1) and a sperm-coating glycoprotein gene are closely related, but distinct, genes belonging to the same gene family. The predicted Tpx-1 protein of a t mutant mouse CRO437 differs from that of BALB/c mice by one amino acid insertion in the putative signal peptide. TPX1 was mapped to 6p21-qter by Southern blot analysis of interspecies somatic hybrid cell lines.

Protein Folding Monitored at Individual Residues During a Two-Dimensional NMR Experiment

An approach is described to monitor directly at the level of individual residues the formation of structure during protein folding. A two-dimensional heteronuclear nuclear magnetic resonance (NMR) spectrum was recorded after the rapid initiation of the refolding of a protein labeled with nitrogen-15. The intensities and line shapes of the cross peaks in the spectrum reflected the kinetic time course of the folding events that occurred during the spectral accumulation. The method was used to demonstrate the cooperative nature of the acquisition of the native main chain fold of apo bovine α-lactalbumin. The general approach, however, should be applicable to the investigation of a wide range of chemical reactions.

Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E. coli

Methods are described for producing an active amino‐terminal domain of tissue inhibitor of metalloproteinases‐1 (N‐TIMP‐1) from inactive protein expressed as inclusion bodies in E. coli. Yields exceed 20 mg per litre of bacterial culture. Activity measurements, CD spectroscopy and NMR spectroscopy of the 15N‐labeled protein show that it is fully active, homogeneous in conformation and suitable for high‐resolution structural analysis. The affinity of N‐TIMP‐1 for matrix metalloproteinases 1, 2 and 3 is 6–8‐fold less than that of the recombinant full‐length protein, indicating that deletion of the C‐terminal domain reduces the free energy of interaction by < 10%.

Mutational Study of the Amino-terminal Domain of Human Tissue Inhibitor of Metalloproteinases 1 (TIMP-1) Locates an Inhibitory Region for Matrix Metalloproteinases*

A bacterial expression system for the inhibitory N-terminal domain of human tissue inhibitor of metalloproteinases 1 (N-TIMP-1) (Huang, W., Suzuki, K., Nagase, H., Arumugam, S., Van Doren, S. R., and Brew, K. (1996) FEBS Lett. 384, 155–161) has been used to produce 20 single- and double-site mutants that probe the roles of different residues in its inhibitory action on metalloproteinases. Mutations that produce the largest increases in theK i for a C-terminally truncated form of stromelysin 1, MMP-3(ΔC), but do not disturb the conformation involve substitutions of residues that are located in a ridge that is centered around the disulfide bond between Cys1 and Cys70. Specific residues that have a large influence on activity include Cys1, Thr2, Met66, Val69, and Cys70. Of the mutations introduced, the greatest functional disturbances, reflected inK i increases of 2–4 orders of magnitude, are generated by changes that disrupt the Cys1–Cys70 disulfide bond and by substitution of Ala for Thr2. Most mutations that perturb the interaction with MMP-3 have parallel effects on the affinity of N-TIMP-1 for MMP-1 (interstitial collagenase) and MMP-2 (gelatinase A). However, the Thr2 to Ala mutation produces an inhibitor that is 17-fold more effective against MMP-3 than MMP-1, suggesting that it is feasible to engineer TIMP-1 variants that are more specifically targeted to selected matrix metalloproteinases. The reactive site identified by these studies is a structurally constrained but elongated region of TIMP that can fit the matrix metalloproteinase substrate-binding site.

Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1' residue of substrate.

The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1·MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys1 and Cys70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1′ specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases.

Primary structure of the major isomorph of the crustacean hyperglycemic hormone (CHH-I) from the sinus gland of the Mexican crayfish Procambarus bouvieri (Ortmann): Interspecies comparison

The amino acid sequence of this neuropeptide was elucidated by means of a combined approach of enzymatic digestions, manual and automatic Edman degradations, and mass spectrometry. It is a 72 residue peptide (molecular mass 8388 Da), with six cysteines forming three disulfide bridges connecting residues 7-43, 23-39, and 26-52, with blocked N- and C-termini, and lacking the amino acids histidine, methionine, and tryptophan. The CHH-I of Procambarus bouvieri is compared with the other known CHHs from Orconectes limosus (98.6% identity), Homarus americanus isomorph A (83.3% identity), Homarus americanus isomorph B (79.2% identity), and Carcinus maenas (61.1% identity).