A new structural insight into XPA–DNA interactions

XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98–219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA–DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA–DNA junction interactions.     

Mass spectrometric analysis of the HIV-1 integrase-pyridoxal 5'-phosphate complex reveals a new binding site for a nucleotide inhibitor

HIV-1 integrase (IN) is an important target for designing new antiviral therapies. Screening of potential inhibitors using recombinant IN-based assays has revealed a number of promising leads including nucleotide analogs such as pyridoxal 5'-phosphate (PLP). Certain PLP derivatives were shown to also exhibit antiviral activities in cell-based assays. To identify an inhibitory binding site of PLP to IN, we used the intrinsic chemical property of this compound to form a Schiff base with a primary amine in the protein at the nucleotide binding site. The amino acid affected was then revealed by mass spectrometric analysis of the proteolytic peptide fragments of IN. We found that an IC(50) concentration (15 mum) of PLP modified a single IN residue, Lys(244), located in the C-terminal domain. In fact, we observed a correlation between interaction of PLP with Lys(244) and the compound's ability to impair formation of the IN.DNA complex. Site-directed mutagenesis studies confirmed an essential role of Lys(244) for catalytic activities of recombinant IN and viral replication. Molecular modeling revealed that Lys(244) together with several other DNA binding residues provides a plausible pocket for a nucleotide inhibitor-binding site. To our knowledge, this is the first report indicating that a small molecule inhibitor can impair IN activity through its binding to the protein C terminus. At the same time, our findings highlight the importance of structural analysis of the full-length protein.     

Interactions of HIV-1 Gag with assembly cofactors

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.     

Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

Transportin 3 (TNPO3 or TRN-SR2) has been shown to be an important cellular factor for early steps of lentiviral replication. However, separate studies have implicated distinct mechanisms for TNPO3 either through its interaction with HIV-1 integrase or capsid. Here we have carried out a detailed biophysical characterization of TNPO3 and investigated its interactions with viral proteins. Biophysical analyses including circular dichroism, analytical ultracentrifugation, small-angle x-ray scattering, and homology modeling provide insight into TNPO3 architecture and indicate that it is highly structured and exists in a monomer-dimer equilibrium in solution. In vitro biochemical binding assays argued against meaningful direct interaction between TNPO3 and the capsid cores. Instead, TNPO3 effectively bound to the functional intasome but not to naked viral DNA, suggesting that TNPO3 can directly engage the HIV-1 IN tetramer prebound to the cognate DNA. Mass spectrometry-based protein footprinting and site-directed mutagenesis studies have enabled us to map several interacting amino acids in the HIV-1 IN C-terminal domain and the cargo binding domain of TNPO3. Our findings provide important information for future genetic analysis to better understand the role of TNPO3 and its interacting partners for HIV-1 replication.     

Human T-lymphotropic virus type 1 p30 interacts with REGgamma and modulates ATM (ataxia telangiectasia mutated) to promote cell survival

Human T-lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia/lymphoma and a variety of inflammatory disorders. HTLV-1 encodes a nuclear localizing protein, p30, that selectively alters viral and cellular gene expression, activates G(2)-M cell cycle checkpoints, and is essential for viral spread. Here, we used immunoprecipitation and affinity pulldown of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ATM (ataxia telangiectasia mutated) and REGγ (a nuclear 20 S proteasome activator). Under conditions of genotoxic stress, p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or overexpression of REGγ paralleled p30 expression, suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular mass complex along with ATM and REGγ. On the basis of our findings, we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.     

Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein

The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.     

Akt Pathway Activation by Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

Human T-cell leukemia virus (HTLV) type 1, the etiological agent of adult T-cell leukemia, expresses the viral oncoprotein Tax1. In contrast, HTLV-2, which expresses Tax2, is non-leukemogenic. One difference between these homologous proteins is the presence of a C-terminal PDZ domain-binding motif (PBM) in Tax1, previously reported to be important for non-canonical NFκB activation. In contrast, this study finds no defect in non-canonical NFκB activity by deletion of the Tax1 PBM. Instead, Tax1 PBM was found to be important for Akt activation. Tax1 attenuates the effects of negative regulators of the PI3K-Akt-mammalian target of rapamycin pathway, phosphatase and tensin homologue (PTEN), and PHLPP. Tax1 competes with PTEN for binding to DLG-1, unlike a PBM deletion mutant of Tax1. Forced membrane expression of PTEN or PHLPP overcame the effects of Tax1, as measured by levels of Akt phosphorylation, and rates of Akt dephosphorylation. The current findings suggest that Akt activation may explain the differences in transforming activity of HTLV-1 and -2.     

A Critical Role of the C-terminal Segment for Allosteric Inhibitor-induced Aberrant Multimerization of HIV-1 Integrase

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising class of antiretroviral agents for clinical development. Although ALLINIs promote aberrant IN multimerization and inhibit IN interaction with its cellular cofactor LEDGF/p75 with comparable potencies in vitro, their primary mechanism of action in infected cells is through inducing aberrant multimerization of IN. Crystal structures have shown that ALLINIs bind at the IN catalytic core domain dimer interface and bridge two interacting subunits. However, how these interactions promote higher-order protein multimerization is not clear. Here, we used mass spectrometry-based protein footprinting to monitor surface topology changes in full-length WT and the drug-resistant A128T mutant INs in the presence of ALLINI-2. These experiments have identified protein-protein interactions that extend beyond the direct inhibitor binding site and which lead to aberrant multimerization of WT but not A128T IN. Specifically, we demonstrate that C-terminal residues Lys-264 and Lys-266 play an important role in the inhibitor induced aberrant multimerization of the WT protein. Our findings provide structural clues for exploiting IN multimerization as a new, attractive therapeutic target and are expected to facilitate development of improved inhibitors.