Search tips
Search criteria

Results 1-25 (1376117)

Clipboard (0)

Related Articles

1.  Increasing stability of water-soluble PQQ glucose dehydrogenase by increasing hydrophobic interaction at dimeric interface 
BMC Biochemistry  2005;6:1.
Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B.
Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3–4°C higher over wild type. All mutants showed 2-fold higher thermal stability at 55°C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities.
We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary structure of the enzyme, we selected residues located at the dimer interface that do not contribute to the intersubunit interaction. By substituting these residues to hydrophobic ones, the thermal stability of PQQGDH-B was increased without decreasing its catalytic activity.
PMCID: PMC551599  PMID: 15715904
2.  Crystal structures of MS2 coat protein mutants in complex with wild-type RNA operator fragments. 
Nucleic Acids Research  1998;26(5):1345-1351.
In MS2 assembly of phage particles results from an interaction between a coat protein dimer and a stem-loop of the RNA genome (the operator hairpin). Amino acid residues Thr45, which is universally conserved among the small RNA phages, and Thr59 are part of the specific RNA binding pocket and interact directly with the RNA; the former through a hydrogen bond, the latter through hydrophobic contacts. The crystal structures of MS2 protein capsids formed by mutants Thr45Ala and Thr59Ser, both with and without the 19 nt wild-type operator hairpin bound, are reported here. The RNA hairpin binds to these mutants in a similar way to its binding to wild-type protein. In a companion paper both mutants are shown to be deficient in RNA binding in an in vivo assay, but in vitro the equilibrium dissociation constant is significantly higher than wild-type for the Thr45Ala mutant. The change in binding affinity of the Thr45Ala mutant is probably a direct consequence of removal of direct hydrogen bonds between the protein and the RNA. The properties of the Thr59Ser mutant are more difficult to explain, but are consistent with a loss of non-polar contact.
PMCID: PMC147404  PMID: 9469847
3.  The Binding of Antigenic Peptides to HLA-DR Is Influenced by Interactions between Pocket 6 and Pocket 91 
Peptide binding to class II MHC protein is commonly viewed as a combination of discrete anchor residue preferences for pockets 1, 4, 6/7, and 9. However, previous studies have suggested cooperative effects during the peptide binding process. Investigation of the DRB1*0901 binding motif demonstrated a clear interaction between peptide binding pockets 6 and 9. In agreement with prior studies, pockets 1 and 4 exhibited clear binding preferences. Previously uncharacterized pockets 6 and 7 accommodated a wide variety of residues. However, although it was previously reported that pocket 9 is completely permissive, several substitutions at this position were unable to bind. Structural modeling revealed a probable interaction between pockets 6 and 9 through β9Lys. Additional binding studies with doubly substituted peptides confirmed that the amino acid bound within pocket 6 profoundly influences the binding preferences for pocket 9 of DRB1*0901, causing complete permissiveness of pocket 9 when a small polar residue is anchored in pocket 6 but accepting relatively few residues when a basic residue is anchored in pocket 6. The β9Lys residue is unique to DR9 alleles. However, similar studies with doubly substituted peptides confirmed an analogous interaction effect for DRA1/B1*0301, a β9Glu allele. Accounting for this interaction resulted in improved epitope prediction. These findings provide a structural explanation for observations that an amino acid in one pocket can influence binding elsewhere in the MHC class II peptide binding groove.
PMCID: PMC4061985  PMID: 19648278
4.  A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein 
PLoS Pathogens  2015;11(7):e1005062.
Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.
Author Summary
Influenza A virus nucleoprotein (NP) is a highly conserved multifunctional protein that plays an essential role in infection by all subtypes of influenza A virus, making it an attractive target for new antiviral drugs. NP regulates viral polymerase activity and transport of the viral genome into/from the host cell nucleus by forming the viral ribonucleoprotein complex (vRNP). Because NP regulates replication and transcription of the viral genome in addition to its role in nuclear export (all of which are essential for the production of viral progeny), it is a promising drug target. Here, we used the antiviral compound RK424 to identify a novel pocket structure within NP. This structure encompassed three different functional domains that are involved in the above-mentioned replication steps. RK424 inhibits viral genome replication/transcription and nuclear export of NP by destabilizing the NP oligomer and inhibiting the binding of chromosome region maintenance 1 (CRM1) to NP via nuclear export signal (NES) 3, which is located in close proximity to the NP pocket. Taken together, these findings suggest that this small NP pocket is a novel antiviral target.
PMCID: PMC4519322  PMID: 26222066
5.  Changes in the repertoire of peptides bound to HLA-B27 subtypes and to site-specific mutants inside and outside pocket B 
HLA-B27 subtypes share many structural features, including their pocket B, which interacts with a conserved Arg residue at the second position of B*2705-bound peptides. Subtypes differ among each other at other locations in the peptide binding site. In this study, metabolic labeling and radiochemical pool sequencing were used to address the following issues: (a) presence of the Arg 2 (R2) motif among peptides bound to the various HLA-B27 subtypes; (b) influence of mutations inside and outside pocket B on this motif; and (c) the degree of similarity among the peptide pools bound to the various B27 subtypes. Sequencing of Arg-labeled peptide pools extracted from B*2701 to B*2706, and from two site-directed mutants of B*2705 with changes outside pocket B, indicated that all of these molecules bind peptides with Arg at position 2. Peptides from several mutants with changes altering the structure of pocket B, and from one mutant at the pocket B rim, also retained the R2 motif. However, this was absent in the peptide pool extracted from the M45 mutant, in which the negative charge of pocket B, conferred to HLA-B27 by Glu45, was canceled. These results indicate that alterations outside pocket B, and even disruption of the network of hydrogen bonds that stabilizes Arg binding in pocket B, do not impair binding of peptides bearing the R2 motif, but a nonconservative substitution at position 45 does. As a substantial fraction of anti-B*2705 cytotoxic T lymphocyte (CTL) clones crossreact with the M45 mutant (Villadangos, J., B. Galocha, D. Lopez, V. Calvo, and J. A. Lopez de Castro. 1992. J. Immunol. 149:505) this result suggest that determinant mimicry by nonidentical peptides may frequently account for unexpected CTL crossreactions. Metabolic labeling with various other amino acids and radiochemical sequencing revealed similarities, but also substantial differences, among the peptide pools from the various HLA-B27 subtypes. This strongly suggests that many peptides bind to multiple subtypes, but significant subsets of peptides bound to a given HLA-B27 subtype do not bind to other subtypes or do so with greatly altered efficiency. These results indicate the importance of polymorphism outside pocket B in modulating peptide binding to HLA-B27.
PMCID: PMC2190930  PMID: 8436905
6.  Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2 
ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca2+-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe85, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr180, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined.
PMCID: PMC4346919  PMID: 25667979
adaptor protein; calcium-binding protein; COPII; crystal structure; EF-hand; motif; protein-protein interaction
7.  Investigation of allosteric modulation mechanism of metabotropic glutamate receptor 1 by molecular dynamics simulations, free energy and weak interaction analysis 
Scientific Reports  2016;6:21763.
Metabotropic glutamate receptor 1 (mGlu1), which belongs to class C G protein-coupled receptors (GPCRs), can be coupled with G protein to transfer extracellular signal by dimerization and allosteric regulation. Unraveling the dimer packing and allosteric mechanism can be of great help for understanding specific regulatory mechanism and designing more potential negative allosteric modulator (NAM). Here, we report molecular dynamics simulation studies of the modulation mechanism of FITM on the wild type, T815M and Y805A mutants of mGlu1 through weak interaction analysis and free energy calculation. The weak interaction analysis demonstrates that van der Waals (vdW) and hydrogen bonding play an important role on the dimer packing between six cholesterol molecules and mGlu1 as well as the interaction between allosteric sites T815, Y805 and FITM in wild type, T815M and Y805A mutants of mGlu1. Besides, the results of free energy calculations indicate that secondary binding pocket is mainly formed by the residues Thr748, Cys746, Lys811 and Ser735 except for FITM-bound pocket in crystal structure. Our results can not only reveal the dimer packing and allosteric regulation mechanism, but also can supply useful information for the design of potential NAM of mGlu1.
PMCID: PMC4757871  PMID: 26887338
8.  Dissecting the key recognition features of the MS2 bacteriophage translational repression complex. 
Nucleic Acids Research  1998;26(5):1337-1344.
The MS2 RNA operator capsid offers an unparalleled opportunity to study sequence-specific protein-protein and RNA-protein interactions in molecular detail. RNA molecules encompassing the minimal translational operator recognition elements can be soaked into crystals of RNA-free coat protein shells, allowing the RNA to access the interior of the capsids and make contact with the operator binding sites. Correct interpretation of these structural studies depends critically on functional analysis in solution to confirm that the interactions seen in the crystal are not an artefact of the unusual approach used to generate the RNA-protein complexes. Here we present a series of in vivo and in vitro functional assays, using coat proteins carrying single amino acid substitutions at residues which either interact with the operator RNA or are involved in stabilizing the conformation of the FG loop, the site of the major quasi-equivalent conformational change. Variant operator RNAs have been assayed for coat protein affinity in vitro. The results reveal the robustness of the operator-coat protein interaction and the requirement for both halves of a protein dimer to contact RNA in order to achieve tight binding. They also suggest that there may be a direct link between the conformation of the FG loop and RNA binding.
PMCID: PMC147387  PMID: 9469846
9.  The yin–yang of kinase activation and unfolding explains the peculiarity of Val600 in the activation segment of BRAF 
eLife  null;5:e12814.
Many driver mutations in cancer are specific in that they occur at significantly higher rates than – presumably – functionally alternative mutations. For example, V600E in the BRAF hydrophobic activation segment (AS) pocket accounts for >95% of all kinase mutations. While many hypotheses tried to explain such significant mutation patterns, conclusive explanations are lacking. Here, we use experimental and in silico structure-energy statistical analyses, to elucidate why the V600E mutation, but no other mutation at this, or any other positions in BRAF’s hydrophobic pocket, is predominant. We find that BRAF mutation frequencies depend on the equilibrium between the destabilization of the hydrophobic pocket, the overall folding energy, the activation of the kinase and the number of bases required to change the corresponding amino acid. Using a random forest classifier, we quantitatively dissected the parameters contributing to BRAF AS cancer frequencies. These findings can be applied to genome-wide association studies and prediction models.
eLife digest
Mutations in the gene that encodes a protein called BRAF are commonly found in certain cancers, such as melanomas. The same BRAF mutation is found in nearly all of these cancers. This mutation causes the 600th amino acid in the BRAF protein – an amino acid called a valine – to be replaced with another amino acid, a glutamate.
BRAF is a type of enzyme called a kinase, and it transmits signals inside cells to promote cell growth. Kinases work by adding a phosphate group to other proteins to alter their activity. The structure of the BRAF kinase contains a pocket-like shape, and the valine at position 600 sits buried inside this pocket when the enzyme is inactive. The “valine-to-glutamate” mutation (often called V600E for short) disrupts the interactions that create this pocket. This in turn results in a permanently active form of BRAF and uncontrolled cell growth. However, it remains unclear why the valine-to-glutamate mutation is so much more common in cancer cells than any other mutation that could affect the pocket in BRAF.
To address this question, Kiel et al. used a computational tool to generate three-dimensional models for all the different amino acid substitutions that could occur in BRAF’s pocket. Each mutation was then assessed to see how it might destabilize the structure of BRAF. Only the mutations that affected the 600th amino acid were predicted to be able to open the pocket without destabilizing the part of the enzyme that adds phosphate groups to other proteins.
Kiel et al. validated their computational predictions by introducing normal or mutant versions of the BRAF-encoding gene into human cells grown in the laboratory. These experiments showed that a mutation that introduced an amino acid called histidine into position 600 could activate BRAF as much the valine-to-glutamate mutation. Kiel et al. suggest that this “valine-to-histidine” substitution is not found in cancers because it requires three changes to the DNA sequence of the BRAF gene, whereas the valine-to-glutamate substitution only requires one.
The results underscore the importance of considering changes at both the DNA and protein level when attempting to understand why certain cancer-causing mutations are more common than others.
PMCID: PMC4749552  PMID: 26744778
structure-energy calculations; genotype-phenotype association; passenger and driver mutations; Human
10.  Effector-Induced Structural Fluctuation Regulates the Ligand Affinity of an Allosteric Protein 
Biochemistry  2008;47(17):4907-4915.
The present study reports distinct dynamic consequences for the T- and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.1 T, and at 37 °C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy- and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the ·s-ms time scale, with the exception of several amino acid residues, that is, · 109Val and · 132Lys in deoxy-Hb A, and R40Lys in HbCO A. The mobility of R40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between R40Lys and · 146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of ·109Val, which is located in the intradimer (R1· 1 or R2· 2) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify R40Lys in HbCO A, but does not significantly affect the flexibility of · 109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (R1 · 2 or R2 · 1) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal ·92His in the ·-heme pocket, as well as some other residues located in the flexible joint (·C helix-RFG corner) and switch (RC helix-·FG corner) regions that play an important role in the dimer-dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the ·-chain. In contrast, no significant perturbation of the dynamic features on the ms-·s time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the ·-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate (Rex) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.
PMCID: PMC2493540  PMID: 18376851
11.  Molecular dynamics guided study of salt bridge length dependence in both fluorinated and non-fluorinated parallel dimeric coiled-coils 
Proteins  2009;74(3):612-629.
The α-helical coiled-coil is one of the most common oligomerization motifs found in both native and engineered proteins. To better understand the stability and dynamics of coiled-coil motifs, including those modified by fluorination, several fluorinated and non-fluorinated parallel dimeric coiled-coil protein structures were designed and modeled. We also attempt to investigate how changing the length and geometry of the important stabilizing salt bridges influences the coiled-coil protein structure. Molecular dynamics (MD) and free energy simulations with AMBER employed a particle mesh Ewald treatment of the electrostatics in explicit TIP3P solvent with balanced force field treatments. Preliminary studies with legacy force fields (ff94, ff96, ff99) show a profound instability of the coiled-coil structures in short MD simulation. Significantly better behavior is evident with the more balanced ff99SB and ff03 protein force fields. Overall, the results suggest that the coiled-coil structures can readily accommodate the larger acidic arginine or S-2,7-diaminoheptanedoic acid mutants in the salt bridge, whereas substitution of the smaller L-ornithine residue leads to rapid disruption of the coiled-coil structure on the MD simulation time scale. This structural distortion of the secondary structure allows both the formation of large hydration pockets proximal to the charged groups and within the hydrophobic core. Moreover, the increased structural fluctuations and movement lead to a decrease in the water occupancy lifetimes in the hydration pockets. In contrast, analysis of the hydration in the stable dimeric coiled coils shows high occupancy water sites along the backbone residues with no water occupancy in the hydrophobic core, although transitory water interactions with the salt bridge residues are evident. The simulations of the fluorinated coiled-coils suggest that in some cases fluorination electrostatically stabilizes the intermolecular coiled-coil salt bridges. Structural analyses also reveal different side chain rotamer preferences for leucine compared to 5,5,5,5′,5′,5′-hexafluoroleucine mutants. These observed differences in the side chain rotamer populations suggest differential changes in the side chain conformational entropy upon coiled-coil formation when the protein is fluorinated. The free energy of hydration of the isolated 5,5,5,5′,5′,5′-hexafluoroleucine amino acid is calculated to be 1.1 kcal/mol less stable than leucine; this hydrophobic penalty in the monomer may provide a driving force for coiled-coil dimer formation. Estimation of the ellipticity at 222 nm from a series of snapshots from the MD simulations with DicroCalc show distinct increases in the ellipticity when the coiled-coil is fluorinated which suggests that the helicity in the folded coiled-coils is greater when fluorinated.
PMCID: PMC2692595  PMID: 18704948
computational chemistry; free energy of hydration; 5, 5, 5, 5′, 5′, 5′-hexafluoroleucine; thermodynamic integration; rotamers
12.  Deciphering the Arginine-Binding Preferences at the Substrate-Binding Groove of Ser/Thr Kinases by Computational Surface Mapping 
PLoS Computational Biology  2011;7(11):e1002288.
Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P−2 and P−5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P−2/P−5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P−5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P−2 and P−5 positions.
Author Summary
Protein kinases are key signaling enzymes and major drug targets that catalyze the transfer of phosphate group to a phospho-accepting residue in the substrate. Unraveling molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor (substrate consensus sequence, SCS) is important for understanding kinase-substrates specificities and for designing peptidic inhibitors. Current methods used to predict this set of essential residues usually rely on linking between experimentally determined SCSs to kinase sequences. As such, these methods are less sensitive when specificity is dictated by subtle or kinase-unique sequence/structural features. In this study, we took a different approach for studying kinases specificities, by applying a new fragment-based method for mapping amino acid side chains on protein surfaces. We predicted and characterized the preference of Ser/Thr kinases toward Arginine binding, using the unbound kinase structures. The method produced high quality predictions and was able to provide novel insights and interesting departures from the prevailing views regarding the specificity-determining elements governing specificity toward Arginine. This work paves the way for studying the kinase binding preferences for other amino acids, for predicting protein-peptide structures, for facilitating the design of novel inhibitors, and for re-engineering of kinase specificities.
PMCID: PMC3219626  PMID: 22125489
13.  A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11 
Journal of Virology  2016;90(9):4579-4592.
Porcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the “active site loop” (His129 to His144) and “supporting loop” (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs.
IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirales. PRRSV is a major agent of respiratory diseases in pigs, causing tremendous economic losses to the swine industry worldwide. The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood. Here, we report the first dimeric structure of the arterivirus nsp11 from PRRSV at 2.75-Å resolution. Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer. Mutagenesis and structural analysis revealed NendoU active site residues, which are conserved throughout the order Nidovirales (families Arteriviridae and Coronaviridae) and the major determinants of dimerization (Ser74 and Phe76) in Arteriviridae. Importantly, these findings may provide a new structural basis for antiviral drug development.
PMCID: PMC4836315  PMID: 26912626
14.  Structural Analysis of a Rabbit Hemorrhagic Disease Virus Binding to Histo-Blood Group Antigens 
Journal of Virology  2014;89(4):2378-2387.
Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family (Lagovirus genus). RHDV is highly contagious and attaches to epithelial cells in the digestive or respiratory tract, leading to massive lesions with high mortality rates. A new variant of RHDV (termed RHDVb) recently has emerged, and previously vaccinated rabbits appear to have little protection against this new strain. Similar to human norovirus (Caliciviridae, Norovirus genus), RHDV binds histo-blood group antigens (HBGAs), and this is thought to be important for infection. Here, we report the HBGA binding site on the RHDVb capsid-protruding domain (P domain) using X-ray crystallography. The HBGA binding pocket was located in a negatively charged patch on the side of the P domain and at a dimeric interface. Residues from both monomers contributed to the HBGA binding and involved a network of direct hydrogen bonds and water-mediated interactions. An amino acid sequence alignment of different RHDV strains indicated that the residues directly interacting with the ABH-fucose of the HBGAs (Asp472, Asn474, and Ser479) were highly conserved. This result suggested that different RHDV strains also could bind HBGAs at the equivalent pocket. Moreover, several HBGA binding characteristics between RHDVb and human genogroup II norovirus were similar, which indicated a possible convergent evolution of HBGA binding interactions. Further structural studies with other RHDV strains are needed in order to better understand the HBGA binding mechanisms among the diverse RHDV strains.
IMPORTANCE We identified, for the first time, the HBGA binding site on an RHDVb P domain using X-ray crystallography. Our results showed that RHDVb and human genogroup II noroviruses had similar HBGA binding interactions. Recently, it was discovered that synthetic HBGAs or HBGA-expressing enteric bacteria could enhance human genogroup II norovirus infection in B cells. Considering that RHDVb and genogroup II norovirus similarly interacted with HBGAs, it may be possible that a comparable cell culture system also could work with RHDVb. Taken together, these new findings will extend our understanding of calicivirus HBGA interactions and may help to elucidate the specific roles of HBGAs in calicivirus infections.
PMCID: PMC4338867  PMID: 25505081
15.  A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding and drug resistance 
Nucleic Acids Research  2008;36(17):5635-5644.
Human DNA topoisomerase I (hTop1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of the antitumor drug camptothecin (CPT). The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro). Tht729Glu and Thr729Lys mutants show severe CPT resistance and furthermore, Thr729Glu shows a remarkable defect in DNA binding. We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.
PMCID: PMC2553582  PMID: 18772225
16.  Site-directed Mutagenesis Identifies Residues Involved in Ligand Recognition in the Human A2a Adenosine Receptor 
The Journal of biological chemistry  1995;270(23):13987-13997.
The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5–7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nm) and [3H]XAC (4 nm), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5′. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were >30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5’-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.
PMCID: PMC3427751  PMID: 7775460
17.  Effects of an Early Conformational Switch Defect during ϕX174 Morphogenesis Are Belatedly Manifested Late in the Assembly Pathway 
Journal of Virology  2013;87(5):2518-2525.
C-terminal, aromatic amino acids in the ϕX174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B− mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B− assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.
PMCID: PMC3571406  PMID: 23255785
18.  The mechanism of H171T resistance reveals the importance of Nδ-protonated His171 for the binding of allosteric inhibitor BI-D to HIV-1 integrase 
Retrovirology  2014;11:100.
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site.
We have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171.
Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor.
Electronic supplementary material
The online version of this article (doi:10.1186/s12977-014-0100-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4251946  PMID: 25421939
HIV-1 integrase; Allosteric inhibitors; Aberrant multimerization; Drug resistance
19.  Cofolding Organizes Alfalfa Mosaic Virus RNA and Coat Protein for Replication 
Science (New York, N.Y.)  2004;306(5704):2108-2111.
Alfalfa mosaic virus genomic RNAs are infectious only when the viral coat protein binds to the RNA 3´ termini. The crystal structure of an alfalfa mosaic virus RNA-peptide complex reveals that conserved AUGC repeats and Pro-Thr-x-Arg-Ser-x-x-Tyr coat protein amino acids cofold upon interacting. Alternating AUGC residues have opposite orientation, and they base pair in different adjacent duplexes. Localized RNA backbone reversals stabilized by arginine-guanine interactions place the adenosines and guanines in reverse order in the duplex. The results suggest that a uniform, organized 3´ conformation, similar to that found on viral RNAs with transfer RNA-like ends, may be essential for replication.
PMCID: PMC1500904  PMID: 15604410
20.  Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms 
Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor–ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ.
PMCID: PMC4498731  PMID: 26170631
schizophrenia; bioinformatics; modeling; docking; DAOA; G72; DAO; computer-aided drug designing; phylogenetic analysis; D-amino acid oxidase activator
21.  Substitutions of Thr30 provide mechanistic insight into tryptophan-mediated activation of TRAP binding to RNA 
Nucleic Acids Research  2006;34(10):2933-2942.
TRAP is an 11 subunit RNA binding protein that regulates expression of genes involved in tryptophan biosynthesis and transport in Bacillus subtilis. TRAP is activated to bind RNA by binding up to 11 molecules of l-tryptophan in pockets formed by adjacent subunits. The precise mechanism by which tryptophan binding activates TRAP is not known. Thr30 is in the tryptophan binding pocket. A TRAP mutant in which Thr30 is substituted with Val (T30V) does not bind tryptophan but binds RNA constitutively, suggesting that Thr30 plays a key role in the activation mechanism. We have examined the effects of other substitutions of Thr30. TRAP proteins with small β-branched aliphatic side chains at residue 30 bind RNA constitutively, whereas those with a small polar side chain show tryptophan-dependent RNA binding. Several mutant proteins exhibited constitutive RNA binding that was enhanced by tryptophan. Although the tryptophan and RNA binding sites on TRAP are distinct and are separated by ∼7.5 Å, several substitutions of residues that interact with the bound RNA restored tryptophan binding to T30V TRAP. These observations support the hypothesis that conformational changes in TRAP relay information between the tryptophan and RNA binding sites of the protein.
PMCID: PMC1474065  PMID: 16738132
22.  Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 of the cyclic nucleotide binding pocket. 
Nucleic Acids Research  1994;22(15):2894-2901.
The cyclic 3', 5' adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute cysteine or glycine for serine 83; cysteine, glycine, isoleucine, or serine for threonine 127; and threonine or alanine for serine 128. Cells that expressed the binding pocket residue-substituted forms of CRP were characterized by measurements of beta-galactosidase activity. Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity and by their capacity to support lacP activation in vitro. CRP structure was assessed by measurement of sensitivity to protease and DTNB-mediated subunit crosslinking. The results of this study show that cAMP interactions with serine 83, threonine 127 and serine 128 contribute to CRP activation and have little effect on cAMP binding. Amino acid substitutions that introduce hydrophobic amino acid side chain constituents at either position 127 or 128 decrease CRP discrimination of cAMP and cGMP. Finally, cAMP-induced CRP structural change(s) that occur in or near the CRP hinge region result from cAMP interaction with threonine 127; substitution of threonine 127 by cysteine, glycine, isoleucine, or serine produced forms of CRP that contained, independently of cAMP binding, structural changes similar to those of the wild-type CRP:cAMP complex.
PMCID: PMC310252  PMID: 8065899
23.  Substrate Binding Mode and Its Implication on Drug Design for Botulinum Neurotoxin A 
PLoS Pathogens  2008;4(9):e1000165.
The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide 197QRATKM202 and its variant 197RRATKM202 to 1.5 Å and 1.6 Å, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5′ sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1′-Arg198, occupies the S1′ site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2′ subsite is formed by Arg363, Asn368 and Asp370, while S3′ subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4′-Lys201 makes hydrogen bond with Gln162. P5′-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.
Author Summary
Botulinum neurotoxins are the most poisonous substance to humans. The ease with which the bacteria can be grown, its potency and persistence have made it a potential bioterrorism agent, and accordingly, botulinum neurotoxin has been declared as Category A agent by the Centers of Disease Control and Prevention. Since it is both a potential bioweapon and a bioterrorism agent, it is imperative to develop counter measures and therapeutics for these neurotoxins, as none are available so far except experimental vaccines and an FDA-approved equine antitoxin. Our work presented here is an important milestone towards achieving this goal. The best antidote can be developed by blocking the active site of any enzyme. The crystal structures of substrate peptide–enzyme complex presented here map the interactions between the two and provide critical information for designing effective drugs against this toxin.
PMCID: PMC2533696  PMID: 18818739
24.  Nuclear Magnetic Resonance Structure Revealed that the Human Polyomavirus JC Virus Agnoprotein Contains an α-Helix Encompassing the Leu/Ile/Phe-Rich Domain 
Journal of Virology  2014;88(12):6556-6575.
Agnoprotein is a small multifunctional regulatory protein required for sustaining the productive replication of JC virus (JCV). It is a mostly cytoplasmic protein localizing in the perinuclear area and forms highly stable dimers/oligomers through a Leu/Ile/Phe-rich domain. There have been no three-dimensional structural data available for agnoprotein due to difficulties associated with the dynamic conversion from monomers to oligomers. Here, we report the first nuclear magnetic resonance (NMR) structure of a synthetic agnoprotein peptide spanning amino acids Thr17 to Glu55 where Lys23 to Phe39 encompassing the Leu/Ile/Phe-rich domain forms an amphipathic α-helix. On the basis of these structural data, a number of Ala substitution mutations were made to investigate the role of the α-helix in the structure and function of agnoprotein. Single L29A and L36A mutations exhibited a significant negative effect on both protein stability and viral replication, whereas the L32A mutation did not. In addition, the L29A mutant displayed a highly nuclear localization pattern, in contrast to the pattern for the wild type (WT). Interestingly, a triple mutant, the L29A+L32A+L36A mutant, yielded no detectable agnoprotein expression, and the replication of this JCV mutant was significantly reduced, suggesting that Leu29 and Leu36 are located at the dimer interface, contributing to the structure and stability of agnoprotein. Two other single mutations, L33A and E34A, did not perturb agnoprotein stability as drastically as that observed with the L29A and L36A mutations, but they negatively affected viral replication, suggesting that the role of these residues is functional rather than structural. Thus, the agnoprotein dimerization domain can be targeted for the development of novel drugs active against JCV infection.
IMPORTANCE Agnoprotein is a small regulatory protein of JC virus (JCV) and is required for the successful completion of the viral replication cycle. It forms highly stable dimers and oligomers through its hydrophobic (Leu/Ile/Phe-rich) domain, which has been shown to play essential roles in the stability and function of the protein. In this work, the Leu/Ile/Phe-rich domain has been further characterized by NMR studies using an agnoprotein peptide spanning amino acids T17 to Q54. Those studies revealed that the dimerization domain of the protein forms an amphipathic α-helix. Subsequent NMR structure-based mutational analysis of the region highlighted the critical importance of certain amino acids within the α-helix for the stability and function of agnoprotein. In conclusion, this study provides a solid foundation for developing effective therapeutic approaches against the dimerization domain of the protein to inhibit its critical roles in JCV infection.
PMCID: PMC4054343  PMID: 24672035
25.  Function of Phe-259 and Thr-314 within the Substrate Binding Pocket of the Juvenile Hormone Esterase of Manduca sexta† 
Biochemistry  2010;49(17):3733-3742.
Juvenile hormone (JH) is a key insect developmental hormone that is found at low nanomolar levels in larval insects. The methyl ester of JH is hydrolyzed in many insects by an esterase that shows high specificity for JH. We have previously determined a crystal structure of the JH esterase (JHE) of the tobacco hornworm Manduca sexta (MsJHE) [Wogulis, M., Wheelock, C. E., Kamita, S. G., Hinton, A. C., Whetstone, P. A., Hammock, B. D., and Wilson, D. K. (2006) Biochemistry 45, 4045-4057]. Our molecular modeling indicates that JH fits very tightly within the substrate binding pocket of MsJHE. This tight fit places two non-catalytic amino acid residues, Phe-259 and Thr-314, within the appropriate distance and geometry to potentially interact with the α,β-unsaturated ester and epoxide, respectively, of JH. These residues are highly conserved in numerous biologically active JHEs. Kinetic analyses of mutants of Phe-259 or Thr-314 indicate that these residues contribute to the low KM that MsJHE shows for JH. This low KM, however, comes at the cost of reduced substrate turnover. Neither nucleophilic attack of the resonance stabilized ester by the catalytic serine nor the availability of a water molecule for attack of the acyl-enzyme intermediate appear to be a rate-determining step in the hydrolysis of JH by MsJHE. We hypothesize that the release of the JH acid metabolite from the substrate binding pocket limits the catalytic cycle. Our findings also demonstrate that chemical bond strength does not necessarily correlate with how reactive the bond will be to metabolism.
PMCID: PMC3570046  PMID: 20307057

Results 1-25 (1376117)