Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP-2 and membrane-type-1 MMP (MT1-MMP) forms a cell surface located 'receptor' involved in pro-MMP-2 activation. We have solved the 2.75 A crystal structure of the complex between the catalytic domain of human MT1-MMP (cdMT1-MMP) and bovine TIMP-2. In comparison with our previously determined MMP-3-TIMP-1 complex, both proteins are considerably tilted to one another and show new features. CdMT1-MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active-site cleft that might be important for interaction with macromolecular substrates. The TIMP-2 polypeptide chain, as in TIMP-1, folds into a continuous wedge; the A-B edge loop is much more elongated and tilted, however, wrapping around the S-loop and the beta-sheet rim of the MT1-MMP. In addition, both C-terminal edge loops make more interactions with the target enzyme. The C-terminal acidic tail of TIMP-2 is disordered but might adopt a defined structure upon binding to pro-MMP-2; the Ser2 side-chain of TIMP-2 extends into the voluminous S1' specificity pocket of cdMT1-MMP, with its Ogamma pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP-1 for MT1-MMP compared with TIMP-2 might be explained by a reduced number of favourable interactions.     

The use of active noise control (ANC) to reduce acoustic noise generated during MRI scanning: some initial results

The search of reprolysin inhibitors offers the possibility of intervention against both matrixins and ADAMs. Here we report the crystal structure of the complex between adamalysin II, a member of the reprolysin family, and a phosphonate inhibitor modeled on an endogenous venom tripeptide. The inhibitor occupies the primed region of the cleavage site adopting a retro-binding mode. The phosphonate group ligates the zinc ion in an asymmetric bidentate mode and the adjacent Trp indole system partly fills the primary specificity subsite S1'. An adamalysin-based model of tumor necrosis factor-alpha-converting enzyme (TACE) reveals a smaller S1' pocket for this enzyme.     

Crystal structure of human cathepsin S

We have determined the 2.5 A structure (Rcryst = 20.5%, Rfree = 28.5%) of a complex between human cathepsin S and the potent, irreversible inhibitor 4-morpholinecarbonyl-Phe-hPhe-vinyl sulfone-phenyl. Noncrystallographic symmetry averaging and other density modification techniques were used to improve electron density maps which were nonoptimal due to systematically incomplete data. Methods that reduce the number of parameters were implemented for refinement. The refined structure shows cathepsin S to be similar to related cysteine proteases such as papain and cathepsins K and L. As expected, the covalently-bound inhibitor is attached to the enzyme at Cys 25, and enzyme binding subsites S3-S1' are occupied by the respective inhibitor substituents. A somewhat larger S2 pocket than what is found in similar enzymes is consistent with the broader specificity of cathepsin S at this site, while Lys 61 in the S3 site may offer opportunities for selective inhibition of this enzyme. The presence of Arg 137 in the S1' pocket, and proximal to Cys 25 may have implications not only for substrate specificity C-terminal to the scissile bond, but also for catalysis.     

Cleavage efficiency of the novel aspartic protease yapsin 1 (Yap3p) enhanced for substrates with arginine residues flanking the P1 site: correlation with electronegative active-site pockets predicted by molecular modeling

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys23. Molecular modeling of yapsin 1 identified the active-site cleft to have negative residues close to or within the S6, S3, S2, S1, S1', S2', and S3' pockets and is more electronegative than rhizopuspepsin or endothiapepsin. In particular, the S2' subsite has three negative charges in and close to this pocket that can provide strong electrostatic interactions with a basic residue. The model, therefore, predicts that substrates with a basic residue in the P1 position would be favored with additional basic residues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspepsin and other aspartic proteases of known 3D structure, is likely to affect its specificity. The model was tested using CCK13-33 analogues. We report that yapsin 1 preferentially cleaves a CCK13-33 substrate with a basic residue in the P1 position since the substrates with Ala in P1 were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking the P1 position. An alanine residue, substituting for the arginine residue in the P6 position in CCK13-33, resulted in a 50% reduction in the cleavage efficiency. Substitution with arginine residues downstream of the cleavage site at the P2', P3', or P6' position increased the cleavage efficiency by 21-, 3- and 7-fold, respectively. Substitution of Lys23 in CCK13-33 with arginine resulted not only in cleavage after the substituted arginine residue, but also forced a cleavage after Met25, suggesting that an arginine residue in the S2' pocket is so favorable that it can affect the primary specificity of yapsin 1. These results are consistent with the predictions from the molecular model of yapsin 1.     

Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8-A resolution

It has recently been demonstrated that the C-terminal deletion mutant of recombinant human carbonic anhydrase IV (G267X CA IV) converts the normally glycosylphosphatidylinositol-anchored enzyme into a soluble secretory form which has the same catalytic properties as the membrane-associated enzyme purified from human tissues. We have determined the three-dimensional structure of the secretory form of human CA IV by x-ray crystallographic methods to a resolution of 2.8 A. Although the zinc binding site and the hydrophobic substrate binding pocket of CA IV are generally similar to those of other mammalian isozymes, unique structural differences are found elsewhere in the active site. Two disufide linkages, Cys-6-Cys-11G and Cys-23-Cys-203, stabilize the conformation of the N-terminal domain. The latter disulfide additionally stabilizes an active site loop containing a cis-peptide linkage between Pro-201 and Thr-202 (this loop contains catalytic residue Thr-199). On the opposite side of the active site, the Val-131-Asp-136 segment adopts an extended loop conformation instead of an alpha-helix conformation as found in other isozymes. Finally, the C terminus is surrounded by a substantial electropositive surface potential, which is likely to stabilize the interaction of CA IV with the negatively charged phospholipid headgroups of the membrane. These structural features are unique to CA IV and provide a framework for the design of sulfonamide inhibitors selective for this particular isozyme.     

Elucidation of the mode of interaction of thermolysin with a proteinaceous metalloproteinase inhibitor, SMPI, based on a model complex structure and a structural dynamics analysis

SMPI is a proteinaceous microbial metalloproteinase inhibitor that was isolated from Streptomyces nigrescens TK-23 in 1979. SMPI is known to selectively inhibit the metalloproteinases in the gluzincin family, according to the Rawling and Barrett classification. There has been no report on the interaction of a metalloproteinase in the family of gluzincins with its specific proteinaceous inhibitor. We have solved the solution structure of SMPI by NMR. Here, we report the binding mode of SMPI to thermolysin, based on the model complex structure generated using our high-resolution NMR structure of SMPI and the crystal structure of thermolysin. The obtained complex model shows that the extruded loop of SMPI, with the scissile bond Cys64-Val65, is complementary in shape to the active cleft of thermolysin. In the complex, the Cys64 (P1) carbonyl oxygen atom can form a tetrahedral coordination to the active zinc in thermolysin, and simultaneously, the methyl groups of Val65 (P1') are closely located in the hydrophobic S1' pocket in thermolysin. From the electrostatic potential surface calculation, the active loop of SMPI and the active cleft in thermolysin have been shown to be complementary in the surface charge distribution, resulting in the stabilization of the complex. The apparently large active loop is less flexible, but maintains a conformation in the nano- to picosecond time-scale, as elucidated from the 15N spin relaxation analysis. This is a quite different structural feature of SMPI from the flexible binding loop generally found in the serine proteinase inhibitors, such as SSI and eglin c, and can be related to the narrow specificity of SMPI. The present study provides the first insight into the interaction between a proteinaceous inhibitor and a gluzincin metalloproteinase.     

Structure of 3-isopropylmalate dehydrogenase in complex with 3-isopropylmalate at 2.0 A resolution: the role of Glu88 in the unique substrate-recognition mechanism

BACKGROUND: 3-Isopropylmalate dehydrogenase (IPMDH) and isocitrate dehydrogenase (ICDH) belong to a unique family of bifunctional decarboxylating dehydrogenases. Although the ICDH dimer catalyzes its reaction under a closed conformation, known structures of the IPMDH dimer (without substrate) adopt a fully open or a partially closed form. Considering the similarity in the catalytic mechanism, the IPMDH dimer must be in a fully closed conformation during the reaction. A large conformational change should therefore occur upon substrate binding. RESULTS: We have determined the crystal structure of IPMDH from Thiobacillus ferrooxidans (Tf) complexed with 3-isopropylmalate (IPM) at 2.0 A resolution by the molecular replacement method. The structure shows a fully closed conformation and the substrate-binding site is quite similar to that of ICDH except for a region around the gamma-isopropyl group. The gamma group is recognized by a unique hydrophobic pocket, which includes Glu88, Leu91 and Leu92 from subunit 1 and Val193' from subunit 2. CONCLUSIONS: A large movement of domain 1 is induced by substrate binding, which results in the formation of the hydrophobic pocket for the gamma-isopropyl moiety of IPM. A glutamic acid in domain 1, Glu88, participates in the formation of the hydrophobic pocket. The C beta and C gamma atoms of Glu88 interact with the gamma-isopropyl moiety of IPM and are central to the recognition of substrate. The acidic tip of Glu88 is likely to interact with the nicotinamide mononucleotide (NMN) ribose of NAD+ in the ternary complex. This structure clearly explains the substrate specificity of IPMDH.     

Structural mapping of the active site specificity determinants of human tissue-type plasminogen activator. Implications for the design of low molecular weight substrates and inhibitors

The recent structure determination of the catalytic domain of tissue-type plasminogen activator (tPA) suggested residue Arg174 could play a role in P3/P4 substrate specificity. Six synthetic chromogenic tPA substrates of the type R-Xaa-Gly-Arg-p-nitroanilide, in which R is an N-terminal protection group, were synthesized to test this property. Although changing the residue Xaa (in its L or D form) at position P3 from the hydrophobic Phe to an acidic residue, Asp or Glu, gave no improvement in catalytic efficiency, comparative analysis of the substrates indicated a preference for an acidic substituent occupying the S3 site when the S4 site contains a hydrophobic or basic moiety. The 2.9 A structure determination of the catalytic domain of human tPA in complex with the bis-benzamidine inhibitor 2, 7-bis-(4-amidinobenzylidene)-cycloheptan-1-one reveals a three-site interaction, salt bridge formation of the proximal amidino group of the inhibitor with Asp189 in the primary specificity pocket, extensive hydrophobic surface burial, and a weak electrostatic interaction between the distal amidino group of the inhibitor and two carbonyl oxygens of the protein. The latter position was previously occupied by the guanidino group of Arg174, which swings out to form the western edge of the S3 pocket. These data suggest that the side chain of Arg174 is flexible, and does not play a major role in the S4 specificity of tPA. On the other hand, this residue would modulate S3 specificity, and may be exploited to fine tune the specificity and selectivity of tPA substrates and inhibitors.     

Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure

The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins.     

Bioactive conformation of a potent stromelysin inhibitor determined by X-nucleus filtered and multidimensional NMR spectroscopy

The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1' pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2' and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.     

A comparison of the crystallographic structures of two catalytic antibodies with esterase activity

The crystallographic structure of the Fab fragment of the catalytic antibody, 29G11, complexed with an (S)-norleucine phenyl phosphonate transition state analog was determined at 2.2 A resolution. The antibody catalyzes the hydrolysis of norleucine phenyl ester with (S)-enantioselectivity. The shape and charge complementarity of the binding pocket for the hapten account for the preferential binding of the (S)-enantiomer of the substrate. The structure is compared to that of the more catalytically efficient antibody, 17E8, induced by the same hapten transition state analog. 29G11 has different residues from 17E8 at eight positions in the heavy chain, including four substitutions in the hapten-binding pocket: A33V, S95G, S99R and Y100AN, and four substitutions at positions remote from the catalytic site, I28T, R40K, V65G and F91L. The two antibodies show large differences in the orientations of their variable and constant domains, reflected by a 32 degrees difference in their elbow angles. The VL and VH domains in the two antibodies differ by a rotation of 8.8 degrees. The hapten binds in similar orientations and locations in 29G11 and 17E8, which appear to have catalytic groups in common, though the changes in the association of the variable domains affect the precise positioning of residues in the hapten-binding pocket.     

Site-directed mutagenesis combined with chemical modification as a strategy for altering the specificity of the S1 and S1' pockets of subtilisin Bacillus lentus

By combining site-directed mutagenesis with chemical modification, we have altered the S1 and S1' pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation of unnatural amino acid moieties, in the following manner: WT --> Cysmutant + H3CSO2SR --> Cys-SR, where R may be infinitely variable. A paradigm between extent of activity changes and surface exposure of the modified residue has emerged. Modification of M222C, a buried residue in the S1' pocket of SBL, caused dramatic changes in kcat/KM, of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S1 pocket and is partially surface exposed, effected more modest activity changes. Introduction of a positive charge at S166C does not alter kcat/KM, whereas the introduction of a negative charge results in lowered activity, possibly due to electrostatic interference with oxyanion stabilization. Activity is virtually unaltered upon modification of S156C, which is located toward the bottom of the S1 pocket and surface exposed and whose side chain is solvated. An unexpected structure-activity relationship was revealed for S166C-SR enzymes in that the pattern of activity changes observed with increasing steric size of R was not monotonic. Molecular modeling analysis was used to analyze this unprecedented structure-activity relationship and revealed that the position of the beta-carbon of Cys166 modulates binding of the P1 residue of the AAPF product inhibitor.     

Activity of dorsal respiratory group inspiratory neurons during laryngeal-induced fictive coughing and swallowing in decerebrate cats

VP39 is a bifunctional vaccinia virus protein that acts as both an mRNA cap-specific RNA 2'-O-methyltransferase and a poly(A) polymerase processivity factor. Here, we report the 1.85 A crystal structure of a VP39 variant complexed with its AdoMet cofactor. VP39 comprises a single core domain with structural similarity to the catalytic domains of other methyltransferases. Surface features and mutagenesis data suggest two possible RNA-binding sites with novel underlying architecture, one of which forms a cleft spanning the region adjacent to the methyltransferase active site. This report provides a prototypic structure for an RNA methyltransferase, a protein that interacts with the mRNA 5' cap, and an intact poxvirus protein.     

Stromelysin-1: three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.     

Static and dynamic roles of extracellular loops in G-protein-coupled receptors: a mechanism for sequential binding of thyrotropin-releasing hormone to its receptor

Small ligands generally bind within the seven transmembrane-spanning helices of G-protein-coupled receptors, but their access to the binding pocket through the closely packed loops has not been elucidated. In this work, a model of the extracellular loops of the thyrotropin-releasing hormone (TRH) receptor (TRHR) was constructed, and molecular dynamics simulations and quasi-harmonic analysis have been performed to study the static and dynamic roles of the extracellular domain. The static analysis based on curvature and electrostatic potential on the surface of TRHR suggests the formation of an initial recognition site between TRH and the surface of its receptor. These results are supported by experimental evidence. A quasi-harmonic analysis of the vibrations of the extracellular loops suggest that the low-frequency motions of the loops will aid the ligand to access its transmembrane binding pocket. We suggest that all small ligands may bind sequentially to the transmembrane pocket by first interacting with the surface binding site and then may be guided into the transmembrane binding pocket by fluctuations in the extracellular loops.     

Arg278, but not Lys229 or Lys236, plays an important role in the binding of retinoic acid by retinoic acid receptor gamma

The diverse biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, beta and gamma) and retinoid X receptors (RXR alpha, beta, and gamma). Although the ligand-binding domains of RARs share the same novel folding pattern, many RAR subtype-specific retinoids have been synthesized indicating that the ligand-binding pocket of each RAR subtype has unique features. Previously we have demonstrated the importance for RA binding and RA-dependent transactivation of Arg276 of RARalpha alone and in RARbeta Arg269 in conjunction with Lys220. In this study, we have examined the role of the homologous amino acid residues (Lys229 and Arg278) in RARgamma for these activities. Like RARalpha but dissimilar to RARbeta, Arg278 in RARgamma alone was found to play an important role in RA binding and RA-dependent transactivation. Since Lys236 in RARgamma was suggested from the crystal structure of holo-RARgamma to interact with RA, we also examined its role and that of its homologs in RARalpha and RARbeta. Despite the suggestion from the crystal structure, neither Lys236 nor its homologs in RARalpha and RARbeta play a role in the binding of RA or RA-dependent transactivation. It is likely that Lys236 in RARgamma and its homologs in RARalpha and RARbeta are solvent exposed rather than pointing into the RA-binding pocket.