Assessment of the ability to model proteins with leucine-rich repeats in light of the latest structural information

The three-dimensional structures of leucine-rich repeat (LRR) -containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor. using an approach that combined homology-based modeling, structure-based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general. cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, interior/exterior orientations of side chains. and backbone conformation of the LRR structures can be predicted correctly. On the other hand. the analysis revealed that, in some cases. it is difficult to predict correctly the twist of the overall super-helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.

Characterization of the retinoid orphan-related receptor-alpha coactivator binding interface: A structural basis for ligand-independent transcription

The retinoid orphan-related receptor-alpha (RORalpha) is a member of the ROR subfamily of orphan receptors and acts as a constitutive activator of transcription in the absence of exogenous ligands. To understand the basis of this activity, we constructed a homology model of Rill using the closely related TRbeta as a template. Molecular modeling suggested that bulky hydrophobic side chains occupy the RORa ligand cavity leaving a small but distinct cavity that may be involved in receptor stabilization. This model was subject to docking simulation with a receptor-interacting peptide from the steroid receptor coactivator, GR-interacting protein-1, which delineated a coactivator binding surface consisting of the signature motif spanning helices 3-5 and helix 12 [activation function 2 (AF2)]. Probing this surface with scanning alanine mutagenesis showed structural and functional equivalence between homologous residues of RORalpha and TRbeta. This was surprising (given that Rill is a ligand-independent activator, whereas TRbeta has an absolute requirement for ligand) and prompted us to use molecular modeling to identify differences between Rill and TRbeta in the way that the All helix interacts with the rest of the receptor. Modeling highlighted a nonconserved amino acid in helix 11 of RORa (Phe491) and a short-length of 3.10 helix at the N terminus of AF2 which we suggest i) ensures that AF2 is locked permanently in the holoconformation described for other liganded receptors and thus 2) enables ligand-independent recruitment of coactivators. Consistent with this, mutation of RORa Phe491 to either methionine or alanine (methionine is the homologous residue in TRbeta), reduced and ablated transcriptional activation and recruitment of coactivators, respectively. Furthermore, we were able to reconstitute transcriptional activity for both a deletion mutant of Ill lacking All and Phe491 Met, by overexpression of a GAL-AF2 fusion protein, demonstrating ligand-independent recruitment of AF2 and a role for Phe491 in recruiting AF2.

Insights into the structural basis for zinc inhibition of the glycine receptor

Histidines 107 and 109 in the glycine receptor ( GlyR) alpha(1) subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha(1) subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His(107) from one subunit and His(109) from an adjacent subunit. This was tested by co-expressing alpha(1) subunits containing the H107A mutation with alpha(1) subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha(1) homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha(1) subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha(1)beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha(1) homomers. The binding of zinc at the interface between adjacent alpha(1) subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

Structural basis and prediction of substrate specificity in protein serine/threonine kinases

The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally. Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input. We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways. Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serine/threonine kinases and helps in silico construction of signaling pathways. The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches.

Sexual dimorphism of the femoral neck during the adolescent growth spurt: a structural analysis

Before puberty, there are only small sex differences in body shape and composition. During adolescence, sexual dimorphism in bone, lean, and fat mass increases, giving rise to the greater size and strength of the male skeleton. The question remains as to whether there are sex differences in bone strength or simply differences in anthropometric dimensions. To test this, we applied hip structural analysis (HSA) to derive strength and geometric indices of the femoral neck using bone densitometry scans (DXA) from a 6-year longitudinal study in Canadian children. Seventy boys and sixty-eight girls were assessed annually for 6 consecutive years. At the femoral neck, cross-sectional area (CSA, an index of axial strength), subperiosteal width (SPW), and section modulus (Z, an index of bending strength) were determined, and data were analyzed using a hierarchical (random effects) modeling approach. Biological age (BA) was defined as years from age at peak height velocity (PHV). When BA, stature, and total-body lean mass (TB lean) were controlled, boys had significantly higher Z than girls at all maturity levels (P < 0.05). Controlling height and TB lean for CSA demonstrated a significant independent sex by BA interaction effect (P < 0.05). That is, CSA was greater in boys before PHV but higher in girls after PHV The coefficients contributing the greatest proportion to the prediction of CSA, SPW, and Z were height and lean mass. Because the significant sex difference in Z was relatively small and close to the error of measurement, we questioned its biological significance. The sex difference in bending strength was therefore explained by anthropometric differences. In contrast to recent hypotheses, we conclude that the CSA-lean ratio does not imply altered mechanosensitivity in girls because bending dominates loading at the neck, and the Z-lean ratio remained similar between the sexes throughout adolescence. That is, despite the greater CSA in girls, the bone is strategically placed to resist bending; hence, the bones of girls and boys adapt to mechanical challenges in a similar way. (C) 2004 Elsevier Inc. All rights reserved.

Modeling of hardening and softening processes in Mg alloys

The tensile deformation behavior of a range of supersaturated Mg-Al solid solutions and an as-cast magnesium alloy AM60 has been studied. The Mg-Al alloys were tested at room temperature while the alloy AM60 was tested in the temperature range 293-573 K. The differences in the deformation behavior of the alloys is discussed in terms of hardening and softening processes. In order to identify which processes were active, the stress dependence of the strain-hardening coefficient was assessed using Lukac and Balik's model of hardening and softening. The analysis indicates that hardening involves solid solution hardening and interaction with forest dislocations and non-dislocation obstacles such as second phase particles. Cross slip is not a significant recovery process in the temperature range 293-423 K. At temperatures between 473 and 523 K the analysis suggests that softening is controlled by cross slip and climb of dislocations. At temperatures above 523 K softening seems to be controlled by dynamic recrystallisation. (C) 2004 Elsevier B.V. All rights reserved.

Modeling the relationships between adult attachment patterns and borderline personality disorder: The role of impulsivity and aggressiveness

To obtain a better understanding of the associations among Borderline Personality Disorder (BPD), adult attachment patterns, impulsivity, and aggressiveness, we tested four competing models of these relationships: a) BPD is associated with the personality traits of impulsivity and aggressiveness, but adult attachment patterns predict neither BPD nor impulsive/aggressive features; b) adult attachment patterns are significant predictors of BPD but not of impulsive/aggressive traits, although these traits correlate with BPD; c) adult attachment patterns are significant predictors of impulsive and aggressive traits, which in turn predict BPD; and d) adult attachment patterns significantly predict both BPD and impulsive/aggressive traits. We assessed 466 consecutively admitted outpatients using the Structured Clinical Interview for DSM-IV Axis II Personality Disorders (V. 2.0), the Attachment Style Questionnaire, the Barratt Impulsiveness Scale-11, and the Aggression Questionnaire. Maximum likelihood structural equation modeling of the covariance matrix showed that model (c) was the best fitting model (chi(2) (21) = 31.67, p >.05, RMSEA = .023, test of close fit p >.85). This result indicates that adult attachment patterns act indirectly as risk factors for BPD because of their relationships with aggressive/impulsive personality traits.

Off-line signature verification and forgery detection using fuzzy modeling

Automatic signature verification is a well-established and an active area of research with numerous applications such as bank check verification, ATM access, etc. This paper proposes a novel approach to the problem of automatic off-line signature verification and forgery detection. The proposed approach is based on fuzzy modeling that employs the Takagi-Sugeno (TS) model. Signature verification and forgery detection are carried out using angle features extracted from box approach. Each feature corresponds to a fuzzy set. The features are fuzzified by an exponential membership function involved in the TS model, which is modified to include structural parameters. The structural parameters are devised to take account of possible variations due to handwriting styles and to reflect moods. The membership functions constitute weights in the TS model. The optimization of the output of the TS model with respect to the structural parameters yields the solution for the parameters. We have also derived two TS models by considering a rule for each input feature in the first formulation (Multiple rules) and by considering a single rule for all input features in the second formulation. In this work, we have found that TS model with multiple rules is better than TS model with single rule for detecting three types of forgeries; random, skilled and unskilled from a large database of sample signatures in addition to verifying genuine signatures. We have also devised three approaches, viz., an innovative approach and two intuitive approaches using the TS model with multiple rules for improved performance. (C) 2004 Pattern Recognition Society. Published by Elsevier Ltd. All rights reserved.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.