Protein modelling using a chimera reference protein derived from exons.

CD26 or dipeptidyl-peptidase IV (DPPIV) is engaged in immune functions by co-stimulatory effects on activation and proliferation of T lymphocytes, binding to adenosine deaminase, and regulation of various chemokines and cytokines. DPPIV peptidase activity is inhibited by both Tat protein from human immunodeficiency virus (HIV)-1 and its N-terminal nonapeptide Tat-(1-9) with amino acid sequence MDPVDPNIE, suggesting that DPPIV mediates immunosuppressive effects of Tat protein. The 2.0- and 3.15-A resolution crystal structures of the binary complex between human DPPIV and nonapeptide Tat-(1-9) and the ternary complex between the variant MWPVDPNIE, called Trp(2)-Tat-(1-9), and DPPIV bound to adenosine deaminase show that Tat-(1-9) and Trp(2)-Tat-(1-9) are located in the active site of DPPIV. The interaction pattern of DPPIV with Trp(2)-Tat-(1-9) is tighter than that with Tat-(1-9), in agreement with inhibition constants (K(i)) of 2 x 10(-6) and 250 x 10(-6) m, respectively. Both peptides cannot be cleaved by DPPIV because the binding pockets of the N-terminal 2 residues are interchanged compared with natural substrates: the N-terminal methionine occupies the hydrophobic S1 pocket of DPPIV that normally accounts for substrate specificity by binding the penultimate residue. Because the N-terminal sequence of the thromboxane A2 receptor resembles the Trp(2)-Tat-(1-9) peptide, a possible interaction with DPPIV is postulated.

Phosphatases are a diverse group of enzymes that regulate numerous cellular processes. Much of what is known relates to the tyrosine, threonine, and serine phosphatases, whereas the histidine phosphatases have not been studied as much. The structure of phosphohistidine phosphatase (PHPT1), the first identified eukaryotic-protein histidine phosphatase, has been determined to a resolution of 1.9A using multiple-wavelength anomalous dispersion methods. This enzyme can dephosphorylate a variety of proteins (e.g. ATP-citrate lyase and the beta-subunit of G proteins). A putative active site has been identified by its electrostatic character, ion binding, and conserved protein residues. Histidine 53 is proposed to play a major role in histidine dephosphorylation based on these observations and previous mutational studies. Models of peptide binding are discussed to suggest possible mechanisms for substrate recognition.

Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 A resolution. BVP adopts the characteristic cysteine-phosphatase alpha/beta fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif (118)HCTHGXNRT(126) in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.

Carnitine acyltransferases have crucial functions in fatty acid metabolism. Members of this enzyme family show distinctive substrate preferences for short-, medium- or long-chain fatty acids. The molecular mechanism for this substrate selectivity is not clear as so far only the structure of carnitine acetyltransferase has been determined. To further our understanding of these important enzymes, we report here the crystal structures at up to 2.0-A resolution of mouse carnitine octanoyltransferase alone and in complex with the substrate octanoylcarnitine. The structures reveal significant differences in the acyl group binding pocket between carnitine octanoyltransferase and carnitine acetyltransferase. Amino acid substitutions and structural changes produce a larger hydrophobic pocket that binds the octanoyl group in an extended conformation. Mutation of a single residue (Gly-553) in this pocket can change the substrate preference between short- and medium-chain acyl groups. The side chains of Cys-323 and Met-335 at the bottom of this pocket assume dual conformations in the substrate complex, and mutagenesis studies suggest that the Met-335 residue is important for catalysis.

S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.

Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined the crystal structures of apo caspase-2, caspase-2 in complex with peptide inhibitors VDVAD-CHO, ADVAD-CHO, and DVAD-CHO, and a T380A mutant of caspase-2 in complex with VDVAD-CHO. Two residues, Thr-380 and Tyr-420, are identified to be critical for the P5 residue recognition; mutation of the two residues reduces the catalytic efficiency by about 4- and 40-fold, respectively. The structures also provide a series of snapshots of caspase-2 in different catalytic states, shedding light on the mechanism of capase-2 activation, substrate binding, and catalysis. By comparing the apo and inhibited caspase-2 structures, we propose that the disruption of a non-conserved salt bridge between Glu-217 and the invariant Arg-378 is important for the activation of caspase-2. These findings broaden our understanding of caspase-2 substrate specificity and catalysis.

The breakdown of fatty acids, performed by the beta-oxidation cycle, is crucial for plant germination and sustainability. beta-Oxidation involves four enzymatic reactions. The final step, in which a two-carbon unit is cleaved from the fatty acid, is performed by a 3-ketoacyl-CoA thiolase (KAT). The shortened fatty acid may then pass through the cycle again (until reaching acetoacetyl-CoA) or be directed to a different cellular function. Crystal structures of KAT from Arabidopsis thaliana and Helianthus annuus have been solved to 1.5 and 1.8 A resolution, respectively. Their dimeric structures are very similar and exhibit a typical thiolase-like fold; dimer formation and active site conformation appear in an open, active, reduced state. Using an interdisciplinary approach, we confirmed the potential of plant KATs to be regulated by the redox environment in the peroxisome within a physiological range. In addition, co-immunoprecipitation studies suggest an interaction between KAT and the multifunctional protein that is responsible for the preceding two steps in beta-oxidation, which would allow a route for substrate channeling. We suggest a model for this complex based on the bacterial system.

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.

Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division. The yeast Saccharomyces cerevisiae calmodulin Cmd1 and the essential myosin light chain Mlc1p share the ability to interact with the class V myosin Myo2p and Myo4 and the class II myosin Myo1p. These myosins are required for vesicle, organelle, and mRNA transport, spindle orientation, and cytokinesis. We have used the budding yeast model system to study how calmodulin and essential myosin light chain selectively regulate class V myosin function. NMR structural analysis of uncomplexed Mlc1p and interaction studies with the first three IQ motifs of Myo2p show that the structural similarities between Mlc1p and the other members of the EF-hand superfamily of calmodulin-like proteins are mainly restricted to the C-lobe of these proteins. The N-lobe of Mlc1p presents a significantly compact and stable structure that is maintained both in the free and complexed states. The Mlc1p N-lobe interacts with the IQ motif in a manner that is regulated both by the IQ motifs sequence as well as by light chain structural features. These characteristic allows a distinctive interaction of Mlc1p with the first IQ motif of Myo2p when compared with calmodulin. This finding gives us a novel view of how calmodulin and essential light chain, through a differential binding to IQ1 of class V myosin motor, regulate this activity during vegetative growth and cytokinesis.

With the rise of antibody based therapeutics as successful medicines, there is an emerging need to understand the fundamental antibody conformational dynamics and its implications towards stability of these medicines. Both deglycosylation and thermal stress have been shown to cause conformational destabilization and aggregation in monoclonal antibodies. Here, we study instabilities caused by deglycosylation and by elevated temperature (400 K) by performing molecular dynamic simulations on a full length murine IgG2a mAb whose crystal structure is available in the Protein Data bank. C(α)-atom root mean square deviation and backbone root mean square fluctuation calculations show that deglycosylation perturbs quaternary and tertiary structures in the C(H) 2 domains. In contrast, thermal stress pervades throughout the antibody structure and both Fabs and Fc regions are destabilized. The thermal stress applied in this study was not sufficient to cause large scale unfolding within the simulation time and most amino acid residues showed similar average solvent accessible surface area and secondary structural conformations in all trajectories. C(H) 3 domains were the most successful at resisting the conformational destabilization. The simulations helped identify aggregation prone regions, which may initiate cross-β motif formation upon deglycosylation and upon applying thermal stress. Deglycosylation leads to increased backbone fluctuations and solvent exposure of a highly conserved APR located in the edge β-strand A of the C(H) 2 domains. Aggregation upon thermal stress is most likely initiated by two APRs that overlap with the complementarity determining regions. This study has important implications for rational design of antibody based therapeutics that are resistant towards aggregation.

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 A resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 A root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 A) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides alpha-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.

Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

Matriptase, a type II trans-membrane serine protease of the S1 trypsin-like family, is expressed on the surface of nearly all normal human epithelium and found in biological fluid-like human milk. Matriptase overexpression has been implicated in tumor progression in certain epithelium-derived cancer cells. Matriptase is tightly regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). It has been demonstrated that the Kunitz domain I (KD1) but not Kunitz domain II (KD2) of HAI-1 is responsible for the inhibitory activity of HAI-1 against matriptase. To investigate the molecular basis of inhibition of matriptase by HAI-1, we solved several crystal structures of matriptase serine protease domain in complex with the fragments of HAI-1. Based on these structures, we found that the binding of KD1 was different from previously predicted binding mode. The P3 arginine residue occupies the S3 specificity pocket of matriptase, but not the S4 pocket as in the cases of hepatocyte growth factor activator·HAI-1 KD1 and matriptase·sunflower trypsin inhibitor-1 complexes. The long 60-loop of matriptase makes direct contact with HAI-1 but remains flexible even in the complexes, and its apex does not bind with KD1 tightly. The interactions between this unique 60-loop and KD1 may provide an opportunity to increase the specificity and inhibitory activity of KD1 for matriptase. Furthermore, comparison between KD1 and a homology model of HAI-1 KD2 rationalizes the structural basis of why KD1 but not KD2 is responsible for the inhibitory activity of HAI-1 against matriptase.

Unlike human thrombin, murine thrombin lacks Na+ activation due to the charge reversal substitution D222K in the Na+ binding loop. However, the enzyme is functionally stabilized in a Na+-bound form and is highly active toward physiologic substrates. The structural basis of this peculiar property is unknown. Here, we present the 2.2 A resolution x-ray crystal structure of murine thrombin in the absence of inhibitors and salts. The enzyme assumes an active conformation, with Ser-195, Glu-192, and Asp-189 oriented as in the Na+-bound fast form of human thrombin. Lys-222 completely occludes the pore of entry to the Na+ binding site and positions its side chain inside the pore, with the Nzeta atom H-bonded to the backbone oxygen atoms of Lys-185, Asp-186b, and Lys-186d. The same architecture is observed in the 1.75 A resolution structure of a thrombin chimera in which the human enzyme carries all residues defining the Na+ pore in the murine enzyme. These findings demonstrate that Na+ activation in thrombin is linked to the architecture of the Na+ pore. The molecular strategy of Na+ activation mimicry unraveled for murine thrombin is relevant to serine proteases and enzymes activated by monovalent cations in general.