Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael Kokkinidis University of Crete & IMBB, FORTH, Heraklion Crete, Greece ‡ MBG, DUTH, Alexandroupolis, Creece

The type III secretion system: is a multiprotein secretion machinery which is composed of approximately proteins. is a multiprotein secretion machinery which is composed of approximately proteins. is found in many gram negative bacteria i.e. Salmonella, Shigella, Yersinia, is found in many gram negative bacteria i.e. Salmonella, Shigella, Yersinia, Pseudomonas, Xanthomonas, Erwinia. is used to transfer pathogenic proteins from the pathogen to the cytoplasm of the host is used to transfer pathogenic proteins from the pathogen to the cytoplasm of the host → a pathogenic mechanism. has morphological similarities with the bacterial flagellum. has morphological similarities with the bacterial flagellum.

The type III secretion system: Morphology of the secretion apparatus Shigella Tamano et al. 2000

The type III secretion system: Morphology of the secretion apparatus Shigella Flagellum Tamano et al Thomas et al Zhao et al. 1996

The HrcQ B protein: is small (14 kDa), hydrophilic, member of the type ΙΙΙ secretion system of Pseudomonas syringae pv. phaseolicola. is small (14 kDa), hydrophilic, member of the type ΙΙΙ secretion system of Pseudomonas syringae pv. phaseolicola. is a highly conserved member of the secretion machinery. It has homologues in every studied type III secretion system and the bacterial flagellum. is a highly conserved member of the secretion machinery. It has homologues in every studied type III secretion system and the bacterial flagellum. among the HrcQ B homologues among the HrcQ B homologues the conservation is restricted to the carboxy terminus.

Severe proteolysis problems Purification of the HrcQ Β and attempts to crystallize it Stable protein was produced after the use of a second, Ni-NTA agarose column. Numerous crystallisation attempts were unsuccesful.

Is there a crystallizable, functional domain of the protein ?

The conserved carboxy-terminal domain is a promising candidate => HrcQ B -C The conserved carboxy-terminal domain is a promising candidate => HrcQ B -C CLUSTAL W (1.82) multiple sequence alignment HrcQb_Psph MSTEDLYQEDVEMLDDYEDPS TEQHWSEEDGEPSGYATAE 40 HrcQb_Psto MSTEDLYQDDVESLEDYDDETAEQE-----HEHEHEQQWAEPDDE-SEYAEAE 47 HrcQ_Erwi CTEQLQALTAGDLLIPPVSYFTPDGQGSLTVAGQRLYGELQLPHHFLLNHLESTALNSAD 240 FliN_Ecol MSDMNNPADDNNGAMDDLWAEAL SEQKSTSS-KSAAE 36 FliN_Yers MSDPKFPSADGKESVDDLWAYAF NEQQATEKPTATTE 37 FliN_Brad MSDT-----DGQVPLPDLNG PMPPTGTDVGYNE 28 *. :. : HrcQb_Psph PDD--HAAQEEQD---EPPALDSLALDLTLRCGELRLTLAELRRLDAGTILEVTGISPGH 95 HrcQb_Psto PDDDEQEEQEEQQ---APSGLDSLALDLTLRCGELRLTLAELRRLDAGTILEVGGVAPGY 104 HrcQ_Erwi DDALTEGSLPEYTGCEDNPQLASLPLSLEVRCDRTALTLGELQRLQAGSVVTLDNVTPGE 300 FliN_Ecol TVFQQFGGGDVSGTLQDIDLIMDIPVKLTVELGRTRMTIKELLRLTQGSVVALDGLAGEP 96 FliN_Yers GVFKSLEAPEGLGNLQDIDLILDIPVKLSVELGRTKMTIKELLRLSQGSVVSLDGLAGEP 97 FliN_Brad DEYAAR AAADLEAVFDVPVQVSAVLGRSKMDVGELLKLGPGTVLELDRRVGEA 81 :.:.:.:.. : : ** :* *::: : HrcQb_Psph ATLCHGEQVVAEGELVDVEGRLGLQITRLVTRS HrcQb_Psto ATLCHGERVVAEGELVDVDGRLGLQITRLAAQP HrcQ_Erwi AGLYHGDTLIARGELVDVEGHLGLQLTQLLLTSCQEVG FliN_Ecol LDILINGYLIAQGEVVVVADKYGVRITDIITPSERMRRLSR 137 FliN_Yers LDILINGYLIAQGEVVVVADKYGVRITDIITSSERMRRLSR 138 FliN_Brad IDIYVNNKLVARGEVVLVEDKLGVTMTEIIKTERT :. ::*.**:* *.: *: :* :

The HrcQ B -C : was purified by affinity chromatography. was purified by affinity chromatography. is stable for several months. is stable for several months. was readily crystallized. was readily crystallized. The structure was solved by Multiple Anomalous Dispersion (three data sets were collected at Hamburg Outstation (DESY)). The structure was solved by Multiple Anomalous Dispersion (three data sets were collected at Hamburg Outstation (DESY)).

The structure of the HrcQ B -C

The structure of the FliN-C

Crystal structure comparison of the HrcQ B -C (1o9y.pdb) and FliN-C (1o6a.pdb) proteins. The HrcQ B -C and FliN-C proteins have : (i) Similar structures, (ii) Sequence similarities, (iii) Common subcellular location, (iv) Analogous pattern of interactions with other members of the systems. (Francis et al. 1994, Thomas et al. 1999, Lux et al. 2000, Thomas et al. 2001) It has been shown that: (i) FliN is the major component of a ring- shaped cytoplasmic structure called C-ring and (ii) the FliN-C is the functionally essential part of the protein while the N-terminus is dispensable. It has been shown that: (i) FliN is the major component of a ring- shaped cytoplasmic structure called C-ring and (ii) the FliN-C is the functionally essential part of the protein while the N-terminus is dispensable. Comparison between the HrcQ B -C & FliN-C proteins

it is reasonable to suggest that the HrcQ B protein is the major component of a cytoplasmic structure analogous to the flagellar C-ring. …thus it is reasonable to suggest that the HrcQ B protein is the major component of a cytoplasmic structure analogous to the flagellar C-ring. In their crystal structures HrcQ B -C from Pseudomonas syringae and FliN-C from Thermotoga maritima were determined to be homotetrameric and homodimeric respectively while FliN from Escherichia coli was tetrameric in solution (Brown et al., 2005). What is the oligomerization state of the structural unit which is used in the assembly of the C-ring ? Comparison between the HrcQ B -C & FliN-C proteins

We have used molecular dynamics simulations to test the hypothesis that a tetramer analogous to the crystallographically determined HrcQ B -C tetramer could provide the building block of the C-ring. The HrcQ B -C forms a stable tetramer. The dimer of the FliN-C is compatible with a tetrameric arrangement similar to the HrcQ B -C tetramer. Molecular Dynamics Simulations

We did not manage to crystallize the full length HrcQ B protein. The conserved carboxy-terminal domain was crystallized and its structure was determined at 2.3 Å. The common features between the FliN-C and HrcQ B -C proteins including their similar structures indicate that the HrcQ B -C is the major component of a C-ring like assembly. The crystallographically determined HrcQ B -C tetramer could provide the building block of this C-ring ? ►the HrcQ B -C tetramer seems to be stable enough, ►the FliN-C dimer is compatible with a HrcQ B -C like tetramer.Summary

Kokkinidis M. (University of Crete & IMBB) Panopoulos N. (University of Crete & IMBB) Tampakaki N. (University of Crete & IMBB) Bastaki M. (University of Crete & IMBB) Glykos N. (MBG, DUTH, Alexadroupolis) Phillips SEV (University of Leeds) Hadden J. (University of Leeds) Acknowledgements