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1.  The Brucella TIR-like protein TcpB interacts with the death domain of MyD88 
The pathogen Brucella melitensis secretes a Toll/interleukin-1 receptor (TIR) domain containing protein that abrogates host innate immune responses. In this study, we have characterized the biochemical interactions of Brucella TIR-like protein TcpB with host innate immune adaptor proteins. Using protein-fragment complementation assays based on Gaussia luciferase and green fluorescent protein, we find that TcpB interacts directly with MyD88 and that this interaction is significantly stronger than the interaction of TcpB with TIRAP, the only other adaptor protein that detectably interacts with TcpB. Surprisingly, the TcpB–MyD88 interaction depends on the death domain (DD) of MyD88, and TcpB does not interact with the isolated TIR domain of MyD88. TcpB disrupts MyD88DD-MyD88DD, MyD88DD-MyD88TIR and MyD88DD-MyD88 interactions but not MyD88–MyD88 or MyD88TIR-MyD88TIR interactions. Structural models consistent with these results suggest how TcpB might inhibit TLR signaling by targeting MyD88 via a DD–TIR domain interface.
doi:10.1016/j.bbrc.2011.11.104
PMCID: PMC3607435  PMID: 22155231
Death domain; TIR domain; Protein interactions; Host–pathogen; Protein complementation assays
2.  Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling 
The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/ RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron–Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (HT and HL) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.
doi:10.1021/ja2093647
PMCID: PMC3279945  PMID: 22229766
3.  A structural model of the E. coli PhoB Dimer in the transcription initiation complex 
Background
There exist > 78,000 proteins and/or nucleic acids structures that were determined experimentally. Only a small portion of these structures corresponds to those of protein complexes. While homology modeling is able to exploit knowledge-based potentials of side-chain rotomers and backbone motifs to infer structures for new proteins, no such general method exists to extend our understanding of protein interaction motifs to novel protein complexes.
Results
We use a Motif Binding Geometries (MBG) approach, to infer the structure of a protein complex from the database of complexes of homologous proteins taken from other contexts (such as the helix-turn-helix motif binding double stranded DNA), and demonstrate its utility on one of the more important regulatory complexes in biology, that of the RNA polymerase initiating transcription under conditions of phosphate starvation. The modeled PhoB/RNAP/σ-factor/DNA complex is stereo-chemically reasonable, has sufficient interfacial Solvent Excluded Surface Areas (SESAs) to provide adequate binding strength, is physically meaningful for transcription regulation, and is consistent with a variety of known experimental constraints.
Conclusions
Based on a straightforward and easy to comprehend concept, "proteins and protein domains that fold similarly could interact similarly", a structural model of the PhoB dimer in the transcription initiation complex has been developed. This approach could be extended to enable structural modeling and prediction of other bio-molecular complexes. Just as models of individual proteins provide insight into molecular recognition, catalytic mechanism, and substrate specificity, models of protein complexes will provide understanding into the combinatorial rules of cellular regulation and signaling.
doi:10.1186/1472-6807-12-3
PMCID: PMC3348028  PMID: 22433509
4.  Mismatched dNTP incorporation by DNA polymerase β does not proceed via globally different conformational pathways† 
Nucleic Acids Research  2008;36(9):2948-2957.
Understanding how DNA polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dNTP incorporations. Little is known about the latter because mismatched complexes do not crystallize readily. In this report, we employed small-angle X-ray scattering (SAXS) and structural modeling to probe the conformations of different intermediate states of mammalian DNA polymerase β (Pol β) in its wild-type and an error-prone variant, I260Q. Our structural results indicate that the mismatched ternary complex lies in-between the open and the closed forms, but more closely resembles the open form for WT and the closed form for I260Q. On the basis of molecular modeling, this over-stabilization of mismatched ternary complex of I260Q is likely caused by formation of a hydrogen bonding network between the side chains of Gln260, Tyr296, Glu295 and Arg258, freeing up Asp192 to coordinate MgdNTP. These results argue against recent reports suggesting that mismatched dNTP incorporations follow a conformational path distinctly different from that of matched dNTP incorporation, or that its conformational closing is a major contributor to fidelity.
doi:10.1093/nar/gkn138
PMCID: PMC2396427  PMID: 18385153
5.  Escape from the Dominant HLA-B27-Restricted Cytotoxic T-Lymphocyte Response in Gag Is Associated with a Dramatic Reduction in Human Immunodeficiency Virus Type 1 Replication▿  
Journal of Virology  2007;81(22):12382-12393.
Human leukocyte antigen (HLA)-B27-positive subjects are uncommon in their ability to control infection with human immunodeficiency virus type 1 (HIV-1). However, late viral escape from a narrowly directed immunodominant Gag-specific CD8+ T-lymphocyte (CTL) response has been linked to AIDS progression in these individuals. Identifying the mechanism of the immune-mediated control may provide critical insights into HIV-1 vaccine development. Here, we illustrate that the CTL escape mutation R264K in the HLA-B27-restricted KK10 epitope in the capsid resulted in a significant defect in viral replication in vitro. The R264K variant was impaired in generating late reverse transcription products, indicating that replication was blocked at a postentry step. Notably, the R264K mutation was associated in vivo with the development of a rare secondary mutation, S173A, which restored viral replication in vitro. Furthermore, infectivity of the R264K variant was rescued by the addition of cyclosporine A or infection of a cyclophilin A-deficient cell line. These data demonstrate a severe functional defect imposed by the R264K mutation during an early step in viral replication that is likely due to the inability of this variant to replicate efficiently in the presence of normal levels of cyclophilin A. We conclude that the impact of the R264K substitution on capsid structure constrains viral escape and enables long-term maintenance of the dominant CTL response against B27-KK10, providing an explanation for the protective effect of HLA-B27 during HIV infection.
doi:10.1128/JVI.01543-07
PMCID: PMC2169010  PMID: 17804494
6.  DNA–XPA interactions: a 31P NMR and molecular modeling study of dCCAATAACC association with the minimal DNA-binding domain (M98–F219) of the nucleotide excision repair protein XPA 
Nucleic Acids Research  2001;29(12):2635-2643.
Recent NMR-based, chemical shift mapping experiments with the minimal DNA-binding domain of XPA (XPA-MBD: M98–F219) suggest that a basic cleft located in the loop-rich subdomain plays a role in DNA-binding. Here, XPA–DNA interactions are further characterized by NMR spectroscopy from the vantage point of the DNA using a single-stranded DNA nonamer, dCCAATAACC (d9). Up to 2.5 molar equivalents of XPA-MBD was titrated into a solution of d9. A subset of 31P resonances of d9 were observed to broaden and/or shift providing direct evidence that XPA-MBD binds d9 by a mechanism that perturbs the phosphodiester backbone of d9. The interior five residues of d9 broadened and/or shifted before 31P resonances of phosphate groups at the termini, suggesting that when d9 is bound to XPA-MBD the internal residues assume a correlation time that is characteristic of the molecular weight of the complex while the residues at the termini undergo a fraying motion away from the surface of the protein on a timescale such that the line widths are more characteristic of the molecular weight of ssDNA. A molecular model of the XPA-MBD complex with d9 was calculated based on the 15N (XPA-MBD) and 31P (d9) chemical shift mapping studies and on the assumption that electrostatic interactions drive the complex formation. The model shows that a nine residue DNA oligomer fully covers the DNA-binding surface of XPA and that there may be an energetic advantage to binding DNA in the 3′→5′ direction rather than in the 5′→3′ direction (relative to XPA-MBD α-helix-3).
PMCID: PMC55733  PMID: 11410673
7.  Reprogrammable Recognition Codes in Bicoid Homeodomain-DNA Interaction 
Molecular and Cellular Biology  2000;20(20):7673-7684.
We describe experiments to determine how the homeodomain of the Drosophila morphogenetic protein Bicoid recognizes different types of DNA sequences found in natural enhancers. Our chemical footprint analyses reveal that the Bicoid homeodomain makes both shared and distinct contacts with a consensus site A1 (TAATCC) and a nonconsensus site X1 (TAAGCT). In particular, the guanine of X1 at position 4 (TAAGCT) is protected by Bicoid homeodomain. We provide further evidence suggesting that the unique arginine at position 54 (Arg 54) of the Bicoid homeodomain enables the protein to recognize X1 by specifically interacting with this position 4 guanine. We also describe experiments to analyze the contribution of artificially introduced Arg 54 to DNA recognition by other Bicoid-related homeodomains, including that from the human disease protein Pitx2. Our experiments demonstrate that the role of Arg 54 varies depending on the exact homeodomain framework and DNA sequences. Together, our results suggest that Bicoid and its related homeodomains utilize distinct recognition codes to interact with different DNA sequences, underscoring the need to study DNA recognition by Bicoid-class homeodomains in an individualized manner.
PMCID: PMC86337  PMID: 11003663
8.  Requirement for the Kinase Activity of Human DNA-Dependent Protein Kinase Catalytic Subunit in DNA Strand Break Rejoining 
Molecular and Cellular Biology  1999;19(5):3877-3884.
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.
PMCID: PMC84245  PMID: 10207111

Results 1-8 (8)