Crystal structures of the Streptomyces coelicolor TetR-like protein ActR alone and in complex with actinorhodin or the actinorhodin biosynthetic precursor (S)-DNPA.

Actinorhodin, an antibiotic produced by Streptomyces coelicolor, is exported from the cell by the ActA efflux pump. actA is divergently transcribed from actR, which encodes a TetR-like transcriptional repressor. We showed previously that ActR represses transcription by binding to an operator from the actA/actR intergenic region. Importantly, actinorhodin itself or various actinorhodin biosynthetic intermediates can cause ActR to dissociate from its operator, leading to derepression. This suggests that ActR may mediate timely self-resistance to an endogenously produced antibiotic by responding to one of its biosynthetic precursors. Here, we report the structural basis for this precursor-mediated derepression with crystal structures of homodimeric ActR by itself and in complex with either actinorhodin or the actinorhodin biosynthetic intermediate (S)-DNPA [4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho-[2,3-c]-pyran-3-(S)-acetic acid]. The ligand-binding tunnel in each ActR monomer has a striking hydrophilic/hydrophobic/hydrophilic arrangement of surface residues that accommodate either one hexacyclic actinorhodin molecule or two back-to-back tricyclic (S)-DNPA molecules. Moreover, our work also reveals the strongest structural evidence to date that TetR-mediated antibiotic resistance may have been acquired from an antibiotic-producer organism

Structural Characterization of a Novel Chlamydia pneumoniae Type III Secretion-Associated Protein, Cpn0803

Type III secretion (T3S) is an essential virulence factor used by Gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S

Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase

<p>Abstract</p> <p>Background</p> <p>The <it>piggyBac </it>mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of <it>piggyBac</it>, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for <it>piggyBac </it>at amino acid positions D268, D346, and D447.</p> <p>Results</p> <p>This study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the <it>piggyBac </it>transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the <it>piggyBac </it>transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features.</p> <p>Conclusion</p> <p>We found all the designated DDD aspartates reside in clusters of amino acids that conserved among <it>piggyBac </it>family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase.</p

Polymorphisms of XRCC4 are involved in reduced colorectal cancer risk in Chinese schizophrenia patients

<p>Abstract</p> <p>Background</p> <p>Genetic factors related to the regulation of apoptosis in schizophrenia patients may be involved in a reduced vulnerability to cancer. XRCC4 is one of the potential candidate genes associated with schizophrenia which might induce colorectal cancer resistance.</p> <p>Methods</p> <p>To examine the genetic association between colorectal cancer and schizophrenia, we analyzed five SNPs (rs6452526, rs2662238, rs963248, rs35268, rs2386275) covering ~205.7 kb in the region of XRCC4.</p> <p>Results</p> <p>We observed that two of the five genetic polymorphisms showed statistically significant differences between 312 colorectal cancer subjects without schizophrenia and 270 schizophrenia subjects (rs6452536, p = 0.004, OR 0.61, 95% CI 0.44-0.86; rs35268, p = 0.028, OR 1.54, 95% CI 1.05-2.26). Moreover, the haplotype which combined all five markers was the most significant, giving a global <it>p </it>= 0.0005.</p> <p>Conclusions</p> <p>Our data firstly indicate that XRCC4 may be a potential protective gene towards schizophrenia, conferring reduced susceptibility to colorectal cancer in the Han Chinese population.</p

Structural and Biochemical Characterization of SrcA, a Multi-Cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 Å revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS

Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ

The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4–DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1–233) homodimer at 2.3 Å resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex

DNA MISMATCH REPAIR: FROM STRUCTURE TO MECHANISM.

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