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Traian Sulea

Publications and source records attributed to Traian Sulea.

18 recordsLinked to original sources
PubMed2006

Binding site-based classification of coronaviral papain-like proteases.

The coronavirus replicase gene encodes one or two papain-like proteases (termed PL1pro and PL2pro) implicated in the N-terminal processing of the replicase polyprotein and thus contributing to the formation of the viral replicase complex that mediates genome replication. Using consensus fold recognition with the 3D-JURY meta-predictor followed by model building and refinement, we developed a structural model for the single PLpro present in the severe acute respiratory syndrome coronavirus (SCoV) genome, based on significant structural relationships to the catalytic core domain of HAUSP, a ubiquitin-specific protease (USP). By combining the SCoV PLpro model with comparative sequence analyses we show that all currently known coronaviral PLpros can be classified into two groups according to their binding site architectures. One group includes all PL2pros and some of the PL1pros, which are characterized by a restricted USP-like binding site. This group is designated the R-group. The remaining PL1pros from some of the coronaviruses form the other group, featuring a more open papain-like binding site, and is referred to as the O-group. This two-group, binding site-based classification is consistent with experimental data accumulated to date for the specificity of PLpro-mediated polyprotein processing and PLpro inhibition. It also provides an independent evaluation of the similarity-based annotation of PLpro-mediated cleavage sites, as well as a basis for comparison with previous groupings based on phylogenetic analyses.

Amino Acid Sequence↗
PubMed2006

Coupled atomic charge selectivity for optimal ligand-charge distributions at protein binding sites.

Charge optimization as a tool for both analyzing and enhancing binding electrostatics has become an attractive approach over the past few years. An interesting feature of this method for molecular design is that it provides not only the optimal charge magnitudes, but also the selectivity of a particular atomic center for its optimal charge. The current approach to compute the charge selectivity at a given atomic center of a ligand in a particular binding process is based on the binding-energy cost incurred upon the perturbation of the optimal charge distribution by a unit charge at the given atomic center, while keeping the other atomic partial charges at their optimal values. A limitation of this method is that it does not take into account the possible concerted changes in the other atomic charges that may incur a lower energetic cost than perturbing a single charge. Here, we describe a novel approach for characterizing charge selectivity in a concerted manner, taking into account the coupling between the ligand charge centers in the binding process. We apply this novel charge selectivity measure to the celecoxib molecule, a nonsteroidal anti-inflammatory agent binding to cyclooxygenase 2 (COX2), which has been recently shown to also exhibit cross-reactivity toward carbonic anhydrase II (CAII), to which it binds with nanomolar affinity. The uncoupled and coupled charge selectivity profiles over the atomic centers of the celecoxib ligand, binding independently to COX2 and CAII, are analyzed comparatively and rationalized with respect to available experimental data. Very different charge selectivity profiles are obtained for the uncoupled versus coupled selectivity calculations.

Binding Sites↗
PubMed2006

Crystal structure of TDP-fucosamine acetyltransferase (WecD) from Escherichia coli, an enzyme required for enterobacterial common antigen synthesis.

Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence -->3)-alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1-->4)-alpha-d-GlcNAc-(1-->. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Angstrom resolution and bound to acetyl-CoA at a 1.66-Angstrom resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

Acetyl Coenzyme A↗
PubMed2006

Structural aspects of recently discovered viral deubiquitinating activities.

Protein ubiquitination has been identified as a regulatory mechanism in key cellular activities, and deubiquitination is recognized as an important step in processes governed by ubiquitin and ubiquitin-like modifiers. Viruses are known to target ubiquitin and ubiquitin-like modifier pathways using various strategies, including the recruitment of host deubiquitinating enzymes. Deubiquitinating activities have recently been described for proteins from three different virus families (adenovirus, coronavirus and herpesvirus), and predicted for others. This review centers on structural-functional aspects that characterize the confirmed viral deubiquitinating enzymes, and their relationships to established families of cellular deubiquitinating enzymes.

Amino Acid Sequence↗
PubMed2005

Roles of dimerization domain residues in binding and catalysis by aminoacylase-1.

The aminoacylase-1/metallopeptidase 20 (Acy1/M20) family is the largest metallopeptidase family. Several crystal structures feature a metal-binding and a dimerization-mediating domain, both arranged in an extended open conformation. We have recently shown [Lindner et al. (2003) J. Biol. Chem. 278, 44496-44504] that in human Acy1 the invariant residues Glu147 and His206 from the metal-binding and the dimerization domain, respectively, are recruited to the active site from opposite dimer subunits. We hypothesized that, to facilitate this, formation of the binary complex is associated with domain closure, which would also position additional residues in the functional active site of Acy1. These would include two partially conserved dimerization domain residues: an asparagine (Asn263) and an arginine (Arg276) from the same subunit as His206 and Glu147, respectively. In this paper, we investigate the significance of the three dimerization domain residues of human Acy1 His206, Asn263, and Arg276 and, additionally, the nearby Asp274 for catalysis using site-directed mutagenesis. Enzyme complementation assays confirm the putative subunit allocations of these residues, and steady-state kinetics support roles for all of them in catalysis but only involve the Arg276 in substrate-binding. The results are consistent with a model of the closed conformation for the structure of the related enzyme carboxypeptidase G2. This study demonstrates experimentally for the first time for a member of the Acy1/M20 family that several residues outside of the metal-binding domain are involved in binding and catalysis.

Amidohydrolases↗
PubMed2005

Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase HisI.

The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohydrolase, the product of the hisI gene. The three-dimensional structure of PR-AMP cyclohydrolase from Methanobacterium thermoautotrophicum was solved and refined to 1.7 A resolution. The enzyme is a homodimer. The position of the Zn(2+)-binding site that is essential for catalysis was inferred from the positions of bound Cd(2+) ions, which were part of the crystallization medium. These metal binding sites include three cysteine ligands, two from one monomer and the third from the second monomer. The enzyme remains active when Cd(2+) is substituted for Zn(2+). The likely binding site for Mg(2+), also necessary for activity in a homologous cyclohydrolase, was also inferred from Cd(2+) positions and is comprised of aspartic acid side chains. The putative substrate-binding cleft is formed at the interface between the two monomers of the dimer. This fact, combined with the localization of the Zn(2+)-binding site, indicates that the enzyme is an obligate dimer.

Amino Acid Sequence↗
PubMed2005

Carboxy-monopeptidase substrate specificity of human cathepsin X.

Cathepsin X is a papain-like cysteine protease with restricted positional specificity, acting primarily as a carboxy-monopeptidase. We mapped the specificities at the S2, S1, and S1' subsites of human cathepsin X by systematically and independently substituting the P2, P1, and P1' positions of the carboxy-monopeptidase substrate Abz-FRF(4NO(2)) with natural amino acids. Human cathepsin X has broad S2, S1, and S1' specificities within two orders of magnitude in k(cat)/K(M), excluding proline that is not tolerated at these subsites. Glycine is not favored in S2, but is among the preferred residues in S1 and S1', which highlights S2 as the affinity-determinant subsite. The presence of peculiar residues at several binding site positions (Asp76, His234, Asn75, and Glu72) does not translate into a markedly different sequence specificity profile relative to other human cathepsins. These findings suggest that a specific function of human cathepsin X is unlikely to result from sequence specificity, but rather from a combination of its unique positional specificity and the co-localization of enzyme and substrate in a specific cellular environment.

Amino Acid Sequence↗
PubMed2005

The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme.

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.

Amino Acid Sequence↗
PubMed2004

The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery.

Chondroitinase B from Pedobacter heparinus is the only known enzyme strictly specific for dermatan sulfate and is a widely used enzymatic tool for the structural characterization of glycosaminoglycans. This beta-helical polysaccharide lyase belongs to family PL-6 and cleaves the beta(1,4) linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides as the product. The previously reported structure of its complex with a dermatan sulfate disaccharide product identified the -1 and -2 subsites of the catalytic groove. We present here the structure of chondroitinase B complexed with several dermatan sulfate and chondroitin sulfate oligosaccharides. In particular, the soaking of chondroitinase B crystals with a dermatan sulfate hexasaccharide results in a complex with two dermatan sulfate disaccharide reaction products, enabling the identification of the +2 and +1 subsites. Unexpectedly, this structure revealed the presence of a calcium ion coordinated by sequence-conserved acidic residues and by the carboxyl group of the l-iduronic acid at the +1 subsite. Kinetic and site-directed mutagenesis experiments have subsequently demonstrated that chondroitinase B absolutely requires calcium for its activity, indicating that the protein-Ca(2+)-oligosaccharide complex is functionally relevant. Modeling of an intact tetrasaccharide in the active site of chondroitinase B provided a better understanding of substrate specificity and the role of Ca(2+) in enzymatic activity. Given these results, we propose that the Ca(2+) ion neutralizes the carboxyl moiety of the l-iduronic acid at the cleavage site, whereas the conserved residues Lys-250 and Arg-271 act as Brønsted base and acid, respectively, in the lytic degradation of dermatan sulfate by chondroitinase B.

Amino Acid Sequence↗
PubMed2003

Specificity determinants of human cathepsin s revealed by crystal structures of complexes.

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.

Base Sequence↗
PubMed2003

Bridging structural biology and genetics by computational methods: an investigation into how the R774C mutation in the AR gene can result in complete androgen insensitivity syndrome.

Recent structural studies of the ligand-binding domain (LBD) of the androgen receptor (AR) have raised more questions than answers, as most of the known pathogenic mutations of the AR gene causing androgen insensitivity syndrome (AIS) are not in the ligand-binding pocket. In this study, we have investigated one such pathogenic mutation, by examining details of its altered atomic structure using a computational technique of molecular dynamics (MD) simulations extended over 4 ns, effectively creating a 4D structural model. The mutation R774C, which is in the LBD of the AR gene, causes complete AIS (CAIS), producing ARs that have a unique thermolabile profile, being thermostable at 22 degrees C but thermolabile at 37 degrees C. We have therefore investigated this mutation by MD simulations at 293 K (20 degrees C), 300 K (27 degrees C), and 310 K (37 degrees C). The MD simulations indicate that: 1) the mutation causes local structural distortions, which result in changes in the shape of the ligand-binding pocket; 2) the mutation alters the dynamic nature of the protein and results in a more diverse conformational distribution of the ligand-binding pocket; and 3) the effect of the mutation on AR structure could be largely reversed by lowering the temperature at which the MD simulations were conducted. These results therefore strongly support the biochemical data, e.g., the mutants' inability to form AR-ligand complexes at 37 degrees C and its characteristic reversible thermolability, clearly indicating the value of such computational methods.

Androgen-Insensitivity Syndrome↗
PubMed2003

On the transferability of hydration-parametrized continuum electrostatics models to solvated binding calculations.

Using molecular mechanics force field partial atomic charges, we show the nonuniqueness of the parametrization of continuum electrostatics models with respect to solute atomic radii and interior dielectric constant based on hydration (vacuum-to-water transfer) free energy data available for small molecules. Moreover, parameter sets that are optimal and equivalent for hydration free energy calculations lead to large variations of calculated absolute and relative electrostatic binding free energies. Hence, parametrization of solvation effects based on hydration data, although a necessary condition, is not sufficient to guarantee its transferability to the calculation of binding free energies in solution.

Journal Article↗
PubMed2003

Profiling charge complementarity and selectivity for binding at the protein surface.

A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins.

Binding Sites↗
PubMed2002

Design of noncovalent inhibitors of human cathepsin L. From the 96-residue proregion to optimized tripeptides.

A novel series of noncovalent inhibitors of cathepsin L have been designed to mimic the mode of autoinhibition of procathepsin L. Just like the propeptide, these peptide-based inhibitors have a reverse-binding mode relative to a substrate and span both the S' and S subsites of the enzyme active site. In contrast to previous studies in which even moderate truncation of the full-length propeptide led to rapid reduction in potency, these blocked tripeptide-sized inhibitors maintain nanomolar potency. Moreover, these short peptides show higher selectivity (up to 310-fold) for inhibiting cathepsin L over K versus only 2-fold selectivity of the 96-residue propeptide of cathepsin L. A 1.9 A X-ray crystallographic structure of the complex of cathepsin L with one of the inhibitors confirms the designed reverse-binding mode of the inhibitor as well as its noncovalent nature. Enzymatic analysis also shows the inhibitors to be resistant to hydrolysis at elevated concentrations of the enzyme. The mode of inhibition of these molecules provides a general strategy for inhibiting other cathepsins as well as other proteases.

Binding Sites↗
PubMed2002

Implications of the reactive thiol and the proximal non-proline cis-peptide bond in the Structure and function of Vibrio harveyi luciferase.

The role of a highly reactive cysteine residue, Cys106, in Vibrio harveyi luciferase in modulating the substrate-enzyme interactions and in turn affecting the enzyme activity has been extensively investigated over the past three decades. Replacing Cys106 with valine dramatically hinders the ability of luciferase to stabilize the C4a-hydroperoxyflavin intermediate [Abu-Soud, H. M., Clark, A. C., Francisco, W. A., Baldwin, T. O., and Raushel, F. M. (1993) J. Biol. Chem. 268, 7699-7706] and consume aldehyde substrate [Xi, L., Cho, K.-W., Herndon, M. E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203], therefore markedly decreasing enzyme activity. On the basis of the structure-activity relationship of flavin analogues and the location of the phosphate binding site of flavin mononucleotide (FMN) coupled with molecular modeling, the functional part of the isoalloxazine ring of FMN, the thiol side chain of Cys106, the methyl group of Ala75, and the unique non-prolyl cis-peptide bond between Ala74 and Ala75 were found to be closely packed [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. Here, by mutating Ala75 to Gly, we restored key wild-type properties to the C106V mutant, in particular, high enzyme activity and a stable C4a-hydroperoxyflavin intermediate, demonstrating that the primary reason for the dark phenotype of the C106V mutant was the unfavorable steric interaction between Val106 and Ala75 side chains, which could in turn disturb the cis-oriented amide linkage of Ala74 and Ala75. Moreover, significant red shifts in light emission of 3-10 nm were measured for luciferases carrying Val106 with the spectrum of the double mutant C106V/A75G now red shifted to that of Photobacterium phosphoreum luciferase, which also has Val and Gly at positions 106 and 75, respectively. These results strengthen the validity of the binding geometry of the modeled flavin with the re-face of the pyrimidine end of the isoalloxazine ring next to Cys106 and implicate the Ala74-Ala75 cis-peptide as a key component in the bioluminescence reaction.

Enzyme Stability↗
PubMed2002

Site-directed mutagenesis of the type II TGF-beta receptor indicates a ligand-binding site distinct from that of the type II activin receptor.

Site-directed mutagenesis was used to map the ligand-binding surface of the type II transforming growth factor-beta receptor extracellular domain (TbetaRII-ECD). Two putative ligand-binding sites were probed, the first being a predicted hydrophobic patch, the second being the finger 1 surface loop. Nine residues were mutated in the context of full-length TbetaRII and the effect of these mutations on ligand-binding and receptor signaling was analyzed. Complementary information was obtained by examining 'natural' evolutionary TbetaRII mutations. Together, the results indicate that residues within the finger 1 region, but not the hydrophobic patch, of the TbetaRII-ECD are required for productive ligand-binding. We conclude that, surprisingly, the ECDs of TbetaRII and type II activin receptor utilize distinct interacting surfaces for binding their respective ligands.

Activin Receptors, Type II↗
PubMed2002

Identification of a functional site on the type I TGF-beta receptor by mutational analysis of its ectodomain.

Six charged amino acid residues located in the ectodomain of the full-length type I transforming growth factor (TGF)-beta receptor were individually mutated to alanine. Mutation of residues D47, D98, K102 and E104 resulted in functionally impaired receptors as demonstrated by a marked decrease in ligand-dependent signaling and ligand internalization relative to the wild-type receptor. The other two mutants (K39A and K87A) exhibited wild-type-like activity. Molecular modeling indicates that the four functionally important residues are located on the convex face of the ectodomain structure. Since mutation of these four residues affects signaling and ligand internalization but not ligand binding, we propose that this functional site is an interacting site between type I and II receptors.

Activin Receptors, Type I↗