Proteins: Structure, Function, Bioinformatics
Dynamics on human Toll-like receptor 4 complexation to MD-2: The coreceptor stabilizing function
The interaction between human Toll-like receptor 4 (hTLR4) and its coreceptor, myeloid differentiation factor 2 (MD-2), is important in Gram-negative bacteria lipopolysaccharide (LPS) recognition. In this process, MD-2 recognizes LPS and promotes the dimerization of the complex hTLR4–MD-2–LPS, triggering an intracellular immune signaling. In this study, we employed distinct computational methods to explore the dynamical properties of the hTLR4–MD-2 complex and investigated the implications of the coreceptor complexation to the structural biology of hTLR4. We characterized both global and local dynamics of free and MD-2 complexed hTLR4, in both (hTLR4–MD-2)1 and (hTLR4–MD-2)2 states. Both molecular dynamics and normal mode analysis reveled a stabilization of the terminal regions of hTLR4 upon complexation to MD-2. We are able to identify conserved important residues involved on the hTLR4–MD-2 interaction dynamics and disclose C-terminal motions that may be associated to the signaling process upon oligomerization. Proteins 2015; 83:373–382. © 2014 Wiley Periodicals, Inc.
Hierarchical domain-motion analysis of conformational changes in sarcoplasmic reticulum Ca2+-ATPase
Sarco(endo)plasmic reticulum Ca2+-ATPase transports two Ca2+ per ATP-hydrolyzed across biological membranes against a large concentration gradient by undergoing large conformational changes. Structural studies with X-ray crystallography revealed functional roles of coupled motions between the cytoplasmic domains and the transmembrane helices in individual reaction steps. Here, we employed ‘Motion Tree (MT)', a tree diagram that describes a conformational change between two structures, and applied it to representative Ca2+-ATPase structures. MT provides information of coupled rigid-body motions of the ATPase in individual reaction steps. Fourteen rigid structural units, ‘common rigid domains (CRDs)' are identified from seven MTs throughout the whole enzymatic reaction cycle. CRDs likely act as not only the structural units, but also the functional units. Some of the functional importance has been newly revealed by the analysis. Stability of each CRD is examined on the morphing trajectories that cover seven conformational transitions. We confirmed that the large conformational changes are realized by the motions only in the flexible regions that connect CRDs. The Ca2+-ATPase efficiently utilizes its intrinsic flexibility and rigidity to response different switches like ligand binding/dissociation or ATP hydrolysis. The analysis detects functional motions without extensive biological knowledge of experts, suggesting its general applicability to domain movements in other membrane proteins to deepen the understanding of protein structure and function. This article is protected by copyright. All rights reserved.
What are the key building blocks that would have been needed to construct complex protein folds? This is an important issue for understanding protein folding mechanism and guiding de novo protein design. Twenty naturally occurring amino acids and eight secondary structures consist of a 28-letter alphabet to determine folding kinetics and mechanism. Here we predict folding kinetic rates of proteins from many reduced alphabets. We find that a reduced alphabet of 10 letters achieves good correlation with folding rates, close to the one achieved by full 28-letter alphabet. Many other reduced alphabets are not significantly correlated to folding rates. The finding suggests that not all amino acids and secondary structures are equally important for protein folding. The foldable sequence of a protein could be designed using at least ten folding units, which can either promote or inhibit protein folding. Reducing alphabet cardinality without losing key folding kinetic information opens the door to potentially faster machine learning and data mining applications in protein structure prediction, sequence alignment and protein design. This article is protected by copyright. All rights reserved.
Inferring the microscopic surface energy of protein-protein interfaces from mutation data
Mutations at protein-protein recognition sites alter binding strength by altering the chemical nature of the interacting surfaces. We present a simple surface energy model, parameterised with empirical values, yielding mean energies of -48 cal.mol– 1.Å– 2 for interactions between hydrophobic surfaces, -51 to -80 cal.mol– 1.Å– 2 for surfaces of complementary charge, and 66 to 83 cal.mol– 1.Å– 2 for electrostatically repelling surfaces, relative to the aqueous phase. This places the mean energy of hydrophobic surface burial at -24 cal.mol– 1.Å– 2. Despite neglecting configurational entropy and intramolecular changes, the model correlates with empirical binding free energies of a functionally diverse set of rigid-body interactions (r=0.66). When used to rerank docking poses, it can place near-native solutions in the top 10 for 37% of the complexes evaluated, and 82% in the top 100. The method shows that hydrophobic burial is the driving force for protein association, accounting for 50-95% of the cohesive energy. The model is available open-source from http://life.bsc.es/pid/web/surface_energy/and via the CCharpPPI web server http://life.bsc.es/pid/ccharppi/. This article is protected by copyright. All rights reserved.
Identification a novel carbohydrate esterase from Bjerkandera adusta: Structural and function predictions through bioinformatics analysis and molecular modeling
A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide.
Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three-dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p-nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U/mg protein on 2-naphthyl acetate. No activity was detected on p-nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N-acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin. This article is protected by copyright. All rights reserved.
Evaluation of conformational changes in diabetes-associated mutation in insulin A chain: A molecular dynamics study
Insulin plays a central role in the regulation of metabolism in humans. Mutations in the insulin gene can impair the folding of its precursor protein, proinsulin, and cause permanent neonatal-onset diabetes mellitus known as Mutant INS-gene induced Diabetes of Youth (MIDY) with insulin deficiency. To gain insights into the molecular basis of this diabetes-associated mutation, we perform molecular dynamics simulations in wild-type and mutant (CysA7 to Tyr or C(A7)Y) insulin A chain in aqueous solutions. The C(A7)Y mutation is one of the identified mutations that impairs the protein folding by substituting the cysteine residue which is required for the disulfide bond formation. A comparative analysis reveals structural differences between the wild-type and the mutant conformations. The analyzed mutant insulin A chain forms a metastable state with major effects on its N-terminal region. This suggests that MIDY mutant involves formation of a partially folded intermediate with conformational change in N-terminal region in A chain that generates flexible N-terminal domain. This may lead to the abnormal interactions with other proinsulins in the aggregation process. This article is protected by copyright. All rights reserved.
Molecular Dynamics Investigation of the Ionic Liquid/Enzyme Interface: Application to Engineering Enzyme Surface Charge
Molecular simulations of the enzymes Candida rugosa lipase and Bos taurus α-chymotrypsin in aqueous ionic liquids 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium ethyl sulfate were used to study the change in enzyme-solvent interactions induced by modification of the enzyme surface charge. The enzymes were altered by randomly mutating lysine surface residues to glutamate, effectively decreasing the net surface charge by two for each mutation. These mutations resemble succinylation of the enzyme by chemical modification, which has been shown to enhance the stability of both enzymes in ILs. After establishing that the enzymes were stable on the simulated time scales, we focused the analysis on the organization of the ionic liquid substituents about the enzyme surface. Calculated solvent charge densities show that for both enzymes and in both solvents that changing positively charged residues to negative charge does indeed increase the charge density of the solvent near the enzyme surface. The radial distribution of IL constituents with respect to the enzyme reveals decreased interactions with the anion are prevalent in the modified systems when compared to the wild type, which is largely accompanied by an increase in cation contact. Additionally, the radial dependence of the charge density and ion distribution indicates that the effect of altering enzyme charge is confined to short range (≤ 1nm) ordering of the IL. Ultimately, these results, which are consistent with that from prior experiments, provide molecular insight into the effect of enzyme surface charge on enzyme stability in ILs. This article is protected by copyright. All rights reserved.
Conserved movement of TMS11 between occluded conformations of LacY and XylE of the Major Facilitator Superfamily suggests a similar hinge-like mechanism
Δ-distance maps can detect local remodeling that is difficult to accurately determine using superimpositions. TMSs 11 in both LacY and XylE of the Major Facilitator Superfamily (MFS) uniquely contribute the greatest amount of mobile surface area in the outward occluded state, and undergo analogous movements. The intracellular part of TMS11 moves away from the C-terminal domain and into the substrate cavity during the conformational change from the outward occluded to the inward occluded state. Upon releasing the substrate to the inside and assuming the inward open state, a difference was noted between LacY and XylE where TMS11 of LacY moved further into the substrate release space whereas in XylE, TMS11 slightly retracted into the C-terminal domain. Independent movement of the N-terminal half of TMS11 suggests that it is flexible in the middle. Repeat-swapped homology modeling was used to discover that a loop connecting TMSs 10 and 11 in LacY probably moves during the transition between the yet to be solved outward open state and the outward occluded state. TMSs 11 and the other elements displaying a notable domain-independent movement colocalize with the interdomain linker, suggesting that these elements could drive the alternating access movement between the domain halves. Preliminary evidence indicates that analogous movements occur in other members of the MFS. This article is protected by copyright. All rights reserved.
The crystal structure of the catalytic domain of the Ser/Thr kinase PknA from M. tuberculosis shows an Src-like autoinhibited conformation
Signal transduction mediated by Ser/Thr phosphorylation in Mycobacterium tuberculosis has been intensively studied in the last years, as its genome harbors eleven genes coding for eukaryotic-like Ser/Thr kinases. Here we describe the crystal structure and the autophosphorylation sites of the catalytic domain of PknA, one of two protein kinases essential for pathogen's survival. The structure of the ligand-free kinase domain shows an auto-inhibited conformation similar to that observed in human Tyr kinases of the Src-family. These results reinforce the high conservation of structural hallmarks and regulation mechanisms between prokaryotic and eukaryotic protein kinases. This article is protected by copyright. All rights reserved.
Structure of the terminal PCP domain of the non-ribosomal peptide synthetase in teicoplanin biosynthesis
The biosynthesis of the glycopeptide antibiotics, of which teicoplanin and vancomycin are representative members, relies on the combination of non-ribosomal peptide synthesis and modification of the peptide by cytochrome P450 (Oxy) enzymes while the peptide remains bound to the peptide synthesis machinery. We have structurally characterized the final peptidyl carrier protein domain of the teicoplanin non-ribosomal peptide synthetase machinery: this domain is believed to mediate the interactions with tailoring Oxy enzymes in addition to its function as a shuttle for intermediates between multiple non-ribosomal peptide synthetase domains. Using solution state NMR, we have determined structures of this PCP domain in two states, the apo and the post-translationally modified holo state, both of which conform to a four-helix bundle assembly. The structures exhibit the same general fold as the majority of known carrier protein structures, in spite of the complex biosynthetic role that PCP domains from the final non-ribosomal peptide synthetase module must play in glycopeptide antibiotic biosynthesis. These structures thus support the hypothesis that it is subtle rearrangements, rather than dramatic conformational changes, which govern carrier protein interactions and selectivity during non-ribosomal peptide synthesis. This article is protected by copyright. All rights reserved.
The antigen binding site of antibodies forms at the interface of their two variable domains, VH and VL, making VH-VL domain orientation a factor that codetermines antibody specificity and affinity. Preserving VH-VL domain orientation in the process of antibody engineering is important in order to retain the original antibody properties, and predicting the correct VH-VL orientation has also been recognized as an important factor in antibody homology modeling. In this article, we present a fast sequence-based predictor that predicts VH-VL domain orientation with Q2 values ranging from 0.54 to 0.73 on the evaluation set. We describe VH-VL orientation in terms of the six absolute ABangle parameters that have recently been proposed as a means to separate the different degrees of freedom of VH-VL domain orientation. In order to assess the impact of adjusting VH-VL orientation according to our predictions, we use the set of antibody structures of the recently published Antibody Modeling Assessment II (AMAII) study. In comparison to the original AMAII homology models, we find an improvement in the accuracy of VH-VL orientation modeling, which also translates into an improvement in the average root-mean-square deviation (RMSD) with regard to the crystal structures. This article is protected by copyright. All rights reserved.
Constructing a model of a query protein based on its alignment to a homolog with experimentally determined spatial structure (the template) is still the most reliable approach to structure prediction. Alignment errors are the main bottleneck for homology modeling when the query is distantly related to the template. Alignment methods often misalign secondary structural elements by a few residues. Therefore, better alignment solutions can be found within a limited set of local shifts of secondary structures. We present a refinement method to improve pairwise sequence alignments by evaluating alignment variants generated by local shifts of template-defined secondary structures. Our method SFESA is based on a novel scoring function that combines the profile-based sequence score and the structure score derived from residue contacts in a template. Such a combined score frequently selects a better alignment variant among a set of candidate alignments generated by local shifts and leads to overall increase in alignment accuracy. Evaluation of several benchmarks shows that our refinement method significantly improves alignments made by automatic methods such as PROMALS, HHpred and CNFpred. The web server is available at http://prodata.swmed.edu/sfesa. Proteins 2014. © 2014 Wiley Periodicals, Inc.
Conformational dynamics of nonsynonymous variants at protein interfaces reveals disease association
Recent studies have shown that the protein interface sites between individual monomeric units in biological assemblies are enriched in disease-associated non-synonymous single nucleotide variants (nsSNVs). To elucidate the mechanistic underpinning of this observation, we investigated the conformational dynamic properties of protein interface sites through a site-specific structural dynamic flexibility metric (dfi) for 333 multimeric protein assemblies. dfi measures the dynamic resilience of a single residue to perturbations that occurred in the rest of the protein structure and identifies sites contributing the most to functionally critical dynamics. Analysis of dfi profiles of over a thousand positions harboring variation revealed that amino acid residues at interfaces have lower average dfi (31%) than those present at non-interfaces (50%), which means that protein interfaces have less dynamic flexibility. Interestingly, interface sites with disease-associated nsSNVs have significantly lower average dfi (23%) as compared to those of neutral nsSNVs (42%), which directly relates structural dynamics to functional importance. We found that less conserved interface positions show much lower dfi for disease nsSNVs as compared to neutral nsSNVs. In this case, dfi is better as compared to the accessible surface area metric, which is based on the static protein structure. Overall, our proteome-wide conformational dynamic analysis indicates that certain interface sites play a critical role in functionally related dynamics (i.e., those with low dfi values), therefore mutations at those sites are more likely to be associated with disease. Proteins 2014. © 2014 Wiley Periodicals, Inc.
Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa
The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright “on” and dark “off” states via photoisomerization and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0-Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand β8 containing Thr159 as well as an altered A-C dimer interface involving strands β7, β8, β10, and β11. The Met159Thr mutation increases the cavity volume for the p-hydroxybenzylidene-imidazolinone chromophore as a result of both the side chain difference and the backbone positional differences. Proteins 2014. © 2014 Wiley Periodicals, Inc.
During CASP10 in summer 2012 we tested BCL::Fold for prediction of free modeling (FM) and template-based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11 we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold's ability to sample the native topology, identify native-like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE-only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native-like assembly of SSEs for further refinement and submission. It was also observed that for some β-strand proteins model refinement failed as β-strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non-natural topologies that require loop regions to pass through the center of the protein. This article is protected by copyright. All rights reserved.
Characterization of the differences in the cyclopiazonic acid binding mode to mammalian and P. falciparum Ca2+ pumps: a computational study
Despite the investments in malaria research, an effective vaccine has not yet been developed and the causative parasites are becoming increasingly resistant to most of the available drugs. PfATP6, the sarco/endoplasmic reticulum Ca2+ pump (SERCA) of P. falciparum, has been recently genetically validated as a potential antimalarial target and cyclopiazonic acid (CPA) has been found to be a potent inhibitor of SERCAs in several organisms, including P. falciparum. In position 263, PfATP6 displays a leucine residue, whilst the corresponding position in the mammalian SERCA is occupied by a glutamic acid. The PfATP6L263E mutation has been studied in relation to the artemisinin inhibitory effect on P. falciparum and recent studies have provided evidence that the parasite with this mutation is more susceptible to CPA. Here, we characterized, for the first time, the interaction of CPA with PfATP6 and its mammalian counterpart in order to understand similarities and differences in the mode of binding of the inhibitor to the two Ca2+ pumps. We found that, even though CPA does not directly interact with the residue in position 263, the presence of a hydrophobic residue in this position in PfATP6 rather than a negatively charged one, as in the mammalian SERCA, entails a conformational arrangement of the binding pocket which, in turn, determines a relaxation of CPA leading to a different binding mode of the compound. Our findings highlight differences between the plasmodial and human SERCA CPA-binding pockets that may be exploited to design CPA derivatives more selective towards PfATP6. This article is protected by copyright. All rights reserved.
The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. Proteins 2015; 83:389–394. © 2014 Wiley Periodicals, Inc.
Methyltransferases do not work by compression, cratic, or desolvation effects, but by electrostatic preorganization
The enzyme catechol O-methyltransferase (COMT) catalyzes the transfer of a methyl group from S-adenosylmethionine to dopamine and related catechols. The search for the origin of COMT catalysis has led to different proposals and hypothesis, including the entropic, the NAC, and the compression proposals as well as the more reasonable electrostatic idea. Thus, it is important to understand the catalytic power of this enzyme and to examine the validity of different proposals and in particular the repeated recent implication of the compression idea. The corresponding analysis should be done by well-defined physically-based considerations that involve computations rather than circular interpretations of experimental results. Thus, we explore here the origin of the catalytic efficiency of COMT by using the empirical valence bond and the linear response approximation approaches. The results demonstrate that the catalytic effect of COMT is mainly due to electrostatic preorganization effects. It is also shown that the compression, NAC and entropic proposals do not account for the catalytic effect. Proteins 2015; 83:318–330. © 2014 Wiley Periodicals, Inc.
Molecular mechanisms and design principles for promiscuous inhibitors to avoid drug resistance: Lessons learned from HIV-1 protease inhibition
Molecular recognition is central to biology and ranges from highly selective to broadly promiscuous. The ability to modulate specificity at will is particularly important for drug development, and discovery of mechanisms contributing to binding specificity is crucial for our basic understanding of biology and for applications in health care. In this study, we used computational molecular design to create a large dataset of diverse small molecules with a range of binding specificities. We then performed structural, energetic, and statistical analysis on the dataset to study molecular mechanisms of achieving specificity goals. The work was done in the context of HIV-1 protease inhibition and the molecular designs targeted a panel of wild-type and drug-resistant mutant HIV-1 protease structures. The analysis focused on mechanisms for promiscuous binding to bind robustly even to resistance mutants. Broadly binding inhibitors tended to be smaller in size, more flexible in chemical structure, and more hydrophobic in nature compared to highly selective ones. Furthermore, structural and energetic analyses illustrated mechanisms by which flexible inhibitors achieved binding; we found ligand conformational adaptation near mutation sites and structural plasticity in targets through torsional flips of asymmetric functional groups to form alternative, compensatory packing interactions or hydrogen bonds. As no inhibitor bound to all variants, we designed small cocktails of inhibitors to do so and discovered that they often jointly covered the target set through mechanistic complementarity. Furthermore, using structural plasticity observed in experiments, and potentially in simulations, is suggested to be a viable means of designing adaptive inhibitors that are promiscuous binders. Proteins 2015; 83:351–372. © 2014 Wiley Periodicals, Inc.
The structure of S. lividans acetoacetyl-CoA synthetase shows a novel interaction between the C-terminal extension and the N-terminal domain
The adenosine monoposphate-forming acyl-CoA synthetase enzymes catalyze a two-step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ∼110 residue C-terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl-CoA synthetase (AacS) is presented that illustrates a novel aspect of this C-terminal domain. Specifically, several acetyl- and acetoacetyl-CoA synthetases contain a 30-residue extension on the C-terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N-terminal domain. Proteins 2014. © 2014 Wiley Periodicals, Inc.