The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al

Rhesus TRIM5α disrupts the HIV-1 capsid at the inter-hexamer interfaces

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction. © 2011 Zhao et al

Structure of a Novel Shoulder-to-Shoulder p24 Dimer in Complex with the Broad-Spectrum Antibody A10F9 and Its Implication in Capsid Assembly

10.1371/journal.pone.0061314PLoS ONE84

Representing and comparing protein structures as paths in three-dimensional space

BACKGROUND: Most existing formulations of protein structure comparison are based on detailed atomic level descriptions of protein structures and bypass potential insights that arise from a higher-level abstraction. RESULTS: We propose a structure comparison approach based on a simplified representation of proteins that describes its three-dimensional path by local curvature along the generalized backbone of the polypeptide. We have implemented a dynamic programming procedure that aligns curvatures of proteins by optimizing a defined sum turning angle deviation measure. CONCLUSION: Although our procedure does not directly optimize global structural similarity as measured by RMSD, our benchmarking results indicate that it can surprisingly well recover the structural similarity defined by structure classification databases and traditional structure alignment programs. In addition, our program can recognize similarities between structures with extensive conformation changes that are beyond the ability of traditional structure alignment programs. We demonstrate the applications of procedure to several contexts of structure comparison. An implementation of our procedure, CURVE, is available as a public webserver

Association of a germline copy number polymorphism of APOBEC3A and APOBEC3B with burden of putative APOBEC-dependent mutations in breast cancer.

The somatic mutations in a cancer genome are the aggregate outcome of one or more mutational processes operative through the lifetime of the individual with cancer. Each mutational process leaves a characteristic mutational signature determined by the mechanisms of DNA damage and repair that constitute it. A role was recently proposed for the APOBEC family of cytidine deaminases in generating particular genome-wide mutational signatures and a signature of localized hypermutation called kataegis. A germline copy number polymorphism involving APOBEC3A and APOBEC3B, which effectively deletes APOBEC3B, has been associated with modestly increased risk of breast cancer. Here we show that breast cancers in carriers of the deletion show more mutations of the putative APOBEC-dependent genome-wide signatures than cancers in non-carriers. The results suggest that the APOBEC3A-APOBEC3B germline deletion allele confers cancer susceptibility through increased activity of APOBEC-dependent mutational processes, although the mechanism by which this increase in activity occurs remains unknown.We would like to thank the Wellcome Trust for support (grant reference 098051). SN-Z is a Wellcome-Beit Prize Fellow and is supported through a Wellcome Trust Intermediate Fellowship (grant reference WT100183MA). PJC is personally funded through a Wellcome Trust Senior Clinical Research Fellowship (grant reference WT088340MA). NB is an EHA fellow and is supported by a Lady Tata Memorial Trust award. The H.L. Holmes Award from the National Research Council Canada and an EMBO Fellowship supports AS

Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity

Sequential Phosphorylation and Multisite Interactions Characterize Specific Target Recognition by the FHA Domain of Ki-67

[[sponsorship]]生物化學研究所,基因體研究中心[[note]]已出版;[SCI];有審查制度;不具代表性[[note]]http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Drexel&SrcApp=hagerty_opac&KeyRecord=1545-9993&DestApp=JCR&RQ=IF_CAT_BOXPLOT[[note]]http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=RID&SrcApp=RID&DestLinkType=FullRecord&DestApp=ALL_WOS&KeyUT=00023311510001