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PubMed2004

Scoring function for automated assessment of protein structure template quality.

We have developed a new scoring function, the template modeling score (TM-score), to assess the quality of protein structure templates and predicted full-length models by extending the approaches used in Global Distance Test (GDT)1 and MaxSub.2 First, a protein size-dependent scale is exploited to eliminate the inherent protein size dependence of the previous scores and appropriately account for random protein structure pairs. Second, rather than setting specific distance cutoffs and calculating only the fractions with errors below the cutoff, all residue pairs in alignment/modeling are evaluated in the proposed score. For comparison of various scoring functions, we have constructed a large-scale benchmark set of structure templates for 1489 small to medium size proteins using the threading program PROSPECTOR_3 and built the full-length models using MODELLER and TASSER. The TM-score of the initial threading alignments, compared to the GDT and MaxSub scoring functions, shows a much stronger correlation to the quality of the final full-length models. The TM-score is further exploited as an assessment of all 'new fold' targets in the recent CASP5 experiment and shows a close coincidence with the results of human-expert visual assessment. These data suggest that the TM-score is a useful complement to the fully automated assessment of protein structure predictions. The executable program of TM-score is freely downloadable at http://bioinformatics.buffalo.edu/TM-score.

Automation↗
PubMed2007

Enzyme-substrate interactions revealed by the crystal structures of the archaeal Sulfolobus PTP-fold phosphatase and its phosphopeptide complexes.

The P-loop-containing protein phos-phatases are important regulators in signal transduction. These enzymes have structural and functional similarity with a conserved sequence of Dx(25-41)HCxxGxxR(T/S) essential for catalysis. The singular protein tyrosine phosphatase (PTP) from archaeal Sulfolobus solfataricus is one of the smallest known PTPs with extreme thermostability. Here, we report the crystal structure of this phosphatase and its complexes with two tyrosyl phosphopeptides A-(p)Y-R and N-K-(p)Y-G-N. The structure suggests the minimal structural motif of the PTP family, having two variable sequences inserted between the beta2-beta3 and beta3-beta4 strands, respectively. The phosphate of both phosphopeptide substrates is bound to the P-loop through several hydrogen bonds. Comparison of several phosphatase-substrate complexes revealed that Gln135 on the Q-loop has different modes of recognition toward phosphopeptides. The substrate specificity of SsoPTP is primarily localized at the phosphotyrosine, suggesting that this phosphatase may be a prototypical PTP.

Amino Acid Sequence↗
PubMed2000

Novel dimerization fold of RAP30/RAP74 in human TFIIF at 1.7 A resolution.

General transcription factor IIF (TFIIF) is required for transcription by RNA polymerase II; it consists minimally of a heterodimer of RNA polymerase-associated proteins RAP30 and RAP74. According to solution and mutagenesis studies, the multiple domains of RAP30 and RAP74 bind PolII, TFIIB, TAF250 and DNA in interactions that are essential for transcription initiation and elongation. The X-ray structure of the RAP30/RAP74 interaction domains at 1.7 A resolution reveals a novel "triple barrel" dimerization fold and suggests with mutant data that interactions with the transcription apparatus are mediated not only by this tripartite beta-barrel, but also via flexible loops and alpha and beta-structures extending from it.

Amino Acid Motifs↗
PubMed2006

Novel allosteric properties produced by residue substitutions in the subunit interface of yeast NAD+-specific isocitrate dehydrogenase.

Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octamer of four IDH1 and four IDH2 subunits, and the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer. To investigate one aspect of the interaction between IDH1 and IDH2, residues in a hydrophobic region at the heterodimer interface (Val-216, Ser-220, and Val-224 in IDH1; Ile-221, Val-225, and Val-229 in IDH2) were replaced by alanine residues in each and in both subunits. Gel filtration and sedimentation velocity analyses demonstrated that the residue substitutions do not disrupt the octameric structure of IDH. However, these substitutions produce novel kinetic properties including, with respect to cofactor, positive allosteric regulation by AMP and cooperativity in the absence of AMP. These allosteric properties are also apparent in NAD+-binding experiments. Despite substantial measurable activity for the mutant enzyme containing residue substitutions in both subunits, expression of this enzyme produces growth phenotypes indicative of IDH dysfunction in vivo.

Amino Acid Sequence↗
PubMed2003

A putative coiled-coil domain of prohibitin is sufficient to repress E2F1-mediated transcription and induce apoptosis.

Prohibitin is a potential tumor suppressor protein that can repress E2F-mediated transcription and arrest cell proliferation. We had shown previously that prohibitin could bind to the Rb protein as well as E2F and this binding was necessary to suppress cell proliferation. Here we show that the E2F1 binding domain of prohibitin has the potential to fold into a coiled-coil structure. This coiled-coil domain by itself could physically interact with E2F1 and block its transcriptional activity. Like full-length prohibitin, the coiled-coil domain also recruited histone deacetylase 1 to repress E2F1. The coiled-coil domain also exhibited growth suppressive properties and we observed a 64% reduction in colony numbers when transfected into T47D cells. Interestingly, a synthetic peptide corresponding to the coiled-coil domain induced apoptosis in four different human cell lines. It is possible that agents that can mimic this peptide would be of value in controlling proliferative disorders.

Apoptosis↗
PubMed2005

Proteins QSAR with Markov average electrostatic potentials.

Classic physicochemical and topological indices have been largely used in small molecules QSAR but less in proteins QSAR. In this study, a Markov model is used to calculate, for the first time, average electrostatic potentials xik for an indirect interaction between aminoacids placed at topologic distances k within a given protein backbone. The short-term average stochastic potential xi1 for 53 Arc repressor mutants was used to model the effect of Alanine scanning on thermal stability. The Arc repressor is a model protein of relevance for biochemical studies on bioorganics and medicinal chemistry. A linear discriminant analysis model developed correctly classified 43 out of 53, 81.1% of proteins according to their thermal stability. More specifically, the model classified 20/28, 71.4% of proteins with near wild-type stability and 23/25, 92.0% of proteins with reduced stability. Moreover, predictability in cross-validation procedures was of 81.0%. Expansion of the electrostatic potential in the series xi0, xi1, xi2, and xi3, justified the use of the abrupt truncation approach, being the overall accuracy >70.0% for xi0 but equal for xi1, xi2, and xi3. The xi1 model compared favorably with respect to others based on D-Fire potential, surface area, volume, partition coefficient, and molar refractivity, with less than 77.0% of accuracy [Ramos de Armas, R.; González-Díaz, H.; Molina, R.; Uriarte, E. Protein Struct. Func. Bioinf.2004, 56, 715]. The xi1 model also has more tractable interpretation than others based on Markovian negentropies and stochastic moments. Finally, the model is notably simpler than the two models based on quadratic and linear indices. Both models, reported by Marrero-Ponce et al., use four-to-five time more descriptors. Introduction of average stochastic potentials may be useful for QSAR applications; having xik amenable physical interpretation and being very effective.

Markov Chains↗
PubMed2006

Dominant-negative suppression of Cav2.1 currents by alpha(1)2.1 truncations requires the conserved interaction domain for beta subunits.

Episodic ataxia type 2 (EA2) is an autosomal dominant disorder arising from CACNA1A mutations, which commonly predict heterozygous expression of Ca(v)2.1 calcium channels with truncated alpha(1)2.1 pore subunits. We hypothesized that alpha(1)2.1 truncations in EA2 exert dominant-negative effects on the function of wild-type subunits. Wild-type and truncated alpha(1)2.1 subunits with fluorescent protein tags were transiently co-expressed in cells stably expressing Ca(v) auxiliary beta subunits, which facilitate alpha1 subunit functional expression through high-affinity interactions with the alpha interaction domain (AID). Co-expression of wild-type subunits with truncations often resulted in severely reduced whole-cell currents compared to expression of wild-type subunits alone. Cellular image analyses revealed that current suppression was not due to reduced wild-type expression levels. Instead, the current suppression depended on truncations terminating distal to the AID. Moreover, only AID-bearing alpha(1)2.1 proteins co-immunoprecipitated with Ca(v) beta subunits. These results indicate that Ca(v) beta subunits may play a prominent role in EA2 disease pathogenesis.

Analysis of Variance↗
PubMed2004

The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily.

The major facilitator superfamily represents the largest group of secondary active membrane transporters in the cell. The 3.3A resolution structure of a member of this protein superfamily, the glycerol-3-phosphate transporter from the Escherichia coli inner membrane, reveals two domains connected by a long central loop. These N- and C-terminal domains, each containing a six-helix bundle, are related by pseudo-twofold symmetry. A substrate translocation pore is located between the two domains and is open to the cytoplasm. Two arginines at the closed end of the pore comprise the substrate-binding site. Biochemical experiments show that, upon substrate binding, the protein adopts a more compact conformation. The crystal structure suggests that the transporter operates through a single binding site, alternating access mechanism via a rocker-switch type of movement of the N- and C-terminal domains. The structure and mechanism of the glycerol-3-phosphate transporter form a paradigm for other members of the major facilitator superfamily.

Binding Sites↗
PubMed2004

Structure and mechanism of ABC transporters.

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substrates across cell or organelle membranes. The recently determined crystal structures of the vitamin B(12) importer BtuCD and its cognate binding protein BtuF have revealed critical architectural features that are probably shared by other ABC transporters. For example, the arrangement of the ABC domains and their interface with the membrane-spanning domains are probably conserved, whereas the number of transmembrane helices and their arrangement are not. Two distinct mechanistic schemes for how ABC engines couple ATP hydrolysis to substrate transport have been proposed recently and are being explored.

ATP-Binding Cassette Transporters↗
PubMed2003

Hexamerization of p97-VCP is promoted by ATP binding to the D1 domain and required for ATPase and biological activities.

The 97-kDa valosin-containing protein (p97-VCP or VCP), a hexameric AAA ATPase, plays an important role in diverse cell activities, including ubiquitin-proteasome mediated protein degradation. In this report, we studied dissociation-reassembly kinetics to analyze the structure-function relationship in VCP. Urea-dissociated VCP can reassemble by itself, but addition of ATP, ADP, or ATP-gamma S accelerates the reassembly. Mutation in the ATP-binding site of D1, but not D2, domain abolishes the ATP acceleration effect and further delays the reassembly. Using hybrid hexamers of the wild type and ATP-binding site mutant, we show that hexameric structure and proper communication among the subunits are required for the ATPase activity and ubiquitin-proteasome mediated degradation. Thus, ATP-binding site in D1 plays a major role in VCP hexamerization, of which proper inter-subunit interaction is essential for the activities.

Adenosine Triphosphatases↗
PubMed2003

The calcium-saturated cTnI/cTnC complex: structure of the inhibitory region of cTnI.

The contiguous inhibitory and regulatory regions of troponin I in the heterotrimeric troponin complex play a critical role in Ca(2+) activation of striated muscle. Knowledge of the structure of this critical region within the complex will enhance efforts toward understanding regulatory mechanisms. Toward this goal, we have used simulated annealing to study the structure of the inhibitory and regulatory regions of cardiac muscle troponin I in the calcium-saturated complex formed between cardiac troponin C and cardiac troponin I. We have incorporated distances determined experimentally by Förster resonance energy transfer in the full-length complex, rather than using peptides derived from cTnI. For these models, we assume a helix-loop-helix conformation for the inhibitory region. We have found several structures that satisfy the experimental constraints fairly well. Although it is not possible to eliminate any of these models at this time, future studies with additional experimental restraints will yield insights on the mechanisms of calcium regulation in cardiac muscle.

Amino Acid Sequence↗
PubMed2003

Functional activities and cellular localization of the ezrin, radixin, moesin (ERM) and RING zinc finger domains in MIR.

Myosin regulatory light chain interacting protein (MIR) belongs to the ezrin, radixin, moesin (ERM) family of proteins involved in membrane cytoskeleton interactions and cell dynamics. MIR contains, beside the ERM domain, a RING zinc finger region. Immunocytochemistry showed that full-length MIR and the subdomains localize differently in cells. Cell fractionation revealed a similar distribution of full-length MIR and the RING domain protein in the Triton X-100-insoluble fraction. The neurite outgrowth inhibitory activity of MIR was attributed to the RING domain. MIR levels were controlled in the cells depending on the intact RING domain and proteasome activity. The dynamic regulation of MIR contributes to its effects on neurite outgrowth and cell motility.

Animals↗
PubMed2001

Functional specificity of actin isoforms.

Actin, one of the main proteins of muscle and cytoskeleton, exists as a variety of highly conserved isoforms whose distribution in vertebrates is tissue-specific. Synthesis of specific actin isoforms is accompanied by their subcellular compartmentalization, with both processes being regulated by factors of cell proliferation and differentiation. Actin isoforms cannot substitute for each other, and the high-level synthesis of exogenous actins leads to alterations in cell organization and morphology. This indicates that the highly conserved actins are functionally specialized for the tissues in which they predominate. The first goal of this review is to analyze the data on the polymerizability of actin isoforms to show that cytoskeleton isoactins form less stable polymers than skeletal muscle actin. This difference correlates with the dynamics of actin microfilaments versus the stability of myofibrillar systems. The three-dimensional actin structure as well as progress in the analysis of conformational changes in both the actin monomer and the filament allows us to view the data on the structure and polymerization of isoactins in terms of structure-function relationships within the actin molecule. Most of the amino acid substitutions that distinguish actin isoforms are located apart from actin-actin contact sites in the polymer. We suggest that these substitutions can modulate the ability of actin monomers to form more or less stable polymers by long-range (allosteric) regulation of the contact sites.

Actins↗
PubMed2003

Identification of essential amino acids in Humanin, a neuroprotective factor against Alzheimer's disease-relevant insults.

Humanin (HN) is a secretory peptide that inhibits neurotoxicity by various Alzheimer's disease-relevant insults. We have so far identified that the substitution of Leu9 for Arg nullifies the extracellular secretion of HN. Here we comprehensively investigate the amino acid requirement of HN essential for its secretion and for its neuroprotective function. Intracellulary expressed HN-EGFP (EGFP N-terminally fused with HN) was extracellularly secreted, whereas neither EGFP nor (L9R)HN-EGFP was secreted at all. While Ala substitution of neither residue affected HN secretion, Arg substitution revealed that the two structures-Leu9-Leu11 and Pro19-Va120-were essential for the secretion of full-length HN. In the Leu9-Leu11 domain, the Leu10 residue turned out to play a central role in this function, because the Asp substitution of Leu10, but not Leu9 or Leu11, nullified the secretion of HN. Utilizing Ala-scanned HN constructs, we also investigated a comprehensive structure-function relationship for the neuroprotective function of full-length HN, which revealed (i) that Pro3, Ser7, Cys8, Leu9, Leu12, Thr13, Ser14, and Pro19 were essential for this function and (ii) that Ser7 and Leu9 were essential for self-dimerization of HN. These findings indicate that HN has activity similar to a signal peptide, for which the Leu9-Leu11 region, particularly Leu10, functions as a core domain, and suggest that self-dimerization of HN is a process essential for its neuroprotective function.

Alanine↗
PubMed2003

Protein structural alignment for detection of maximally conserved regions.

An algorithm for comparison of homologous protein structures and for study of conformational changes in proteins, has been developed. The method is based on identification of pieces of the two molecules that have similar shapes, as determined by the local conformation of the polypeptide chain. Pieces that superpose within a specified tolerance are assembled into domains based on similar transformations for superposition. The result is sets of pieces that represent conserved structural elements and conserved spatial relationships between structural elements within the proteins being compared. A similarity criterion based on maximum distance rather than on root mean square deviation reduces bias by outliers. The utility of the method is demonstrated by using examples from the protein kinase family.

Algorithms↗
PubMed2003

Mapping functional residues onto integrin crystal structures.

Historical data, available in the literature, concerning the sites responsible for integrin-ligand binding and activation may now be transposed onto recently solved crystal structures of integrin dimers and functional domains. The data were obtained through studies of natural and engineered mutations within integrins, and analyses of the epitopes for function-altering monoclonal antibodies. In the past two years, this combining of structure and function has led to the development of novel hypotheses for the mechanisms of integrin regulation.

Binding Sites↗