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Biomedical subjects

K Brew

Publications and source records attributed to K Brew.

At least 19 recordsLinked to original sources
PubMed2000

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 Sorsby's 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.

Amino Acid Sequence↗
PubMed2000

NMR structure of tissue inhibitor of metalloproteinases-1 implicates localized induced fit in recognition of matrix metalloproteinases.

A high quality solution structure of the matrix metalloproteinase inhibitory N-terminal domain of recombinant human tissue inhibitor of metalloproteinases-1 (N-TIMP-1) has been determined. For the rigidly packed residues, the average RMSD to the mean structure is 0. 57 A for the backbone atoms and 1.00 A for all heavy atoms. Comparison of the solution structure of free N-TIMP-1 with the crystal structure of TIMP-1 bound to the catalytic domain of MMP-3 ( Gomis-R]uth et al., 1997 ) shows that the structural core of the beta barrel flanked by helices is nearly unchanged by the association with MMP-3, evident from a backbone RMSD of 1.15 A. However, clear differences in the conformation of the MMP-binding ridge of free and MMP-bound TIMP-1 suggest induced fit throughout the ridge. The MMP-dependent conformational changes in the ridge include a dramatic bending of AB loop residues Glu28 through Leu34, moderate hinge bending of the CD-loop about residues Ala65 and Cys70, and modest bending of the Cys1 through Pro6 segment. A large number of interresidue Nuclear Overhauser enhancements (NOEs) augmented by stereospecific assignments, torsion restraints, and dipolar couplings (an average of 18 non-trivial restraints per residue) engender confidence in these structural inferences. A tight cluster of three lysine residues and one arginine residue atop beta-strands A and B, and identical among TIMP sequences, form the heart of a highly conserved electropositive patch that may interact with anionic components of the extracellular matrix.

Catalytic Domain↗
PubMed1999

Engineering of selective TIMPs.

Differences in proteinase susceptibility between free TIMP-1 and the TIMP-1-MMP-3 complex and mutagenesis studies suggested that the residues around the disulfide bond between Cys1 and Cys70 in TIMP-1 may interact with MMPs. The crystal structure of the complex between TIMP-1 and the catalytic domain of MMP-3 has revealed that the alpha-amino group of Cys1 bidentately chelates the catalytic zinc of MMP-3 and the Thr2 side chain occupies the S1' pocket. Generation of the N-terminal domain of TIMP-1 (N-TIMP-1) variants with 15 different amino acid substitutions for Thr2 has indicated that the nature of the side chain of residue 2 has a major effect on the affinity of N-TIMP-1 for three different MMPs (MMPs-1, -2 and -3). The results also demonstrate that the mode of binding of N-TIMP-1 residue 2 differs from the binding of the P1' residue of a peptide substrate.

Amino Acid Sequence↗
PubMed1999

Rapid collapse and slow structural reorganisation during the refolding of bovine alpha-lactalbumin.

The refolding of bovine alpha-lactalbumin (BLA) from its chemically denatured state in 6 M GuHCl has been investigated by a variety of complementary biophysical approaches. CD experiments indicate that the species formed in the early stages of refolding of the apo-protein have at least 85 % of the alpha-helical content of the native state, and kinetic NMR experiments show that they possess near-native compactness. Hydrogen exchange measurements using mass spectrometry and NMR indicate that persistent structure in these transient species is located predominantly in the alpha-domain of the native protein and is similar to that present in the partially folded A-state formed by the protein at low pH. The extent of the exchange protection is, however, small, and there is no evidence for the existence of well-defined discrete kinetic intermediates of the type populated in the refolding of the structurally homologous c-type lysozymes. Rather, both mass spectrometric and NMR data indicate that the rate-determining transition from the compact partially structured (molten globule) species to the native state is highly cooperative. The data show that folding in the presence of Ca2+ is similar to that in its absence, although the rate is increased by more than two orders of magnitude. Sequential mixing experiments monitored by fluorescence spectroscopy indicate that this slower folding is not the result of the accumulation of kinetically trapped species. Rather, the data are consistent with a model in which binding of Ca2+ stabilizes native-like contacts in the partially folded species and reduces the barriers for the conversion of the protein to its native state. Taken together the results indicate that folding of BLA, in the presence of its four disulphide bonds, corresponds to one of the limiting cases of protein folding in which rapid collapse to a globule with a native-like fold is followed by a search for native-like side-chain contacts that enable efficient conversion to the close packed native structure.

Animals↗
PubMed1999

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.

Cell Line↗
PubMed1999

Role of a conserved acidic cluster in bovine beta1,4 galactosyltransferase-1 probed by mutagenesis of a bacterially expressed recombinant enzyme.

The truncated catalytic domain of bovine beta1,4 galactosyltransferase-1 was expressed as inclusion bodies in E.coli and folded to generate 10-15 mg of active enzyme per liter of bacterial culture after extraction and purification under denaturing conditions. Mutations were introduced to investigate the roles of Trp312, Asp318, and Asp320, components of a highly conserved region of sequence in all known beta4GT-1 homologues that includes a cluster of acidic residues. Near and far UV CD spectra of the mutants indicate that the substitutions did not perturb the secondary and tertiary structure of beta4GT-1, and steady state kinetic studies indicate only minor effects on the response to an essential metal cofactor. However substitutions for the two aspartyl residues result in a reduction in catalytic efficiency of a magnitude that suggests they are important for catalysis. It seems possible that this anionic center may act in stabilizing a carbocation formed from the galactose component of the donor substrate in the transition state, reflecting a common reaction mechanism for beta-galactosyltransferase reactions.

Amino Acid Sequence↗
PubMed1999

Stability, activity and flexibility in alpha-lactalbumin.

alpha-Lactalbumins and the type-c lysozymes are homologues with similar folds that differ in function and stability. To determine if the lower stability of alpha-lactalbumin results from specific substitutions required for its adaptation to a new function, the effects of lysozyme-based and other substitutions on thermal stability were determined. Unblocking the upper cleft in alpha-lactalbumin by replacing Tyr103 with Ala, perturbs stability and structure but Pro, which also generates an open cleft, is compatible with normal structure and activity. These effects appear to reflect alternative enthalpic and entropic forms of structural stabilization by Tyr and Pro. Of 23 mutations, only three, which involve substitutions for residues in flexible substructures adjacent to the functional site, increase stability. Two are lysozyme-based substitutions for Leu110, a component of a region with alternative helix and loop conformations, and one is Asn for Lys114, a residue whose microenvironment changes when alpha-lactalbumin interacts with its target enzyme. While all substitutions for Leu110 perturb activity, a Lys114 to Asn mutation increases T(m) by more than 10 degrees C and reduces activity, but two other destabilizing substitutions do not affect activity. It is proposed that increased stability and reduced activity in Lys114Asn result from reduced flexibility in the functional site of alpha-lactalbumin.

Amino Acid Sequence↗
PubMed1998

Structural evidence for the presence of a secondary calcium binding site in human alpha-lactalbumin.

The high-resolution X-ray crystal structure of human alpha-lactalbumin (at 1.8 A) in the presence of an elevated level of calcium reveals a new secondary calcium binding site, 7.9 A away from the primary calcium binding site known in all alpha-lactalbumin structures so far. The new calcium binding site is different from the zinc and sulfate binding sites [Ren, J., et al. (1993) J. Biol. Chem. 268, 19292-19298] but shares common features with the manganese binding site as described by Gerkin [Gerkin, T. A. (1984) Biochemistry 23, 4688-4697]. The proximity of the manganese and calcium binding region and the location of the functional site on one side of the charged surface of the alpha-lactalbumin molecule suggest that these binding sites might play a role in the formation of the lactose synthase complex.

Binding Sites↗
PubMed1998

Expression of human pro-matrix metalloproteinase 3 that lacks the N-terminal 34 residues in Escherichia coli: autoactivation and interaction with tissue inhibitor of metalloproteinase 1 (TIMP-1).

Human pro-matrix metalloproteinase 3 (proMMP-3) lacking the N-terminal 34 amino acids and the C-terminal hemopexin-like domain was expressed in E. coli and used to investigate the process of proenzyme activation and its interaction with an endogenous inhibitor TIMP-1 during activation. The truncated precursor was purified from the E. coli extract in the presence of 5mM EGTA. The active 23.5 kDa form was generated simply by exposure to Ca2+ and Zn2+ but not either by Ca2+ alone or by Zn2+ alone. The rate of MMP-3(deltaC) formation was concentration dependent, indicating that autoactivation is a bimolecular reaction. The truncated precursor was able to interact with the N-terminal domain of TIMP-1 without losing the 48 residue-long propeptide. However, upon a longer incubation, the propeptide was slowly processed, indicating that the association of the N-terminally truncated proMMP-3 with TIMP-1 is weaker than that of the fully activated MMP-3 and TIMP-1. These results indicate that the expression of MMP activities is regulated by endogenous inhibitor TIMPs during their activation processes which provide an additional control mechanism of extracellular matrix breakdown.

Enzyme Activation↗
PubMed1997

Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.

Matrix metalloproteinases (MMPs) are zinc endopeptidases that are required for the degradation of extracellular matrix components during normal embryo development, morphogenesis and tissue remodelling. Their proteolytic activities are precisely regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in diseases such as arthritis, atherosclerosis, tumour growth and metastasis. Here we report the crystal structure of an MMP-TIMP complex formed between the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1. TIMP-1, a 184-residue protein, has the shape of an elongated, contiguous wedge. With its long edge, consisting of five different chain regions, it occupies the entire length of the active-site cleft of MMP-3. The central disulphide-linked segments Cys 1-Thr 2-Cys 3-Val 4 and Ser 68-Val 69 bind to either side of the catalytic zinc. Cys 1 bidentally coordinates this zinc, and the Thr-2 side chain extends into the large specificity pocket of MMP-3. This unusual architecture of the interface between MMP-3 and TIMP-1 suggests new possibilities for designing TIMP variants and synthetic MMP inhibitors with potential therapeutic applications.

Binding Sites↗
PubMed1997

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 the Ki for a C-terminally truncated form of stromelysin 1, MMP-3(DeltaC), 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 in Ki 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.

Binding Sites↗
PubMed1997

Involvement of a region near valine-69 of tissue inhibitor of metalloproteinases (TIMP)-1 in the interaction with matrix metalloproteinase 3 (stromelysin 1).

Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs) by forming a 1:1 stoichiometric complex, but the inhibition mechanism of these inhibitors is not known. Here we have investigated the reactive site of TIMP-1 by its proteinase susceptibility before and after forming a complex with MMP-3 (stromelysin 1). When TIMP-1 was allowed to react with human neutrophil elastase, its inhibitory activity was destroyed. This resulted from cleavage of the Val69-Cys70 bond. However, cleavage of this bond by neutrophil elastase was prevented when TIMP-1 formed a complex with the catalytic domain of MMP-3, and full TIMP-1 activity was restored after dissociation of the complex at pH 3.0 in the presence of EDTA. These results indicate that the region around Val69 closely associates with an active MMP. The three-dimensional structure of the N-terminal domain of TIMP-2 elucidated by NMR studies [Williamson, Martorell, Carr, Murphy, Docherty, Freedman and Feeney (1994) Biochemistry 33, 11745-11759] reveals that Val69 and Cys70 form part of an extended ridge that also includes the N-terminal section of the inhibitor. This region is probably involved in the interaction with the catalytic domains of MMPs.

Binding Sites↗
PubMed1996

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 alpha-lactalbumin. The general approach, however, should be applicable to the investigation of a wide range of chemical reactions.

Circular Dichroism↗
PubMed1996

Functional site in alpha-lactalbumin encompasses a region corresponding to a subsite in lysozyme and parts of two adjacent flexible substructures.

Aromatic cluster 1 of alpha-lactalbumin (LA), a substructure adjacent to the cleft, is important for its interaction with galactosyltransferase (GT) and effects on glucose binding in the lactose synthase complex [Grobler, J. A., Wang, M., Pike, A. K., & Brew, K. (1994) J. Biol. Chem. 269, 5106-5114]. The full extent of the functional region in LA has been probed by mutagenesis of residues that are near aromatic cluster 1 or within the cleft that corresponds to the active site in the homologous type c lysozymes. The conserved residues Val42, Gln54, and Ile59, which correspond to residues of lysozyme that act in substrate binding in subsites C to E, together with residues adjacent to aromatic cluster 1, were found to be not required for activity. In contrast, replacing Leu110, a component of the region corresponding to lysozyme subsite F, with His or Glu greatly reduces the affinity of LA for GT while the introduction of Arg lowers the synergism of LA and glucose binding to GT and also reduces the affinity of LA for GT. Substitutions for Ala106, which is adjacent to Leu110 in the structure, also perturb activity. The region of the cleft corresponding to subsite F is important for function in LA as well as in lysozyme since other components of this subsite, His32 and Phe31, are also crucial for LA activity. The qualitatively different effects of various substitutions for Leu110 may be mediated by their influence on His32 or by changes in the structure of the lactose synthase complex.

Amino Acid Sequence↗
PubMed1996

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 LA's modulatory properties are located in a region of the structure that has relatively high thermal parameters and is therefore probably flexible in vivo. CONCLUSIONS: LA's 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.

Animals↗
PubMed1996

Mutational analysis of the catalytic subunit of muscle protein phosphatase-1.

A mutational analysis of rabbit skeletal muscle protein phosphatase-1 was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli. The selection of the sites to be mutated was based on sequence alignments which showed the existence of a number of invariant residues when eukaroytic Ser/Thr protein phosphatases were compared with bacteriophage phosphatases and adenosinetetraphosphatase [Barton et al. (1995) Eur. J. Biochem. 220, 225-237]. In other studies, it had been shown that PP1 is a metalloprotein [Chu et al. (1996) J. Biol. Chem. 271, 2574-2577], and in this study, we have largely focused on invariant histidine and aspartate residues which may be involved in metal binding. The residues which were mutated were H66, H125, H173, H248, D64, D71, D92, D95, N124, and R96E. The results showed that mutation of H66, H248, D64, and D92 resulted in severe loss of catalytic function. Mutation of D95, N124, and R96 also led to loss of function, while attempts to mutate H125 and H173 led to production of insoluble, inactive proteins. The results of the mutational analysis are consistent with the involvement of conserved His and Asp residues in metal binding, and are discussed in the context of the recently described crystal structure of PP1 [Goldberg et al. (1995) Nature, 376, 745-753], which reveals that PP1 possesses a bimetallic center at the active site. The behavior of the D95, R96, and N124 mutants supports a catalytic mechanism involving nucleophilic attack by a hydroxide ion with H125 functioning as a proton donor to the leaving alcohol group.

Amino Acid Sequence↗
PubMed1996

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%.

Amino Acid Sequence↗