BackgroundType III secretion systems are used by Gram-negative bacteria as “macromolecular syringes” to inject effector proteins into eukaryotic cells. Two hydrophobic proteins called translocators form the necessary pore in the host cell membrane. Both translocators depend on binding to a single chaperone in the bacterial cytoplasm to ensure their stability and efficient transport through the secretion needle. It was suggested that the conserved chaperones bind the more divergent translocators via a hexapeptide motif that is found in both translocators and conserved between species.ResultsWe crystallized a synthetic decapeptide from the Yersinia enterocolitica minor type III secretion translocator YopD bound to its cognate chaperone SycD and determined the complex structure at 2.5 Å resolution. The structure of peptide-bound SycD is almost identical to that of apo SycD with an all helical fold consisting of three tetratricopeptide repeats (TPRs) and an additional C-terminal helix. Peptide-bound SycD formed a kinked head-to-head dimer that had previously been observed for the apo form of SycD. The homodimer interface comprises both helices of the first tetratricopeptide repeat. The YopD peptide bound in extended conformation into a mainly hydrophobic groove on the concave side of SycD. TPRs 1 and 2 of SycD form three hydrophobic pockets that accommodated the conserved hydrophobic residues at position 1, 3 and 6 of the translocator hexapeptide sequence. Two tyrosines that are highly conserved among translocator chaperones contribute to the hydrophobic patches but also form hydrogen bonds to the peptide backbone.ConclusionsThe interaction between SycD and YopD is very similar to the binding of the Pseudomonas minor translocator PopD to its chaperone PcrH and the Shigella major translocator IpaB to its chaperone IpgC. This confirms the prediction made by Kolbe and co-workers that a hexapeptide with hydrophobic residues at three positions is a conserved chaperone binding motif. Because the hydrophobic groove on the concave side of translocator chaperones is involved in binding of the major and the minor translocator, simultaneous binding of both translocators to a single type III secretion class II chaperone appears unlikely.

Several gram-negative bacteria employ type III secretion systems (T3SS) to inject effector proteins into eukaryotic host cells directly from the bacterial cytoplasm. The export gate SctV (YscV in Yersinia) binds substrate:chaperone complexes such as YscX:YscY, which are essential for formation of a functional T3SS. Here, we present structures of the YscX:YscY complex alone and bound to nonameric YscV. YscX binds its chaperone YscY at two distinct sites, resembling the heterotrimeric complex of the T3SS needle subunit with its chaperone and co-chaperone. In the ternary complex the YscX N-terminus, which mediates YscX secretion, occupies a binding site within one YscV that is also used by flagellar chaperones, suggesting the interaction’s importance for substrate recognition. The YscX C-terminus inserts between protomers of the YscV ring where the stalk protein binds to couple YscV to the T3SS ATPase. This primary YscV–YscX interaction is essential for the formation of a secretion-competent T3SS.

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The transmembrane pump halorhodopsin in halophilic archaea translocates chloride ions from the extracellular to the cytoplasmic side upon illumination. In the ground state a tightly bound chloride ion occupies the primary chloride-binding site (CBS I) close to the protonated Schiff base that links the retinal chromophore to the protein. The light-triggered trans-cis isomerization of retinal causes structural changes in the protein associated with movement of the chloride ion. In reverse, chemical depletion of CBS I in Natronomonas pharaonis halorhodopsin (NpHR) through deprotonation of the Schiff base results in conformational changes of the protein: a state thought to mimic late stages of the photocycle. Here, crystals of Halobacterium salinarum halorhodopsin (HsHR) were soaked at high pH to provoke deprotonation of the Schiff base and loss of chloride. The crystals changed colour from purple to yellow and the occupancy of CBS I was reduced from 1 to about 0.5. In contrast to NpHR, this chloride depletion did not cause substantial conformational changes in the protein. Nevertheless, two observations indicate that chloride depletion could eventually result in structural changes similar to those found in NpHR. Firstly, the partially chloride-depleted form of HsHR has increased normalized B factors in the region of helix C that is close to CBS I and changes its conformation in NpHR. Secondly, prolonged soaking of HsHR crystals at high pH resulted in loss of diffraction. In conclusion, the conformation of the chloride-free protein may not be compatible with this crystal form of HsHR despite a packing arrangement that hardly restrains helices E and F that presumably move during ion transport.

Poster Sessions C478 functional and structural studies were performed, focusing on SmeDEF, the most relevant antibiotic-and triclosan-removing multidrug efflux pump of S. maltophilia. Expression of smeDEF is regulated by the repressor SmeT. Triclosan released SmeT from its operator and induces the expression of smeDEF, thus reducing the susceptibility of S. maltophilia to antibiotics in the presence of the biocide. The structure of SmeT bound to triclosan is described. Two molecules of triclosan were found to bind to one subunit of the SmeT homodimer. The binding of the biocide stabilizes the N terminal domain of both subunits in a conformation unable to bind DNA. This complex structure is the first structural evidence of the ability of triclosan to act as an effector via its binding to a transcriptional regulator (SmeT). Given that SmeT mediate the susceptibility of Stenotrophomonas maltophilia to antibiotics by repressing SmeDEF expression, the present results provide information that aids our understanding of the molecular basis of biocide-induced antibiotic resistance. Keywords: complex, ligand, antibacterial MS36.P25Interaction of the type III secretion chaperone SycD with YscY Madeleine Schreiner,, Hartmut Niemann, Department of Chemistry, Bielefeld University, Bielefeld (Germany). E-mail: madeleine. schreiner@uni-bielefeld.deThe type III secretion system (T3SS) is used by several Gramnegative pathogenic bacteria to inject cytotoxins, so called effector proteins, into the host cell to manipulate their host for their own benefit. The effector translocation occurs via a needle-like nanomachine, the injectisome, which spans the whole bacterial envelope. The effectors enter the cell through a pore within the host cell membrane formed by bacterial translocator proteins. Both the effectors and translocators need chaperones for their efficient translocation. T3S chaperones work without the need for ATP hydrolysis and are divided into three subclasses: class I chaperones interacting with effector proteins, class II chaperones interacting with translocator proteins and class III chaperones interacting with needle components. [1] SycD (specific yop chaperone D) is the class II chaperone of the translocators YopB (Yersinia outer protein B) and YopD from the enteropathogen Yersinia enterocolitica. Additionally, SycD plays an important role in diverse regulatory processes of the T3SS and interacts with several other T3S proteins like TyeA, YscM2 or YscY. Like all structurally characterised class II chaperones SycD comprises an overall α-helical fold consisting of three tetratricopeptide repeats (TPR) providing a concave hydrophobic groove for translocator binding. SycD is known to form homodimers in solution involving the residues A61 and L65 of the first TPR as binding platform and mutations within these dimerisation-mediating residues lead to a stable monomeric chaperone that is not able to rescue a sycD null mutant of Y. enterocolitica.[2] Thus the dimerisation is either essential for the chaperone function or the T...