Structural insights into the activation of the RhoA GTPase by the lymphoid blast crisis (Lbc) oncoprotein.

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and (1)H,(13)C,(15)N-based NMR spectroscopy. The two domains assume a "chair" shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.

The Nursing Home Falls Self-Efficacy Scale: development and testing.

We examined a brief measure of falls self-efficacy in nursing home residents participating in a pilot randomized controlled trial to study the effects of hip protectors on the prevention of fractures (N = 116, mean age 82 ± 8, 72% female). Internal consistency reliability was acceptable with Cronbach's alpha of .79. Factor analysis supported two factors representing self-efficacy expectations and outcome expectancy. Contrasted groups comparisons and construct validity were examined. We found lower falls self-efficacy in participants who needed help with mobility, in people with lower executive function, and in participants who reported fear of falling. Scores were not associated with prospective falls or adherence with hip protector use. The findings of this study provide preliminary support for the reliability and validity of the scale for future research.

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.

Backbone and sidechain 1H, 13C and 15N resonance assignments of the Bright/ARID domain from the human JARID1C (SMCX) protein.

We have assigned 1H, 13C and 15N resonances of the Bright/ARID DNA-binding domain from the human JARID1C protein, a newly discovered histone demethylase belonging to the JmjC domain-containing protein family.

The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins.

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein-ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.

Backbone and sidechain 1H, 13C and 15N resonance assignments of the RGS domain from human RGS14.

We have assigned 1H, 13C and 15N resonances of the RGS domain from the human RGS14 protein, a multi-domain member of the RGS (Regulators of G-protein signalling) family of proteins, important in the down-regulation of specific G-protein signalling pathways.

Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination.

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.

Structural and biochemical study of effector molecule recognition by the E.coli glyoxylate and allantoin utilization regulatory protein AllR.

The interaction of Escherichia coli AllR regulator with operator DNA is disrupted by the effector molecule glyoxylate. This is a general, yet uncharacterized regulatory mechanism for the large IclR family of transcriptional regulators to which AllR belongs. The crystal structures of the C-terminal effector-binding domain of AllR regulator and its complex with glyoxylate were determined at 1.7 and 1.8 A, respectively. Residues involved in glyoxylate binding were explored in vitro and in vivo. Altering the residues Cys217, Ser234 and Ser236 resulted in glyoxylate-independent repression by AllR. Sequence analysis revealed low conservation of amino acid residues participating in effector binding among IclR regulators, which reflects potential chemical diversity of effector molecules, recognized by members of this family. Comparing the AllR structure to that of Thermotoga maritima TM0065, the other representative of the IclR family that has been structurally characterized, indicates that both proteins assume similar quaternary structures as a dimer of dimers. Mutations in the tetramerization region, which in AllR involve the Cys135-Cys142 region, resulted in dissociation of AllR tetramer to dimers in vitro and were functionally inactive in vivo. Glyoxylate does not appear to function through the inhibition of tetramerization. Using sedimentation velocity, glyoxylate was shown to conformationally change the AllR tetramer as well as monomer and dimer resulting in altered outline of AllR molecules.

Quantitative study of the effects of chemical shift tolerances and rates of SA cooling on structure calculation from automatically assigned NOE data.

The calculation of protein structures from nuclear magnetic resonance (NMR) data has been greatly facilitated by improvements in software for the automatic assignment of NOESY spectra. Nevertheless, for larger proteins, resonance overlap may lead to an overwhelming number of assignment options per peak. Although most software for automatic NOESY assignment can deal with a certain level of assignment ambiguity, structure calculations fail when this becomes too high. Reducing the number of assignment options per peak by reducing the chemical shift tolerances can lead to correct assignments being excluded, and thus also to incorrect structures. We have investigated, systematically, for three proteins of different size, the influence of the chemical shift tolerance limits (Delta) and of the number of simulated annealing (SA) cooling steps on the performance of the software ARIA. Large tolerance windows, and the correspondingly high levels of ambiguity, did not cause problems when appropriately slower cooling was used in our SA protocol. In cases where a high percentage of well-converged structures was not achieved, we demonstrate that it is more productive to calculate fewer structures whilst applying slow cooling, than to calculate many structures with fast cooling. In this way, high-quality structures were obtained even for proteins whose NMR spectra showed great degeneracy, and where there was much inconsistency in peak alignment between different samples. The method described herein opens the way to the automated structure determination of larger proteins from NMR data.

Recognition of proline-rich motifs by protein-protein-interaction domains.

Protein-protein interactions are essential in every aspect of cellular activity. Multiprotein complexes form and dissociate constantly in a specifically tuned manner, often by conserved mechanisms. Protein domains that bind proline-rich motifs (PRMs) are frequently involved in signaling events. The unique properties of proline provide a mechanism for highly discriminatory recognition without requiring high affinities. We present herein a detailed, quantitative assessment of the structural features that define the interfaces between PRM-binding domains and their target PRMs, and investigate the specificity of PRM recognition. Together with the analysis of peptide-library screens, this approach has allowed the identification of several highly conserved key interactions found in all complexes of PRM-binding domains. The inhibition of protein-protein interactions by using small-molecule agents is very challenging. Therefore, it is important to first pinpoint the critical interactions that must be considered in the design of inhibitors of PRM-binding domains.

Solution structure, backbone dynamics, and association behavior of the C-terminal BRCT domain from the breast cancer-associated protein BRCA1.

BRCA1 is a tumor suppressor protein associated with breast and ovarian cancer. The C-terminal region of BRCA1 consists of two closely spaced BRCT domains which mediate essential biological functions, including regulation of transcription and control of cell-cycle progression by their interaction with phosphorylated effector proteins. Here we report the NMR structure of the isolated C-terminal BRCT domain (BRCT-c) from human BRCA1. BRCT-c is well-structured in solution, folding independently in the absence of its BRCT-n counterpart. Ultracentrifugation experiments and size exclusion chromatography reveal that BRCT-c exists as a monomer under near-physiological conditions. Dynamics measurements from NMR data show three loops which coincide with the most variable sequence regions in BRCT domains, to be genuinely flexible in solution. The solution structure of BRCT-c shows subtle conformational changes when compared to the crystal structure of BRCT-c in the tandem repeat of BRCA1. These affect sites involved in formation of the BRCT-n-BRCT-c interface and the binding to phosphoserine-containing peptides. The results suggest that the presence of native BRCT-n and a properly aligned BRCT-n-BRCT-c interface are essential if BRCT-c is to adopt a biologically active conformation. Structural consequences of cancer-associated mutations and biological implications of the dynamic behavior are discussed.

The SEP domain of p47 acts as a reversible competitive inhibitor of cathepsin L.

The solution structure of the human p47 SEP domain in a construct comprising residues G1-S2-p47(171-270) was determined by NMR spectroscopy. A structure-derived hypothesis about the domains' function was formulated and pursued in binding experiments with cysteine proteases. The SEP domain was found to be a reversible competitive inhibitor of cathepsin L with a Ki of 1.5 microM. The binding of G1-S2-p47(171-270) to cathepsin L was mapped by biochemical assays and the binding interface was investigated by NMR chemical shift perturbation experiments.

Solution structure of human cofilin: actin binding, pH sensitivity, and relationship to actin-depolymerizing factor.

Human actin-depolymerizing factor (ADF) and cofilin are pH-sensitive, actin-depolymerizing proteins. Although 72% identical in sequence, ADF has a much higher depolymerizing activity than cofilin at pH 8. To understand this, we solved the structure of human cofilin using nuclear magnetic resonance and compared it with human ADF. Important sequence differences between vertebrate ADF/cofilins were correlated with unique structural determinants in the F-actin-binding site to account for differences in biochemical activities of the two proteins. Cofilin has a short beta-strand at the C terminus, not found in ADF, which packs against strands beta3/beta4, changing the environment around Lys96, a residue essential for F-actin binding. A salt bridge involving His133 and Asp98 (Glu98 in ADF) may explain the pH sensitivity of human cofilin and ADF; these two residues are fully conserved in vertebrate ADF/cofilins. Chemical shift perturbations identified residues that (i) differ in their chemical environments between wild type cofilin and mutants S3D, which has greatly reduced G-actin binding, and K96Q, which does not bind F-actin; (ii) are affected when G-actin binds cofilin; and (iii) are affected by pH change from 6 to 8. Many residues affected by G-actin binding also show perturbation in the mutants or in response to pH. Our evidence suggests the involvement of residues 133-138 of strand beta5 in all of the activities examined. Because residues in beta5 are perturbed by mutations that affect both G-actin and F-actin binding, this strand forms a "boundary" or "bridge" between the proposed F- and G-actin-binding sites.

Design of N-substituted peptomer ligands for EVH1 domains.

Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes. Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs. The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein. Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity. We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 microm. We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions. These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility.

Prevention of brain aging and dementia.

The brain is subjected to multiple factors that result in damage to its cellular constituents, the neuron and supporting cells, and the neural networks that form the bases of cognitive ability. Like other systems, the brain has remarkable capacity to repair that damage and to adapt or compensate for the loss of neurons and the disruption of the neural architecture. Brain aging and dementia can be conceptualized as a balance between neuronal injury and repair. This balance can be affected not only by genetic and age-related factors but also by multiple environmental factors. The latter includes many factors, including education, nutrition, exercise, socialization, and stress. As individuals, we have the potential to modify these factors through lifestyle choices. Advances in neuroscience have led to the development of pharmacologic agents that can ameliorate the effects of even genetic (e.g., statins and antihypertensive agents) and age-related (e.g., antioxidants and estrogen replacement) factors. By altering the balance between neuronal injury and repair, we can delay the expression and progression of the neurodegenerative processes of brain aging, AD, and related dementias.