Structures of the tricorn-interacting aminopeptidase F1 with different ligands explain its catalytic mechanism

F1 is a 33.5 kDa serine peptidase of the alpha/beta-hydrolase family from the archaeon Thermoplasma acidophilum. Subsequent to proteasomal protein degradation, tricorn generates small peptides, which are cleaved by F1 to yield single amino acids. We have solved the crystal structure of F1 with multiwavelength anomalous dispersion (MAD) phasing at 1.8 A resolution. In addition to the conserved catalytic domain, the structure reveals a chiefly alpha-helical domain capping the catalytic triad. Thus, the active site is accessible only through a narrow opening from the protein surface. Two structures with molecules bound to the active serine, including the inhibitor phenylalanyl chloromethylketone, elucidate the N-terminal recognition of substrates and the catalytic activation switch mechanism of F1. The cap domain mainly confers the specificity for hydrophobic side chains by a novel cavity system, which, analogously to the tricorn protease, guides substrates to the buried active site and products away from it. Finally, the structure of F1 suggests a possible functional complex with tricorn that allows efficient processive degradation to free amino acids for cellular recycling.     

Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conformational change of methionine 260 involved in substrate recognition

Aminopeptidase N from Escherichia coli is a broad specificity zinc exopeptidase belonging to aminopeptidase clan MA, family M1. The structures of the ligand-free form and the enzyme-bestatin complex were determined at 1.5- and 1.6-A resolution, respectively. The enzyme is composed of four domains: an N-terminal beta-domain (Met(1)-Asp(193)), a catalytic domain (Phe(194)-Gly(444)), a middle beta-domain (Thr(445)-Trp(546)), and a C-terminal alpha-domain (Ser(547)-Ala(870)). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal-binding motif (HEXXHX(18)E) is found in the domain. The zinc ion is coordinated by His(297), His(301), Glu(320), and a water molecule. The groove on the catalytic domain that contains the active site is covered by the C-terminal alpha-domain, and a large cavity is formed inside the protein. However, there exists a small hole at the center of the C-terminal alpha-domain. The N terminus of bestatin is recognized by Glu(121) and Glu(264), which are located in the N-terminal and catalytic domains, respectively. Glu(298) and Tyr(381), located near the zinc ion, are considered to be involved in peptide cleavage. A difference revealed between the ligand-free form and the enzyme-bestatin complex indicated that Met(260) functions as a cushion to accept substrates with different N-terminal residue sizes, resulting in the broad substrate specificity of this enzyme.     

Crystal structure of the cold-active aminopeptidase from Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene A4 hydrolase

The crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal alpha-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX(18)H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate.     

The C-terminal domain of aminopeptidase A is an intramolecular chaperone required for the correct folding, cell surface expression, and activity of this monozinc aminopeptidase

Aminopeptidase A (APA, EC 3.4.11.7) is a type II integral membrane glycoprotein responsible for the conversion of angiotensin II to angiotensin III in the brain. Previous site-directed mutagenesis studies and the recent molecular modeling of the APA zinc metallopeptidase domain have shown that all the amino acids involved in catalysis are located between residues 200 and 500. The APA ectodomain is cleaved in the kidney into an N-terminal fragment corresponding to the zinc metallopeptidase domain, and a C-terminal fragment of unknown function. We investigated the function of this C-terminal domain, by expressing truncated APAs in Chinese hamster ovary and AtT-20 cells. Deletion of the C-terminal domain abolished the maturation and enzymatic activity of the N-terminal domain, which was retained in the endoplasmic reticulum as an unfolded protein bound to calnexin. Expression in trans of the C-terminal domain resulted in association of the N- and C-terminal domains soon after biosynthesis, allowing folding rescue, maturation, cell surface expression, and activity of the N-terminal zinc metallopeptidase domain. We also show that the C-terminal domain is not required for the catalytic activity of APA but is essential for its activation. Moreover, we show that the C-terminal domain of aminopeptidase N (EC 3.4.11.2, APN) also promotes maturation and cell surface expression of the N-terminal domain of APN, suggesting a common role of the C-terminal domain in the monozinc aminopeptidase family. Our data provide the first demonstration that the C-terminal domain of an eukaryotic exopeptidase acts as an intramolecular chaperone.     

Crystal structure of XoLAP, a leucine aminopeptidase, from Xanthomonas oryzae pv. oryzae

Aminopeptidases are metalloproteinases that degrade N-terminal residues from protein and play important roles in cell growth and development by controlling cell homeostasis and protein maturation. We determined the crystal structure of XoLAP, a leucyl aminopeptidase, at 2.6 Å resolution from Xanthomonas oryzae pv. oryzae, causing the destructive rice disease of bacterial blight. It is the first crystal structure of aminopeptidase from phytopathogens as a drug target. XoLAP existed as a hexamer and the monomer structure consisted of an N-terminal cap domain and a C-terminal peptidase domain with two divalent zinc ions. XoLAP structure was compared with BlLAP and EcLAP (EcPepA) structures. Based on the structural comparison, the molecular model of XoLAP in complex with the natural aminopeptidase inhibitor of microginin FR1 was proposed. The model structure will be useful to develop a novel antibacterial drug against Xoo.     

β-Propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection

Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain beta-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed beta-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the beta-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.     

Structure of peptidase T from Salmonella typhimurium.

The structure of peptidase T, or tripeptidase, was determined by multiple wavelength anomalous dispersion (MAD) methodology and refined to 2.4 A resolution. Peptidase T comprises two domains; a catalytic domain with an active site containing two metal ions, and a smaller domain formed through a long insertion into the catalytic domain. The two metal ions, presumably zinc, are separated by 3.3 A, and are coordinated by five carboxylate and histidine ligands. The molecular surface of the active site is negatively charged. Peptidase T has the same basic fold as carboxypeptidase G2. When the structures of the two enzymes are superimposed, a number of homologous residues, not evident from the sequence alone, could be identified. Comparison of the active sites of peptidase T, carboxypeptidase G2, Aeromonas proteolytica aminopeptidase, carboxypeptidase A and leucine aminopeptidase reveals a common structural framework with interesting similarities and differences in the active sites and in the zinc coordination. A putative binding site for the C-terminal end of the tripeptide substrate was found at a peptidase T specific fingerprint sequence motif.     

Structural basis for the dynamics of human methionyl-tRNA synthetase in multi-tRNA synthetase complexes

In mammals, eight aminoacyl-tRNA synthetases (AARSs) and three AARS-interacting multifunctional proteins (AIMPs) form a multi-tRNA synthetase complex (MSC). MSC components possess extension peptides for MSC assembly and specific functions. Human cytosolic methionyl-tRNA synthetase (MRS) has appended peptides at both termini of the catalytic main body. The N-terminal extension includes a glutathione transferase (GST) domain responsible for interacting with AIMP3, and a long linker peptide between the GST and catalytic domains. Herein, we determined crystal structures of the human MRS catalytic main body, and the complex of the GST domain and AIMP3. The structures reveal human-specific structural details of the MRS, and provide a dynamic model for MRS at the level of domain orientation. A movement of zinc knuckles inserted in the catalytic domain is required for MRS catalytic activity. Depending on the position of the GST domain relative to the catalytic main body, MRS can either block or present its tRNA binding site. Since MRS is part of a huge MSC, we propose a dynamic switching between two possible MRS conformations; a closed conformation in which the catalytic domain is compactly attached to the MSC, and an open conformation with a free catalytic domain dissociated from other MSC components.     

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.     

Crystal structure of Thermotoga maritima 4-alpha-glucanotransferase and its acarbose complex: implications for substrate specificity and catalysis

4-alpha-Glucanotransferase (GTase) is an essential enzyme in alpha-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-alpha-D-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6A and 2.5A resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (beta/alpha)(8) barrel topology (domain A), a 65 residue domain, domain B, inserted between strand beta3 and helix alpha6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel beta-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (-2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (beta/alpha)(8) barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic "clamp" that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from -2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from -2 to +2, play the dominant role in enzyme-substrate recognition, consistent with the observation that the smallest donor for T.maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.     

Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.     

The crystal structure of AF1521 a protein from Archaeoglobus fulgidus with homology to the non-histone domain of macroH2A

MacroH2A is an unusual histone H2A variant that has an extensive C-terminal tail that comprises approximately two thirds of the protein. The C-terminal non-histone domain of macroH2A is also found in a number of other proteins and has been termed the macro domain. Here we report the crystal structure to 1.7A of AF1521, a protein consisting of a stand-alone macro domain from Archaeoglobus fulgidus. The structure has a mixed alpha/beta fold that closely resembles the N-terminal DNA binding domain of the Escherichia coli leucine aminopeptidase PepA. The structure also shows some similarity to members of the P-loop family of nucleotide hydrolases.     

Domain swapping in the low-similarity isomerase/hydratase superfamily: the crystal structure of rat mitochondrial Delta3, Delta2-enoyl-CoA isomerase

Two monofunctional Delta(3), Delta(2)-enoyl-CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low-similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl-CoA substrate for re-entry into the beta-oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C-terminal domain of a given monomer interacts with its own N-terminal domain. This differs from that of crotonase and 1,4-dihydroxy-2-naphtoyl-CoA synthase, whose C-terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro-R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel-shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl-chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments.     

Crystal structure of Rv2118c: an AdoMet-dependent methyltransferase from Mycobacterium tuberculosis H37Rv

Rv2118c belongs to the class of conserved hypothetical proteins from Mycobacterium tuberculosis H37Rv. The crystal structure of Rv2118c in complex with S-adenosyl-l-methionine (AdoMet) has been determined at 1.98 A resolution. The crystallographic asymmetric unit consists of a monomer, but symmetry-related subunits interact extensively, leading to a tetrameric structure. The structure of the monomer can be divided functionally into two domains: the larger catalytic C-terminal domain that binds the cofactor AdoMet and is involved in the transfer of methyl group from AdoMet to the substrate and a smaller N-terminal domain. The structure of the catalytic domain is very similar to that of other AdoMet-dependent methyltransferases. The N-terminal domain is primarily a beta-structure with a fold not found in other methyltransferases of known structure. Database searches reveal a conserved family of Rv2118c-like proteins from various organisms. Multiple sequence alignments show several regions of high sequence similarity (motifs) in this family of proteins. Structure analysis and homology to yeast Gcd14p suggest that Rv2118c could be an RNA methyltransferase, but further studies are required to establish its functional role conclusively. Copyright 12001 Academic Press.     

Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein.

Retroviral integrase, an essential enzyme for replication of human immunodeficiency virus type-1 (HIV-1) and other retroviruses, contains three structurally distinct domains, an N-terminal domain, the catalytic core and a C-terminal domain. To elucidate their spatial arrangement, we have solved the structure of a fragment of HIV-1 integrase comprising the N-terminal and catalytic core domains. This structure reveals a dimer interface between the N-terminal domains different from that observed for the isolated domain. It also complements the previously determined structure of the C-terminal two domains of HIV-1 integrase; superposition of the conserved catalytic core of the two structures results in a plausible full-length integrase dimer. Furthermore, an integrase tetramer formed by crystal lattice contacts bears structural resemblance to a related bacterial transposase, Tn5, and exhibits positively charged channels suitable for DNA binding.     

Crystal structure of a heat and protease-stable part of the bacteriophage T4 short tail fibre.

Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres. After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipopolysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube. The short tail fibres consist of a parallel, in-register, trimer of gene product 12 (gp12). The 1.9 A crystal structure of a heat and protease-stable fragment of gp12 reveals three new folds: a central right-handed triple beta-helix, a globular C-terminal domain containing a beta-sandwich and an N-terminal beta-structure reminiscent of but different from the adenovirus triple beta-spiral. The centre of the C-terminal domain shows weak homology to gp11, a trimeric protein connecting the short fibre to the base-plate, suggesting that the trimerisation motifs of gp11 and gp12 are similar. Repeating sequence motifs suggest that the N-terminal beta-structure extends further towards the N terminus and is conserved in the long tail fibre proteins gp34 and gp37.     

Molecular basis for bre5 cofactor recognition by the ubp3 deubiquitylating enzyme

Yeast Ubp3 and its co-factor Bre5 form a deubiquitylation complex to regulate protein transport between the endoplasmic reticulum and Golgi compartments of the cell. A novel N-terminal domain of the Ubp3 catalytic subunit forms a complex with the NTF2-like domain of the Bre5 regulatory subunit. Here, we report the X-ray crystal structure of an Ubp3-Bre5 complex and show that it forms a symmetric hetero-tetrameric complex in which the Bre5 NTF2-like domain dimer interacts with two L-shaped beta-strand-turn-alpha-helix motifs of Ubp3. The Ubp3 N-terminal domain binds within a hydrophobic cavity on the surface of the Bre5 NTF2-like domain subunit with conserved residues within both proteins interacting predominantly through antiparallel beta-sheet hydrogen bonds and van der Waals contacts. Structure-based mutagenesis and functional studies confirm the significance of the observed interactions for Ubp3-Bre5 association in vitro and Ubp3 function in vivo. Comparison of the structure to other protein complexes with NTF2-like domains shows that the Ubp3-Bre5 interface is novel. Together, these studies provide new insights into Ubp3 recognition by Bre5 and into protein recognition by NTF2-like domains.     

Structure of human cytosolic X-prolyl aminopeptidase: a double Mn(II)-dependent dimeric enzyme with a novel three-domain subunit

X-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides. Mammalian X-prolyl aminopeptidases are shown to be responsible for the degradation of bradykinin, a blood pressure regulator peptide, and have been linked to myocardial infarction. The x-ray crystal structure of human cytosolic X-prolyl aminopeptidase (XPN-PEP1) was solved at a resolution of 1.6 angstroms. The structure reveals a dimer with a unique three-domain organization in each subunit, rather than the two domains common to all other known structures of X-prolyl aminopeptidase and prolidases. The C-terminal catalytic domain of XPNPEP1 coordinates two metal ions and shares a similar fold with other prolyl aminopeptidases. Metal content analysis and activity assays confirm that the enzyme is double Mn(II) dependent for its activity, which contrasts with the previous notion that each XPNPEP1 subunit contains only one Mn(II) ion. Activity assays on an E41A mutant demonstrate that the acidic residue, which was considered as a stabilizing factor in the protonation of catalytic residue His498, plays only a marginal role in catalysis. Further mutagenesis reveals the significance of the N-terminal domain and dimerization for the activity of XPNPEP1, and we provide putative structural explanations for their functional roles. Structural comparisons further suggest mechanisms for substrate selectivity in different X-prolyl peptidases.