Key Residues (key + residue)

Distribution by Scientific Domains

Selected Abstracts

Structural, Dynamic Properties of Key Residues in A, Amyloidogenesis: Implications of an Important Role of Nanosecond Timescale Dynamics

CHEMBIOCHEM, Issue 11 2007
Kwang Hun Lim Prof. Dr.
Flexibility exercise. NMR studies of the A,(1,40) and -(1,42) peptides demonstrated that structural, dynamic properties of soluble monomeric A, peptides are highly correlated to the amyloid fibril structure. In particular, fast-timescale dynamics were found to be an additional critical parameter in identifying the more crucial aggregation-prone segments of A, peptides (see figure). This method can be applied to investigate amyloidogenic and nonamyloidogenic properties of disordered states of proteins. [source]

Structure of CDP- d -glucose 4,6-dehydratase from Salmonella typhi complexed with CDP- d -xylose

Nicole M. Koropatkin
Tyvelose is a unique 3,6-dideoxyhexose found in the O antigens of some pathogenic species of Yersinia and Salmonella. It is produced via a complex biochemical pathway that employs CDP- d -glucose as the starting ligand. CDP- d -­glucose 4,6-dehydratase catalyzes the first irreversible step in the synthesis of this 3,6-dideoxysugar by converting CDP- d -­glucose to CDP-4-keto-6-deoxyglucose via an NAD+ -dependent intramolecular oxidation,reduction reaction. Here, the cloning, protein purification and X-ray crystallographic analysis of CDP- d -glucose 4,6-dehydratase from Salmonella typhi complexed with the substrate analog CDP- d -­xylose are described. Each subunit of the tetrameric enzyme folds into two domains. The N-terminal region contains a Rossmann fold and provides the platform for NAD(H) binding. The C-terminal motif is primarily composed of ,-­helices and houses the binding pocket for the CDP portion of the CDP- d -xylose ligand. The xylose moiety extends into the active-site cleft that is located between the two domains. Key residues involved in anchoring the sugar group to the protein include Ser134, Tyr159, Asn197 and Arg208. Strikingly, Ser134,O, and Tyr159,O, sit within 2.9,Å of the 4,-­hydroxyl group of xylose. Additionally, the side chains of Asp135 and Lys136 are located at 3.5 and 3.2,Å, respectively, from C-5 of xylose. In the structurally related dTDP- d -glucose 4,6-dehydratase, the Asp/Lys pair is replaced with an Asp/Glu couple. On the basis of this investigation, it can be speculated that Tyr159 serves as the catalytic base to abstract the 4,-­hydroxyl proton from the sugar and that Asp135 and Lys136 play critical roles in the subsequent dehydration step that leads to the final product. [source]

N-terminal residues regulate proteasomal degradation of AANAT

Zheping Huang
Abstract:, Serotonin N -acetyltransferase (AANAT) catalyzes the conversion of serotonin to N -acetylserotonin, which is the immediate precursor for formation of melatonin. Although it is known that AANAT is degraded via the proteasomal proteolysis, detailed mechanisms are not defined. In this paper, we tested the in vivo role of proteasome inhibition on AANAT activity and melatonin release and examined the amino acid residues in AANAT that contribute to the proteasome degradation. We have shown that inhibition of proteasome activities in vivo in the intact pineal gland fails to prevent the light-induced suppression of melatonin secretion. Furthermore, in cell lines stably expressing AANAT, inhibition of proteasomal proteolysis, which resulted in a large accumulation of AANAT protein, similarly failed to increase AANAT enzyme activity proportional to the amount of proteins accumulated. Site-directed mutagenesis analysis of AANAT revealed that the AANAT degradation is independent of lysine and the two surface cysteine residues. Deletion analysis of N-terminus identified the second amino acid leucine (L2) as the key residue that contributes to the proteasomal proteolysis of AANAT protein. These results suggest that rat AANAT protein is degraded via the N-end rule pathway of proteasomal proteolysis and the leucine at the N-terminus appears to be the key residue recognized by N-end rule pathway. [source]

Low-temperature Spectroscopy of Met100Ala Mutant of Photoactive Yellow Protein,

Yasushi Imamoto
The trans -to- cis photoisomerization of the p -coumaroyl chromophore of photoactive yellow protein (PYP) triggers the photocycle. Met100, which is located in the vicinity of the chromophore, is a key residue for the cis -to- trans back-isomerization of the chromophore, which is a rate-determining reaction of the PYP photocycle. Here we characterized the photocycle of the Met100Ala mutant of PYP (M100A) by low temperature UV,visible spectroscopy. Irradiation of M100A at 80 K yielded a 380 nm species (M100ABL), while the corresponding intermediate of wild type (WT; PYPBL) is formed above 90 K. The amounts of redshifted intermediates produced from M100A (M100AB, and M100AL) were substantially less than those from WT. While the near-UV intermediate (PYPM) is not formed from WT in glycerol samples at low temperature, M100AM was clearly observed above 190 K. These alterations of the photocycle of M100A were explained by the shift in the equilibrium between the intermediates. The carbonyl oxygen of the thioester linkage of the cis -chromophore in the photocycle intermediates is close to the phenyl ring of Phe96 (<3.5 Å), which would be displaced by the mutation of Met100. These findings imply that the interaction between chromophore and amino acid residues near Met100 is altered during the early stage of the PYP photocycle. [source]

Induced-fit or preexisting equilibrium dynamics?

Lessons from protein crystallography, MD simulations on acetylcholinesterase, implications for structure-based drug design
Abstract Crystal structures of acetylcholinesterase complexed with ligands are compared with side-chain conformations accessed by native acetylcholinesterase in molecular dynamics (MD) simulations. Several crystallographic conformations of a key residue in a specific binding site are accessed in a simulation of native acetylcholinesterase, although not seen in rotomer plots. Conformational changes upon ligand binding thus involve preexisting equilibrium dynamics. Consequently, rational drug design could benefit significantly from conformations monitored by MD simulations of native targets. [source]

Conformational changes induced by a single amino acid substitution in the trans -membrane domain of Vpu: Implications for HIV-1 susceptibility to channel blocking drugs

PROTEIN SCIENCE, Issue 10 2007
Sang Ho Park
Abstract The channel-forming trans -membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight ,-helix spanning residues 6,27 with an average tilt angle of 41° in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11° compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers. [source]

Structure of photoactive yellow protein (PYP) E46Q mutant at 1.2,Å resolution suggests how Glu46 controls the spectroscopic and kinetic characteristics of PYP

Masakazu Sugishima
Photoactive yellow protein from Ectothiorhodospira halophila is a photoreceptor protein involved in the negative phototaxis of this bacterium. Its chromophore (p -coumaric acid) is deprotonated in the ground state, which is stabilized by a hydrogen-bond network between Tyr42, Glu46 and Thr50. Glu46 is a key residue as it has been suggested that the proton at Glu46 is transferred to the chromophore during its photoconversion from the dark state to the signalling state. The structure of E46Q mutant protein was determined at 1.2,Å resolution, revealing that the phenolic O atom of p -­coumaric acid is hydrogen bonded to NH2 of Gln46 in E46Q with a longer distance (2.86 ± 0.02,Å) than its distance (2.51,Å) to Glu46,OH in the wild type. This and the decreased thermal stability of E46Q relative to the wild type show that this hydrogen bond is weakened in the E46Q mutant compared with the corresponding bond in the wild type. Several characteristic features of E46Q such as an alkali shift in the pKa and the rapid photocycle can be explained by this weakened hydrogen bond. Furthermore, the red shift in the absorption maximum in E46Q can be explained by the delocalization of the electron on the phenolic oxygen of p -­coumaric acid owing to the weakening of this hydrogen bond. [source]

Structures of three diphtheria toxin repressor (DtxR) variants with decreased repressor activity

Ehmke Pohl
The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae regulates the expression of the gene on corynebacteriophages that encodes diphtheria toxin (DT). Other genes regulated by DtxR include those that encode proteins involved in siderophore-mediated iron uptake. DtxR requires activation by divalent metals and holo-DtxR is a dimeric regulator with two distinct metal-binding sites per three-domain monomer. At site 1, three side chains and a sulfate or phosphate anion are involved in metal coordination. In the DtxR,DNA complex this anion is replaced by the side chain of Glu170 provided by the third domain of the repressor. At site 2 the metal ion is coordinated exclusively by constituents of the polypeptide chain. In this paper, five crystal structures of three DtxR variants focusing on residues Glu20, Arg80 and Cys102 are reported. The resolution of these structures ranges from 2.3 to 2.8,Å. The side chain of Glu20 provided by the DNA-binding domain forms a salt bridge to Arg80, which in turn interacts with the anion. Replacing either of the salt-bridge partners with an alanine reduces repressor activity substantially and it has been inferred that the salt bridge could possibly control the wedge angle between the DNA-binding domain and the dimerization domain, thereby modulating repressor activity. Cys102 is a key residue of metal site 2 and its substitution into a serine abolishes repressor activity. The crystal structures of Zn-Glu20Ala-DtxR, Zn-Arg80Ala-DtxR, Cd-Cys102Ser-DtxR and apo-Cys102Ser-DtxR in two related space groups reveal that none of these substitutions leads to dramatic rearrangements of the DtxR fold. However, the five crystal structures presented here show significant local changes and a considerable degree of flexibility of the DNA-binding domain with respect to the dimerization domain. Furthermore, all five structures deviate significantly from the structure in the DtxR,DNA complex with respect to overall domain orientation. These results confirm the importance of the hinge motion for repressor activity. Since the third domain has often been invisible in previous crystal structures of DtxR, it is also noteworthy that the SH3-like domain could be traced in four of the five crystal structures. [source]

A mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometry

FEBS JOURNAL, Issue 17 2009
Elizabeth Diago-Navarro
Kid, the toxin of the parD (kis, kid) maintenance system of plasmid R1, is an endoribonuclease that preferentially cleaves RNA at the 5, of A in the core sequence 5,-UA(A/C)-3,. A model of the Kid toxin interacting with the uncleavable mimetic 5,-AdUACA-3, is available. To evaluate this model, a significant collection of mutants in some of the key residues proposed to be involved in RNA binding (T46, A55, T69 and R85) or RNA cleavage (R73, D75 and H17) were analysed by mass spectrometry in RNA binding and cleavage assays. A pair of substrates, 5,-AUACA-3,, and its uncleavable mimetic 5,-AdUACA-3,, used to establish the model and structure of the Kid,RNA complex, were used in both the RNA cleavage and binding assays. A second RNA substrate, 5,-UUACU-3, efficiently cleaved by Kid both in vivo and in vitro, was also used in the cleavage assays. Compared with the wild-type protein, mutations in the residues of the catalytic site abolished RNA cleavage without substantially altering RNA binding. Mutations in residues proposed to be involved in RNA binding show reduced binding efficiency and a corresponding decrease in RNA cleavage efficiency. The cleavage profiles of the different mutants were similar with the two substrates used, but RNA cleavage required much lower protein concentrations when the 5,-UUACU-3, substrate was used. Protein synthesis and growth assays are consistent with there being a correlation between the RNase activity of Kid and its inhibitory potential. These results give important support to the available models of Kid RNase and the Kid,RNA complex. [source]

The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies

FEBS JOURNAL, Issue 5 2008
Christiane Goedl
Schizophyllum commune,,,-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into ,- d -glucose 1-phosphate and ,- d -glucose. Recruitment of phosphate by the free enzyme induces ,,,-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (kcat/Km = 21 000 m,1·s,1) was dramatic (,107 -fold) in purified Arg507,Ala (R507A) and Lys512,Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in kcat (Arg507) and Km (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H2PO4,, the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with ,,,-trehalose (Ki = 0.4 ,m) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (Ki = 300 ,m). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward ,,,-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively. [source]

Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidase

FEBS JOURNAL, Issue 4 2008
Hua Zhang
The nicotinamidase/pyrazinamidase (PncA) of Mycobacterium tuberculosis is involved in the activation of the important front-line antituberculosis drug pyrazinamide by converting it into the active form, pyrazinoic acid. Mutations in the pncA gene cause pyrazinamide resistance in M. tuberculosis. The properties of M. tuberculosis PncA were characterized in this study. The enzyme was found to be a 20.89 kDa monomeric protein. The optimal pH and temperature of enzymatic activity were pH 7.0 and 40 °C, respectively. Inductively coupled plasma-optical emission spectrometry revealed that the enzyme was an Mn2+/Fe2+ -containing protein with a molar ratio of [Mn2+] to [Fe2+] of 1 : 1; furthermore, the external addition of either type of metal ion had no apparent effect on the wild-type enzymatic activity. The activity of the purified enzyme was determined by HPLC, and it was shown that it possessed similar pyrazinamidase and nicotinamidase activity, by contrast with previous reports. Nine PncA mutants were generated by site-directed mutagenesis. Determination of the enzymatic activity and metal ion content suggested that Asp8, Lys96 and Cys138 were key residues for catalysis, and Asp49, His51, His57 and His71 were essential for metal ion binding. Our data show that M. tuberculosis PncA may bind metal ions in a manner different from that observed in the case of Pyrococcus horikoshii PncA. [source]

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

FEBS JOURNAL, Issue 13 2007
Ronnie A. Böck
Dihydrofolate reductase (EC is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80,90%, and D31A and D31L by >,90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in Km for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in kcat/Km. Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in Km; 21-fold decrease in kcat/Km). Only minor variations in the Km for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim. [source]

The structural comparison of the bacterial PepX and human DPP-IV reveals sites for the design of inhibitors of PepX activity

FEBS JOURNAL, Issue 8 2005
Pascal Rigolet
X-prolyl dipeptidyl aminopeptidases (X-PDAP) are enzymes catalysing the release of dipeptides from the amino termini of polypeptides containing a proline or an alanine at the penultimate position. Involved in various mammalian regulation processes, as well as in chronic human diseases, they have been proposed to play a role in pathogenicity for Streptococci. We compared the structure of X-PDAP from Lactococcus lactis (PepX) with its human counterpart DPP-IV. Despite very different overall folds, the residues most implicated for X-PDAP activity are conserved in the same positions and orientations in both enzymes, thus defining a structural signature for the X-PDAP specificity that crosses the species frontiers of evolution. Starting from this observation, we tested some inhibitors of DPP-IV on PepX activity, for which no specific inhibitor is known. We thus found that PepX was highly sensitive to valine-pyrrolidide with a KI of 9.3 µm, close to that reported in DPP-IV inhibition. We finally used the structure of PepX from L. lactis as a template for computer-based homology modeling of PepX from the pathogenic Streptococcus gordonii. Docking simulations of valine-pyrrolidide into the active site of PepX led to the identification of key residues for a rational drug design against PepX from Streptococci. These results could have applications in human health giving new perspectives to the struggle against pathogens. [source]

A novel method for enzyme design

Xiaolei Zhu
Abstract Rational design of enzymes is a stringent test of our understanding of protein structure and function relationship, which also has numerous potential applications. We present a novel method for enzyme design that can find good candidate protein scaffolds in a protein-ligand database based on vector matching of key residues. Residues in the vicinity of the active site were also compared according to a similarity score between the scaffold protein and the target enzyme. Suitable scaffold proteins were selected, and the side chains of residues around the active sites were rebuilt using a previously developed side-chain packing program. Triose phosphate isomerase (TIM) was used as a validation test for enzyme design. Selected scaffold proteins were found to accommodate the enzyme active sites and successfully form a good transition state complex. This method overcomes the limitations of the current enzyme design methods that use limited number of protein scaffold and based on the position of ligands. As there are a large number of protein scaffolds available in the Protein Data Band, this method should be widely applicable for various types of enzyme design. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2009 [source]

Identification of key residues involved in mediating the in vivo anti-tumor/anti-endothelial activity of Alphastatin

Summary., Background :,We have recently shown that Alphastatin, a 24-amino-acid peptide (ADSGEGDFLAEGGGVRGPRVVERH) derived from human fibrinogen has anti-endothelial properties in vitro and in vivo. Objectives:, The aim of this study was to determine the activity of a terminally modified (stabilized) form of Alphastatin in vitro and in vivo and to identify the key residues required for this activity. Methods:, The in vitro activity of modified Alphastatin, truncates and mutants was determined by endothelial cell (HuDMEC) tubule formation and migration. Active peptides were then assessed in vivo using syngeneic murine subcutaneous 4T1 mammary carcinomas. Results:, Modified Alphastatin-inhibited HuDMEC migration and tubule formation in response to multiple growth factors and caused a 45% inhibition in tumor growth when administered intravenously at 0.25 mg kg,1 (three times per week). Intravenous (i.v.) administration proved non-toxic at all doses investigated, whereas oral and intraperitoneal (i.p.) administration demonstrated neither anti-tumor activity nor toxicity. Truncations of Alphastatin revealed an 11-amino-acid peptide (DFLAEGGGVRG), termed AHN419, which inhibited endothelial cell activity in vitro; however, intravenous AHN419 caused a non-significant growth inhibition in vivo. Single amino acid substitutions to alanine along the entire length of Alphastatin indicated that additional residues outside the AHN419 sequence were required for full activity. Conclusions:, Terminal modification of Alphastatin altered the in vivo efficacy and these studies suggest that a hydrophobic cluster (Phe8, Leu9, Ala10 and Val15) is essential for the biological activity, but additional residues, including Ser3-Gly14, Pro18-Val20 and Arg23 are required for full inhibitory activity of Alphastatin. [source]

Co-regulation of Xanthomonas campestris virulence by quorum sensing and a novel two-component regulatory system RavS/RavR

Ya-Wen He
Summary Xanthomonas campestris pv. campestris (Xcc) is known to regulate virulence through a quorum-sensing mechanism. Detection of the quorum-sensing signal DSF by sensor RpfC leads to activation of the response regulator RpfG, which influences virulence by degrading cyclic-di-GMP and by subsequent increasing expression of the global regulator Clp. In this study, we show that mutation of a response regulator RavR containing the GGDEF,EAL domains decreases Xcc virulence factor production. The functionality of RavR is dependent on its EAL domain-associated cyclic-di-GMP phosphodiesterase activity. Deletion of a multidomain sensor gene ravS, which shares the same operon with ravR, results in similar phenotype changes as the ravR mutant. In addition, the sensor mutant phenotypes can be rescued by in trans expression of the response regulator, supporting the notion that RavS and RavR constitute a two-component regulatory system. Significantly, mutation of either the PAS domain or key residues of RavS implicated in sensing low-oxygen tension abrogates the sensor activity in virulence regulation. Moreover, similar to the DSF signalling system, RavS/RavR regulates virulence gene expression through the global regulator Clp. These results outline a co-regulation mechanism that allows Xcc to integrate population density and environmental cues to modulate virulence factor production and adaptation. [source]

The Drosophila G protein-coupled receptor, Methuselah, exhibits a promiscuous response to peptides

PROTEIN SCIENCE, Issue 11 2009
William W. Ja
Abstract Methuselah (Mth) is a G protein-coupled receptor (GPCR) associated with longevity in Drosophila melanogaster. Previously, Stunted (Sun) was identified as a peptide agonist of Mth. Here, we identify two additional activators of Mth signaling: Drosophila Sex Peptide (SP) and a novel peptide (Serendipitous Peptide Activator of Mth, SPAM). Minimal functional sequences and key residues were identified from Sun and SPAM by studying truncation and alanine-scanning mutations. These peptide agonists share little sequence homology and illustrate the promiscuity of Mth for activation. mth mutants exhibit no defects in behaviors controlled by SP, casting doubt on the biological significance of Mth activation by any of these agonists, and illustrating the difficulty in applying in vitro studies to their relevance in vivo. Future studies of Mth ligands will help further our understanding of the functional interaction of agonists and GPCRs. [source]

Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis

Dalei Wu
Abstract Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr. [source]

The MRG domain of human MRG15 uses a shallow hydrophobic pocket to interact with the N-terminal region of PAM14

PROTEIN SCIENCE, Issue 10 2006
Peng Zhang
Abstract MRG15 is a transcription factor expressed in a variety of human tissues, and its orthologs have been found in many other eukaryotes which constitute the MRG protein family. It plays a vital role in embryonic development and cell proliferation, and is involved in cellular senescence. The C-terminal part of MRG15 forms a conserved MRG domain which is involved in interactions with the tumor suppressor protein retinoblastoma and a nucleoprotein PAM14 during transcriptional regulation. We report here the characterization of the interaction between the MRG domain of human MRG15 and PAM14 using both yeast two-hybrid and in vitro binding assays based on the crystal structure of the MRG domain. The MRG domain is predominantly hydrophobic, and consists of mainly ,-helices that are arranged in a three-layer sandwich topology. The hydrophobic core is stabilized by interactions among a number of conserved hydrophobic residues. The molecular surface is largely hydrophobic, but contains a few hydrophilic patches. Structure-based site-directed mutagenesis studies identified key residues involved in the binding of PAM14. Structural and biochemical data together demonstrate that the PAM14 binding site is consisted of residues Ile160, Leu168, Val169, Trp172, Tyr235, Val268, and Arg269 of MRG15, which form a shallow hydrophobic pocket to interact with the N-terminal 50 residues of PAM14 through primarily hydrophobic interactions. These results provide the molecular basis for the interaction between the MRG domain and PAM14, and reveal insights into the potential biological function of MRG15 in transcription regulation and chromatin remodeling. [source]

Automatic generation and evaluation of sparse protein signatures for families of protein structural domains

Matthew J. Blades
Abstract We identified key residues from the structural alignment of families of protein domains from SCOP which we represented in the form of sparse protein signatures. A signature-generating algorithm (SigGen) was developed and used to automatically identify key residues based on several structural and sequence-based criteria. The capacity of the signatures to detect related sequences from the SWISSPROT database was assessed by receiver operator characteristic (ROC) analysis and jack-knife testing. Test signatures for families from each of the main SCOP classes are described in relation to the quality of the structural alignments, the SigGen parameters used, and their diagnostic performance. We show that automatically generated signatures are potently diagnostic for their family (ROC50 scores typically >0.8), consistently outperform random signatures, and can identify sequence relationships in the "twilight zone" of protein sequence similarity (<40%). Signatures based on 15%,30% of alignment positions occurred most frequently among the best-performing signatures. When alignment quality is poor, sparser signatures perform better, whereas signatures generated from higher-quality alignments of fewer structures require more positions to be diagnostic. Our validation of signatures from the Globin family shows that when sequences from the structural alignment are removed and new signatures generated, the omitted sequences are still detected. The positions highlighted by the signature often correspond (alignment specificity >0.7) to the key positions in the original (non-jack-knifed) alignment. We discuss potential applications of sparse signatures in sequence annotation and homology modeling. [source]

Modeling of the bacterial luciferase-flavin mononucleotide complex combining flexible docking with structure-activity data

Leo Yen-Cheng Lin
FMN, flavin mononucleotide; FMNH2, reduced FMN Abstract Although the crystal structure of Vibrio harveyi luciferase has been elucidated, the binding sites for the flavin mononucleotide and fatty aldehyde substrates are still unknown. The determined location of the phosphate-binding site close to Arg 107 on the , subunit of luciferase is supported here by point mutagenesis. This information, together with previous structure-activity data for the length of the linker connecting the phosphate group to the isoalloxazine ring represent important characteristics of the luciferase-bound conformation of the flavin mononucleotide. A model of the luciferase,flavin complex is developed here using flexible docking supplemented by these structural constraints. The location of the phosphate moiety was used as the anchor in a flexible docking procedure performed by conformation search by using the Monte Carlo minimization approach. The resulting databases of energy-ranked feasible conformations of the luciferase complexes with flavin mononucleotide, ,-phosphopentylflavin, ,-phosphobutylflavin, and ,-phosphopropylflavin were filtered according to the structure-activity profile of these analogs. A unique model was sought not only on energetic criteria but also on the geometric requirement that the isoalloxazine ring of the active flavin analogs must assume a common orientation in the luciferase-binding site, an orientation that is also inaccessible to the inactive flavin analog. The resulting model of the bacterial luciferase,flavin mononucleotide complex is consistent with the experimental data available in the literature. Specifically, the isoalloxazine ring of the flavin mononucleotide interacts with the Ala 74,Ala 75 cis -peptide bond as well as with the Cys 106 side chain in the , subunit of luciferase. The model of the binary complex reveals a distinct cavity suitable for aldehyde binding adjacent to the isoalloxazine ring and flanked by other key residues (His 44 and Trp 250) implicated in the active site. [source]

The structure of the genomic Bacillus subtilis dUTPase: novel features in the Phe-lid

Javier García-Nafría
dUTPases are a ubiquitous family of enzymes that are essential for all organisms and catalyse the breakdown of 2-deoxyuridine triphosphate (dUTP). In Bacillus subtilis there are two homotrimeric dUTPases: a genomic and a prophage form. Here, the structures of the genomic dUTPase and of its complex with the substrate analogue dUpNHpp and calcium are described, both at 1.85,Å resolution. The overall fold resembles that of previously solved trimeric dUTPases. The C-terminus, which contains one of the conserved sequence motifs, is disordered in both structures. The crystal of the complex contains six independent protomers which accommodate six dUpNHpp molecules, with three triphosphates in the trans conformation and the other three in the active gauche conformation. The structure of the complex confirms the role of several key residues that are involved in ligand binding and the position of the catalytic water. Asp82, which has previously been proposed to act as a general base, points away from the active site. In the complex Ser64 reorients in order to hydrogen bond the phosphate chain of the substrate. A novel feature has been identified: the position in the sequence of the `Phe-lid', which packs against the uracil moiety, is adjacent to motif III, whereas in all other dUTPase structures the lid is in a conserved position in motif V of the flexible C-terminal arm. This requires a reconsideration of some aspects of the accepted mechanism. [source]

Refined structure of bovine carboxypeptidase A at 1.25,Å resolution

Alexandra Kilshtain-Vardi
The crystal structure of the bovine zinc metalloproteinase carboxypeptidase A (CPA) has been refined to 1.25,Å resolution based on room-temperature X-ray synchrotron data. The significantly improved structure of CPA at this resolution (anisotropic temperature factors, R factor = 10.4%, Rfree = 14.5%) allowed the modelling of conformational disorders of side chains, improved the description of the protein solvent network (375 water molecules) and provided a more accurate picture of the interactions between the active-site zinc and its ligands. The calculation of standard uncertainties in individual atom positions of the refined model of CPA allowed the deduction of the protonation state of some key residues in the active site and confirmed that Glu72 and Glu270 are negatively charged in the resting state of the enzyme at pH 7.5. These results were further validated by theoretical calculations that showed significant reduction of the pKa of these side chains relative to solution values. The distance between the zinc-bound solvent molecule and the metal ion is strongly suggestive of a neutral water molecule and not a hydroxide ion in the resting state of the enzyme. These findings could support both the general acid/general base mechanism, as well as the anhydride mechanism suggested for CPA. [source]

Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

William J. McKinstry
Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5,Å, and diffracted to 2.0,Å resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone. [source]

Mechanisms of constitutive activation of Janus kinase 2-V617F revealed at the atomic level through molecular dynamics simulations

CANCER, Issue 8 2009
Tai-Sung Lee PhD
Abstract BACKGROUND: The tyrosine kinase Janus kinase 2 (JAK2) is important in triggering nuclear translocation and regulation of target genes expression through signal transducer and activator of transcription pathways. The valine-to-phenylalanine mutation at amino acid 617 (V617F), which results in the deregulation of JAK2, has been implicated in the oncogenesis of chronic myeloproliferative disease. However, both the mechanism of JAK2 autoinhibition and the mechanism of V617F constitutive activation remain unclear. METHOD: In this work, the authors used molecular dynamics simulation techniques to establish plausible mechanisms of JAK2 autoinhibition and V617F constitutive activation at the atomic level. RESULTS: In wild-type JAK2, the activation loop of JAK2-homology domain 1 (JH1) is pulled toward the JH1/JH2 interface through interactions with key residues of JH2, especially S591, F595, and V617, and stabilizes the inactivated form of JH1. In the case of V617F, through the aromatic ring-ring stacking interaction, F617 blocks the interaction of JH1 the activation loop, S591, and F595, thus causing the JH1 activation loop to move back to its activated form. CONCLUSIONS: The current results indicated that this simulation-derived mechanism of JAK2 autoregulation is consistent with current available experimental evidence and may lead to a deeper understanding of JAK2 and other kinase systems that are regulated by pseudokinases. Cancer 2009. © 2009 American Cancer Society. [source]

Structure-Based Calculation of Binding Affinities of ,2A -Adrenoceptor Agonists

CHEMMEDCHEM, Issue 6 2007
Balázs Balogh
An atomic resolution structure of ,2a -adrenoceptor was constructed and 15 known agonists were docked into the optimized model and experimental binding free energies were estimated. The figure shows the binding of the agonist clonidine (sticks) to the core binding pocket of the adrenoceptor (blue cartoon, key residues are marked with sticks). [source]