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1.  Temperature-dependent conformational change affecting Tyr11 and sweetness loops of brazzein 
Proteins  2013;81(6):919-925.
The sweet protein brazzein, a member of the Csβα fold family, contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Nevertheless, a variable temperature study has revealed that the protein undergoes a local, reversible conformational change between 37 and 3°C with a midpoint about 27°C that changes the orientations and side-chain hydrogen bond partners of Tyr8 and Tyr11. To test the functional significance of this effect, we used NMR saturation transfer to investigate the interaction between brazzein and the amino terminal domain of the sweet receptor subunit T1R2; the results showed a stronger interaction at 7°C than at 37°C. Thus the low temperature conformation, which alters the orientations of two loops known to be critical for the sweetness of brazzein, may represent the bound state of brazzein in the complex with the human sweet receptor.
PMCID: PMC3982881  PMID: 23349025
human sweet receptor; sweet protein; NMR spectroscopy; three-dimensional solution structures; saturation transfer difference spectroscopy
2.  Multi-constraint Computational Design Suggests that Native Sequences of Germline Antibody H3 Loops are Nearly Optimal for Conformational Flexibility 
Proteins  2009;75(4):846-858.
The limited size of the germline antibody repertoire has to recognize a far larger number of potential antigens. The ability of a single antibody to bind multiple ligands due to conformational flexibility in the antigen-binding site can significantly enlarge the repertoire. Among the six hyper-variable complementarity determining regions (CDRs) that comprise the binding site, the CDR H3 loop is particularly flexible. Computational protein design studies showed that predicted low energy sequences compatible with a given backbone structure often have considerable similarity to the corresponding native sequences of naturally occurring proteins, indicating that native protein sequences are close to optimal for their structures. Here, we take a step forward to determine whether conformational flexibility, believed to play a key functional role in germline antibodies, is also central in shaping their native sequence. In particular, we use a multi-constraint computational design strategy, along with the Rosetta energy function, to propose that the native sequences of CDR H3 loops from germline antibodies are nearly optimal for conformational flexibility. Moreover, we find that antibody maturation may lead to sequences with a higher degree of optimization for a single conformation, while disfavoring sequences that are intrinsically flexible. In addition, this computational strategy allows us to predict mutations in the CDR H3 loop to stabilize the antigen-bound conformation, a computational mimic of affinity maturation, that may increase antigen binding affinity by pre-organizing the antigen binding loop. In vivo affinity maturation data are consistent with our predictions. The method described here can be useful to design antibodies with higher selectivity and affinity by reducing conformational diversity.
PMCID: PMC3978785  PMID: 19194863
antibody flexibility; computational structural biology; computational design; multi-constraint design; affinity maturation
3.  Conformational flexibility and binding interactions of the G protein βγ heterodimer 
Proteins  2011;79(2):518-527.
Previous NMR experiments on unbound G protein βγ heterodimer suggested that particular residues in the binding interface are mobile on the nanosecond timescale. In this work we performed nanosecond-timescale molecular dynamics simulations to investigate conformational changes and dynamics of Gβγ in the presence of several binding partners: a high-affinity peptide (SIGK), phosducin, and the GDP-bound α subunit. In these simulations, the high mobility of GβW99 was reduced by SIGK, and it appeared that a tyrosine might stabilize GβW99 by hydrophobic or aromatic stacking interactions in addition to hydrogen bonds. Simulations of the phosducin-Gβγ complex showed that the mobility of GβW99 was restricted, consistent with inferences from NMR. However, large-scale conformational changes of Gβγ due to binding, which were hypothesized in the NMR study, were not observed in the simulations, most likely due to their short (nanosecond) duration.
A pocket consisting of hydrophobic amino acids on Gα appears to restrict GβW99 mobility in the crystal structure of the Gαβγ heterotrimer. The simulation trajectories are consistent with this idea. However, local conformational changes of residues GβW63, GβW211, GβW297, GβW332 and GβW339 were detected during the MD simulations. As expected, the magnitude of atomic fluctuations observed in simulations was greater for α than for the βγ subunits, suggesting that α has greater flexibility. These observations support the notion that to maintain the high mobility of GβW99 observed by solution NMR requires that the Gβ−α interface must open up on time scale longer than can be observed in nanosecond scale simulations.
PMCID: PMC3974715  PMID: 21064128
G-protein alpha beta gamma subunits; molecular dynamics; hot spot; subunit interactions
4.  Assessment of 3D models for allergen research 
Proteins  2013;81(4):545-554.
Allergenic proteins must cross-link specific IgE molecules, bound to the surface of mast cells and basophils, to stimulate an immune response. A structural understanding of the allergen-IgE interface is needed to predict cross-reactivities between allergens and to design hypoallergenic proteins. However, there are less than 90 experimentally determined structures available for the approximately 1500 sequences of allergens and isoallergens catalogued in the Structural Database of Allergenic Proteins (SDAP). To provide reliable structural data for the remaining proteins, we previously produced over 500 3D-models using an automated procedure, with strict controls at template choice and model quality evaluation. Here we assessed how well the fold and residue surface exposure of 10 of these models correlated with recently published experimental 3D structures determined by X-ray crystallography or NMR. We also discuss the impact of intrinsically disordered regions on the structural comparison and epitope prediction. Overall, for seven allergens with sequence identities to the original templates higher than 27%, the backbone root-mean square deviations were less than 2Å between the models and the subsequently determined experimental structures for ordered regions. Further, the surface exposure of known IgE epitopes on the models of three major allergens, from peanut (Ara h 1), latex (Hev b 2) and soy (Gly m 4) was very similar to the experimentally determined structures. For three remaining allergens with lower sequence identities to the modeling templates, the 3D folds were correctly identified. However the accuracy of those models is not sufficient for a reliable epitope mapping.
PMCID: PMC3593753  PMID: 23239464
template based modeling; allergenic proteins; IgE epitopes; Structural Database of Allergenic Proteins (SDAP)
5.  Evidence of π-stacking Interactions in the Self-Assembly of hIAPP22–29† 
Proteins  2013;81(4):690-703.
The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self-assembly of hIAPP22–29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe-23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman, and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe-23 virtually abolish the ability of hIAPP22–29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β-sheet secondary structure. TEM images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the -C=C- ring mode (ca. 1600 cm−1) upon aggregation that has previously been shown to be indicative of π-stacking. While previous work has demonstrated that π-stacking is not an absolute requirement for fibrillization, our findings indicate that Phe-23 also contributes to fibril formation through π-stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self-assembly of hIAPP22–29.
PMCID: PMC3594381  PMID: 23229921
islet amyloid polypeptide; self-assembly; phenylalanine; pentafluorophenylalanine; Raman spectroscopy; fluorescence; π-stacking
6.  A Broad Specificity Nucleoside Kinase from Thermoplasma acidophilum 
Proteins  2013;81(4):568-582.
The crystal structure of Ta0880, determined at 1.91 A resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β /α sandwich domain and a smaller lid domain. The overall fold belongs to the PfkB family of carbohydrate kinases (a family member of the Ribokinase clan) which include ribokinases, 1-phosphofructokinases, 6-phosphofructo-2-kinase, inosine/guanosine kinases, frutokinases, adenosine kinases, and many more. Based on its general fold, Ta0880 had been annotated as a ribokinase-like protein. Using a coupled pyruvate kinase/lactate dehydrogenase assay, the activity of Ta0880 was assessed against a variety of ribokinase/pfkB-like family substrates; activity was not observed for ribose, fructose-1-phosphate, or fructose-6-phosphate. Based on structural similarity with nucleoside kinases (NK) from Methanocaldococcus jannaschii (MjNK, PDB 2C49 and 2C4E) and Burkholderia thailandensis (BtNK, PDB 3B1O), nucleoside kinase activity was investigated. Ta0880 (TaNK) was confirmed to have nucleoside kinase activity with an apparent KM for guanosine of 0.21 μM and catalytic efficiency of 345,000 M−1 s−1. These three NKs have significantly different substrate, phosphate donor, and cation specificities and comparisons of specificity and structure identified residues likely responsible for the nucleoside substrate selectivity. Phylogenetic analysis identified three clusters within the PfkB family and indicates that TaNK represents a new sub-family with broad nucleoside specificities.
PMCID: PMC3595323  PMID: 23161756
ribokinase; PfkB-like superfamily; kinetics; structure-function relationship; nucleoside kinase
7.  Solution Structures of Mycobacterium tuberculosis Thioredoxin C and Models of the Intact Thioredoxin System Suggest New Approaches to Inhibitor and Drug Design 
Proteins  2013;81(4):675-689.
Here we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C-terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared to the solution structure. Based on these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti-mycobacterial agents, or as chemical genetic probes of function.
PMCID: PMC3620657  PMID: 23229911
Thioredoxin; Thioredoxin reductase; Mycobacterium tuberculosis; NMR; solution structure
8.  Molecular dynamics simulations of transitions for ECD epidermal growth factor receptors show key differences between human and drosophila forms of the receptors 
Proteins  2013;81(7):1113-1126.
Recent X-ray structural work on the Drosophila epidermal growth factor receptor (EFGR) has suggested an asymmetric dimer that rationalizes binding affinity measurements that go back decades (Alvarado et al., Cell 2010;142:568–579; Dawson et al., Structure 2007;15:942–954; Lemmon et al., Embo J 1997;16:281–294; Mattoon et al., Proc Natl Acad Sci USA 2004;101:923–928; Mayawala et al., Febs Lett 2005;579:3043–3047; Ozcan et al., Proc Natl Acad Sci USA 2006;103:5735–5740). This type of asymmetric structure has not been seen for the human EGF receptor family and it may or may not be important for function in that realm. We hypothesize that conformational changes in the Drosophila system have been optimized for the transition, whereas the barrier for the same transition is much higher in the human forms. To address our hypothesis we perform dynamic importance sampling (DIMS) (Perilla et al., J Comput Chem 2010;32:196–209) for barrier crossing transitions in both Drosophila and human EFGRs. For each set of transitions, we work from the hypothesis, based on results from the AdK system, that salt-bridge pairs making and breaking connections are central to the conformational change. To evaluate the effectiveness of the salt-bridges as drivers for the conformational change, we use the effective transfer entropy based on stable state MD calculations (Kamberaj and Der Vaart, Biophys J 2009;97:1747–1755) to define a reduced subset of degrees of freedom that seem to be important for driving the transition (Perilla and Woolf, J Chem Phys 2012;136:164101). Our results suggest that salt-bridge making and breaking is not the dominant factor in driving the symmetric to asymmetric transition, but that instead it is a result of more concerted and correlated functional motions within a subset of the dimer structures. Furthermore, the analysis suggests that the set of residues involved in the transitions from the Drosophila relative to the human forms differs and that this difference in substate distributions relates to why the asymmetric form may be more common to Drosophila than to the human forms. We close with a discussion about the residues that may be changed in the human and the Drosophila forms to potentially shift the kinetics of the symmetric to asymmetric transition.
PMCID: PMC3968921  PMID: 23348956
epidermal growth factor receptor; molecular dynamics; conformational change; order parameters; extra-cellular domain
9.  Thumb inhibitor binding eliminates functionally important dynamics in the hepatitis C virus RNA polymerase 
Proteins  2012;81(1):40-52.
Hepatitis C virus (HCV) has infected almost 200 million people worldwide, typically causing chronic liver damage and severe complications such as liver failure. Currently, there are few approved treatments for viral infection. Thus, the HCV RNA-dependent RNA polymerase (gene product NS5B) has emerged as an important target for small molecule therapeutics. Potential therapeutic agents include allosteric inhibitors that bind distal to the enzyme active site. While their mechanism of action is not conclusively known, it has been suggested that certain inhibitors prevent a conformational change in NS5B that is crucial for RNA replication. To gain insight into the molecular origin of long-range allosteric inhibition of NS5B, we employed molecular dynamics simulations of the enzyme with and without an inhibitor bound to the thumb domain. These studies indicate that the presence of an inhibitor in the thumb domain alters both the structure and internal motions of NS5B. Principal components analysis identified motions that are severely attenuated by inhibitor binding. These motions may have functional relevance by facilitating interactions between NS5B and RNA template or nascent RNA duplex, with presence of the ligand leading to enzyme conformations with narrower and thus less accessible RNA binding channels. This study provides the first evidence for a mechanistic basis of allosteric inhibition in NS5B. Moreover, we present evidence that allosteric inhibition of NS5B results from intrinsic features of the enzyme free energy landscape, suggesting a common mechanism for the action of diverse allosteric ligands.
PMCID: PMC3943204  PMID: 22855387
allostery; non-nucleoside inhibitor; conformational change; NS5B polymerase; molecular simulation
10.  Structural Insight for the Roles of Fas Death Domain Binding to FADD and Oligomerization Degree of the Fas - FADD complex in the Death Inducing Signaling Complex Formation: A Computational Study 
Proteins  2012;81(3):377-385.
Fas binding to Fas-associated death domain (FADD) activates FADD-caspase-8 binding to form death-inducing signaling complex (DISC) that triggers apoptosis. The Fas-Fas association exists primary as dimer in the Fas-FADD complex and the Fas-FADD tetramer complexes have the tendency to form higher order oligomer. The importance of the oligomerized Fas-FADD complex in DISC formation has been confirmed. This study sought to provide structural insight for the roles of Fas death domain (Fas DD) binding to FADD and the oligomerization of Fas DD-FADD complex in activating FADD-procaspase-8 binding. Results show Fas DD binding to FADD stabilized the FADD conformation, including the increased stability of the critical residues in FADD death effector domain (FADD DED) for FADD-procaspase-8 binding. Fas DD binding to FADD resulted in the decreased degree of both correlated and anti-correlated motion of the residues in FADD and caused the reversed correlated motion between FADD DED and FADD death domain (FADD DD). The exposure of procaspase-8 binding residues in FADD that allows FADD to interact with procaspase-8 was observed with Fas DD binding to FADD. We also observed different degrees of conformational and motion changes of FADD in the Fas DD-FADD complex with different degrees of oligomerization. The increased conformational stability and the decreased degree of correlated motion of the residues in FADD in Fas DD-FADD tetramer complex were observed compared to those in Fas DD-FADD dimer complex. This study provides structural evidence for the roles of Fas DD binding to FADD and the oligomerization degree of Fas DD-FADD complex in DISC formation to signal apoptosis.
PMCID: PMC3556372  PMID: 23042204
Fas-FADD binding; DISC; oligomeric Fas-FADD complex; molecular dynamics; conformational and dynamical motion analysis
11.  Prediction, Refinement and Persistency of Transmembrane Helix Dimers in Lipid Bilayers using Implicit and Explicit Solvent/Lipid Representations: Microsecond Molecular Dynamics Simulations of ErbB1/B2 and EphA1 
Proteins  2012;81(3):365-376.
All-atom simulations are carried out on ErbB1/B2 and EphA1 transmembrane helix dimers in lipid bilayers starting from their solution/DMPC bicelle NMR structures. Over the course of microsecond trajectories, the structures remain in close proximity to the initial configuration and satisfy the great majority of experimental tertiary contact restraints. These results further validate CHARMM protein/lipid force fields and simulation protocols on Anton. Separately, dimer conformations are generated using replica exchange in conjunction with an implicit solvent and lipid representation. The implicit model requires further improvement, and this study investigates whether lengthy all-atom molecular dynamics simulations can alleviate the shortcomings of the initial conditions. The simulations correct many of the deficiencies. For example excessive helix twisting is eliminated over a period of hundreds of nanoseconds. The helix tilt, crossing angles and dimer contacts approximate those of the NMR derived structure, although the detailed contact surface remains off-set for one of two helices in both systems. Hence, even microsecond simulations are not long enough for extensive helix rotations. The alternate structures can be rationalized with reference to interaction motifs and may represent still sought after receptor states that are important in ErbB1/B2 and EphA1 signaling.
PMCID: PMC3557542  PMID: 23042146
structure prediction; implicit solvent and lipid; Generalized Born model; replica exchange; receptor tyrosine kinases; solution NMR
12.  Structural Features of Cholesteryl Ester Transfer Protein: A Molecular Dynamics Simulation Study 
Proteins  2012;81(3):415-425.
Cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl esters from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins or very-low-density lipoproteins. Inhibition of CETP raises high-density lipoprotein cholesterol (good cholesterol) levels and reduces low-density lipoprotein cholesterol (bad cholesterol) levels, making it a promising drug target for the prevention and treatment of coronary heart disease. Although the crystal structure of CETP has been determined, the molecular mechanism mediating cholesteryl esters transfer is still unknown, even the structural features of CETP in a physiological environment remain elusive. We performed molecular dynamics simulations to explore the structural features of CETP in an aqueous solution. Results show that the distal portion flexibility of N-terminal β-barrel domain is considerably greater in solution than in crystal; conversely, the flexibility of helix X is slightly less. During the simulations the distal end of C-terminal β-barrel domain expanded while the hydrophilic surface increasing more than the hydrophobic surface. In addition a new surface pore was generated in this domain. This surface pore and all cavities in CETP are stable. These results suggest that the formation of a continuous tunnel within CETP by connecting cavities is permitted in solution.
PMCID: PMC3557553  PMID: 23042613
CETP; MD simulations; structural flexibility; cavity; hydrophobicity; salt bridge
13.  Crystal Structure of Decaprenylphosphoryl-β-D-Ribose 2’-Epimerase from Mycobacterium smegmatis 
Proteins  2012;81(3):538-543.
Decaprenylphosphoryl-β-D-ribose 2’-epimerase (DprE1) is an essential enzyme in the biosynthesis of cell wall components and a target for development of anti-tuberculosis drugs. We determined the crystal structure of a truncated form of DprE1 from Mycobacterium smegmatis in two crystal forms to up to 2.35 Å resolution. The structure extends from residue 75 to the C-terminus and shares homology with FAD-dependent oxidoreductases of the vanillyl-alcohol oxidase family including the DprE1 homologue from M. tuberculosis. The M. smegmatis DprE1 structure reported here provides further insights into the active site geometry of this tuberculosis drug target.
PMCID: PMC3557725  PMID: 23184707
X-ray crystallography; tuberculosis; benzothiazinone; FAD; oxidoreductase
14.  Starting-structure dependence of nanosecond timescale inter-substate transitions and reproducibility of MD-derived order parameters 
Proteins  2012;81(3):499-509.
Factors affecting the accuracy of molecular dynamics (MD) simulations are investigated by comparing generalized order parameters for backbone NH vectors of the B3 immunoglobulin-binding domain of streptococcal protein G (GB3) derived from simulations with values obtained from NMR spin relaxation (Yao L, Grishaev A, Cornilescu G, Bax A. J Am Chem Soc 2010; 132: 4295–309.). Choices for many parameters of the simulations, such as buffer volume, water model, or salt concentration, have only minor influences on the resulting order parameters. In contrast, seemingly minor conformational differences in starting structures, such as orientations of sidechain hydroxyl groups, resulting from applying different protonation algorithms to the same structure, have major effects on backbone dynamics. Some, but not all, of these effects are mitigated by increased sampling in simulations. Most discrepancies between simulated and experimental results occur for residues located at the ends of secondary structures and involve large amplitude nanosecond timescale transitions between distinct conformational substates. These transitions result in autocorrelation functions for bond vector reorientation that do not converge when calculated over individual simulation blocks, typically of length similar to the overall rotational diffusion time. A test for convergence before averaging the order parameters from different blocks results in better agreement between order parameters calculated from different sets of simulations and with NMR-derived order parameters. Thus, MD-derived order parameters are more strongly affected by transitions between conformational substates than by fluctuations within individual substates themselves, while conformational differences in the starting structures affect the frequency and scale of such transitions.
PMCID: PMC3557728  PMID: 23161667
protein dynamics; molecular dynamics simulation; B3 domain of protein G; NMR spin relaxation; generalized order parameter; starting structure; autocorrelation function; force field; hydrogen bond
15.  Sequence Recombination Improves Target Specificity in a Redesigned Collagen Peptide abc-type Heterotrimer 
Proteins  2012;81(3):386-393.
Stability of the collagen triple helix is largely governed by its imino acid content, namely the occurrence of proline and 4R-hydroxyproline at the X and Y positions respectively of the periodic (Gly-X-Y)n sequence. Although other amino acids at these positions reduce stability of the triple helix, this can be partially compensated by introducing intermolecular side chain salt bridges. This approach was previously used to design an abc-type heterotrimer composed of one basic, one acidic and one neutral imino acid rich chain (Gauba & Hartgerink, 2007). In this study, an abc-type heterotrimer was designed to be the most stable species using a sequence recombination strategy that preserved both the amino acid composition and the network of interchain salt-bridges of the original design. The target heterotrimer had the highest Tm of 50°C, 7°C greater than the next most stable species. Stability of the heterotrimer decreased with increasing ionic strength, consistent with the role of intermolecular salt bridges in promoting stability. Quantitative meta-analysis of these results and published stability measurements on closely related peptides was used to discriminate the contributions of backbone propensity and side chain electrostatics to collagen stability.
PMCID: PMC3557756  PMID: 23042255
backbone propensity; proline; hydroxyproline; electrostatics; salt bridge; folding; circular dichroism; triple-helix; energy landscape
16.  Understanding the Basis of a Class of Paradoxical Mutations in AraC through Simulations 
Proteins  2012;81(3):490-498.
Most mutations at position 15 in the N-terminal arm of the regulatory protein AraC leave the protein incapable of responding to arabinose and inducing the proteins required for arabinose catabolism. Mutations at other positions of the arm do not have this behavior. Simple energetic analysis of the interactions between the arm and bound arabinose do not explain the uninducibility of AraC with mutations at position 15. Extensive molecular dynamics simulations, carried out largely on the Open Science Grid, were done of the wild type protein with and without bound arabinose and of all possible mutations at position 15, many of which were constructed and measured for this work. Good correlation was found for deviation of arm position during the simulations and inducibility as measured in vivo of the same mutant proteins. Analysis of the molecular dynamics trajectories revealed that preservation of the shape of the arm is critical to inducibility. To maintain the correct shape of the arm, the strengths of three interactions observed to be strong in simulations of the wild type AraC protein need to be preserved. These interactions are between arabinose and residue 15, arabinose and residues 8–9, and residue 13 and residue 15.The latter interaction is notable because residues L9, Y13, F15, W95, and Y97 form a hydrophobic cluster which needs to be preserved for retention of the correct shape.
PMCID: PMC3557760  PMID: 23150197
Langevin dynamics; molecular dynamics; hydrophobic cluster; in vivo measurements; AraC protein; gene regulation
17.  Performance of ZDOCK and ZRANK in CAPRI Rounds 13 - 19 
Proteins  2010;78(15):3104-3110.
We report the performance of the ZDOCK and ZRANK algorithms in CAPRI rounds 13-19, and introduce a novel measure Atom Contact Frequency (ACF). To compute ACF, we identify the residues that most often make contact with the binding partner in the complete set of ZDOCK predictions for each target. We used ACF to predict the interface of the proteins, which in combination with biological data available in the literature, is a valuable addition to our docking pipeline. Furthermore, we incorporated a straightforward and efficient clustering algorithm with two purposes: 1) to determine clusters of similar docking poses (corresponding to energy funnels), and 2) to remove redundancies from the final set of predictions. With these new developments, we achieved at least one acceptable prediction for targets 29 and 36, at least one medium quality prediction for targets 41 and 42, and at least one high quality prediction for targets 37 and 40; thus we succeded for six out of a total of 12 targets.
PMCID: PMC3936321  PMID: 20936681
18.  Thermodynamic basis of selectivity in guide-target-mismatched RNA interference 
Proteins  2012;80(5):1283-1298.
Silencing in RNAi is strongly affected by guide-strand/target-mRNA mismatches. Target nucleation is thought to occur at positions 2–8 of the guide (“seed region”); successful hybridization in this region is the primary determinant of target binding affinity and hence target cleavage. To define a molecular basis for the target sequence selectivity in RNAi, we studied all possible distinct single mismatches in seven positions of the seed region — a total of 21 substitutions. We report results from soft-core thermodynamic integration simulations to determine changes in target binding free energies to Argonaute due to single mismatches in the guide strand, which arise during binding of an imperfectly matched target mRNA. In agreement with experiment, most mismatches impair target binding, consistent with a prominent role for binding affinity changes in RNAi sequence selectivity. Individual Argonaute residues located near the mismatched base pair are found to contribute significantly to binding affinity changes. We also employ this methodology to analyze the mismatch-dependent free energy changes for dissociation of a DNA·RNA hybrid from Argonaute, as a model for the escape of miRNAs from the silencing pathway. Several mismatched sequences of the miRNA have increased affinity to Argonaute, implying that some mismatches may reduce the probability for escape. Furthermore, calculations of base-substitution-dependent free energy changes for binding ssDNA reveal mild sequence sensitivity as expected for guide strand binding to Argonaute. Our findings give a thermodynamic basis for RNAi target sequence selectivity and suggest that miRNA mismatches may increase silencing effectiveness and thus could be evolutionarily advantageous.
PMCID: PMC3935976  PMID: 22275138
molecular dynamics; RNAi; Argonaute; guide-target mismatch; binding free energy
19.  Toward optimal fragment generations for ab initio protein structure assembly 
Proteins  2012;81(2):229-239.
Fragment assembly using structural motifs excised from other solved proteins has shown to be an efficient method for ab initio protein-structure prediction. However, how to construct accurate fragments, how to derive optimal restraints from fragments, and what the best fragment length is are the basic issues yet to be systematically examined. In this work, we developed a gapless-threading method to generate position-specific structure fragments. Distance profiles and torsion angle pairs are then derived from the fragments by statistical consistency analysis, which achieved comparable accuracy with the machine-learning-based methods although the fragments were taken from unrelated proteins. When measured by both accuracies of the derived distance profiles and torsion angle pairs, we come to a consistent conclusion that the optimal fragment length for structural assembly is around 10, and at least 100 fragments at each location are needed to achieve optimal structure assembly. The distant profiles and torsion angle pairs as derived by the fragments have been successfully used in QUARK for ab initio protein structure assembly and are provided by the QUARK online server at
PMCID: PMC3551984  PMID: 22972754
ab initio folding; contact prediction; secondary structure prediction; structural fragments
20.  Prediction of phenotypes of missense mutations in human proteins from biological assemblies 
Proteins  2012;81(2):199-213.
Single nucleotide polymorphism (SNPs) are the most frequent variation in the human genome. Non-synonymous SNPs that lead to missense mutations can be neutral or deleterious, and several computational methods have been presented that predict the phenotype of human missense mutations. These methods employ sequence-based and structure-based features in various combinations, relying on different statistical distributions of these features for deleterious and neutral mutations. One structure-based feature that has not been studied significantly is the accessible surface area within biologically relevant oligomeric assemblies. These assemblies are different from the crystallographic asymmetric unit for more than half of X-ray crystal structures. We find that mutations in the core of proteins or in the interfaces in biological assemblies are significantly more likely to be disease-associated than those on the surface of the biological assemblies. For structures with more than one protein in the biological assembly (whether the same sequence or different), we find the accessible surface area from biological assemblies provides a statistically significant improvement in prediction over the accessible surface area of monomers from protein crystal structures (p=6e-5). When adding this information to sequence-based features such as the difference between wildtype and mutant position-specific profile scores, the improvement from biological assemblies is statistically significant but much smaller (p=0.018). Combining this information with sequence-based features in a support vector machine leads to 82% accuracy on a balanced data set of 50% disease-associated mutations from SwissVar and 50% neutral mutations from human/primate sequence differences in orthologous proteins.
PMCID: PMC3552143  PMID: 22965855
missense mutations; phenotype prediction; protein structure; biological assemblies; machine learning
21.  Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the Rous sarcoma virus capsid protein C-terminal domain 
Proteins  2012;81(2):316-325.
An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C-terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y-CTD, F167Y-CTD) and suppressor mutations (R185W-CTD, F167Y-CTD). The stabilities of three double mutant proteins (D155Y/R185W-CTD, F167Y/R185W-CTD and F167Y/I190V-CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT-CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation.
PMCID: PMC3657841  PMID: 23011855
Retroviral capsid protein; protein mutagenesis; protein dimerization; protein stability
22.  HIV-1 Env gp120 Structural Determinants for Peptide Triazole Dual Receptor Site Antagonism 
Proteins  2012;81(2):271-290.
Despite advances in HIV therapy, viral resistance and side-effects with current drug regimens require targeting new components of the virus. Dual antagonist peptide triazoles (PT) are a novel class of HIV-1 inhibitors that specifically target the gp120 component of the viral spike and inhibit its interaction with both of its cell surface protein ligands, namely the initial receptor CD4 and the co-receptor (CCR5/CXCR4), thus preventing viral entry. Following an initial survey of 19 gp120 alanine mutants by ELISA, we screened 11 mutants for their importance in binding to, and inhibition by the PT KR21 using surface plasmon resonance. Key mutants were purified and tested for their effects on the peptide’s affinity and its ability to inhibit binding of CD4 and the co-receptor surrogate mAb 17b. Effects of the mutations on KR21 viral neutralization were measured by single-round cell infection assays. Two mutations, D474A and T257A, caused large-scale loss of KR21 binding, as well as losses in both CD4/17b and viral inhibition by KR21. A set of other Ala mutants revealed more moderate losses in direct binding affinity and inhibition sensitivity to KR21. The cluster of sensitive residues defines a PT functional epitope. This site is in a conserved region of gp120 that overlaps the CD4 binding site and is distant from the co-receptor/17b binding site, suggesting an allosteric mode of inhibition for the latter. The arrangement and sequence conservation of the residues in the functional epitope explain the breadth of antiviral activity, and improve the potential for rational inhibitor development.
PMCID: PMC3731144  PMID: 23011758
entry inhibitor; KR21; dual antagonism; binding site
23.  Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure 
Proteins  2012;80(3):935-945.
Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nano-machine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC-bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.
PMCID: PMC3903301  PMID: 22423359
Shigella; dysentery; invasion plasmid antigens; invasion; bile salts
24.  Crystal structure of SsfS6, the putative C-glycosyltransferase involved in SF2575 biosynthesis 
Proteins  2013;81(7):1277-1282.
The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C-glycosyltransferase (C-GT) that forms the C-C glycosidic bond between the sugar and the SF2575 tetracycline-like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT-B fold wherein the TDP and docked putative aglycon are consistent with the overall C-glycosylation reaction. As one of only a few existing structures for C-glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C-glycosylation.
PMCID: PMC3896073  PMID: 23526584
natural product; antitumor; GT-B fold; X-ray diffraction; molecular docking; biosynthesis; carbohydrate
25.  Native cysteine residues are dispensable for the structure and function of all five yeast mitotic septins 
Proteins  2013;81(11):10.1002/prot.24345.
Budding yeast septins assemble into hetero-octamers and filaments required for cytokinesis. Solvent-exposed cysteine (Cys) residues provide sites for attaching substituents useful in assessing assembly kinetics and protein interactions. To introduce Cys at defined locations, site-directed mutagenesis was used, first, to replace the native Cys residues in Cdc3 (C124 C253 C279), Cdc10 (C266), Cdc11 (C43 C137 C138), Cdc12 (C40 C278), and Shs1 (C29 C148) with Ala, Ser, Val, or Phe. When plasmid-expressed, each Cys-less septin mutant rescued the cytokinesis defects caused by absence of the corresponding chromosomal gene. When integrated and expressed from its endogenous promoter, the same mutants were fully functional, except Cys-less Cdc12 mutants (which were viable, but exhibited slow growth and aberrant morphology) and Cdc3(C124V C253V C279V) (which was inviable). No adverse phenotypes were observed when certain pairs of Cys-less septins were co-expressed as the sole source of these proteins. Cells grew less well when three Cys-less septins were co-expressed, suggesting some reduction in fitness. Nonetheless, cells chromosomally expressing Cys-less Cdc10, Cdc11, and Cdc12, and expressing Cys-less Cdc3 from a plasmid, grew well at 30ºC. Moreover, recombinant Cys-less septins—or where one of the Cys-less septins contained a single Cys introduced at a new site—displayed assembly properties in vitro indistinguishable from wild-type.
PMCID: PMC3880206  PMID: 23775754
Saccharomyces cerevisiae; site-directed mutagenesis; genetic complementation; gene replacement; protein purification; electron microscopy

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