The type III secretion system :

1. Crystal structure of theHrcQB-C protein from the Type ΙΙΙ secretion systemof Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael KokkinidisUniversity of Crete & IMBB, FORTH, Heraklion Crete, Greece‡ MBG, DUTH, Alexandroupolis, Creece

2. The type III secretion system: • is a multiprotein secretion machinery which is composed of approximately 20-25proteins. • 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 → a pathogenic mechanism. • has morphological similarities with the bacterial flagellum.

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

4. The type III secretion system: Morphology of the secretion apparatus Shigella Tamano et al. 2000 Flagellum Thomas et al. 1999 Zhao et al. 1996

5. TheHrcQB protein: • 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 inevery studied type III secretion system and the bacterial flagellum. • among the HrcQB homologues the conservation is restricted to the carboxy terminus.

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

7. Is there a crystallizable, functional domain of the protein ?

8. The conserved carboxy-terminal domain is a promising candidate => HrcQB-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-------- 128 HrcQb_Psto ATLCHGERVVAEGELVDVDGRLGLQITRLAAQP-------- 137 HrcQ_Erwi AGLYHGDTLIARGELVDVEGHLGLQLTQLLLTSCQEVG--- 338 FliN_Ecol LDILINGYLIAQGEVVVVADKYGVRITDIITPSERMRRLSR 137 FliN_Yers LDILINGYLIAQGEVVVVADKYGVRITDIITSSERMRRLSR 138 FliN_Brad IDIYVNNKLVARGEVVLVEDKLGVTMTEIIKTERT------ 116 : . ::*.**:* * .: *: :* :

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

10. The structure of the HrcQB-C

11. The structure of the HrcQB-C

12. The structure of the HrcQB-C

13. The structure of the FliN-C

14. Comparison between the HrcQB-C & FliN-C proteins Crystal structure comparison of the HrcQB-C (1o9y.pdb) and FliN-C (1o6a.pdb) proteins. The HrcQB-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.

15. Comparison between the HrcQB-C & FliN-C proteins …thus it is reasonable to suggest that the HrcQB protein is the major component of a cytoplasmic structure analogous to the flagellar C-ring. What is the oligomerization state of the structural unit which is used in the assembly of the C-ring ? In their crystal structures HrcQB-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).

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

17. Summary • We did not manage to crystallize the full length HrcQB 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 HrcQB-C proteins including their similar structures indicate that the HrcQB-C is the major component of a C-ring like assembly. • The crystallographically determined HrcQB-C tetramer could provide the building block of this C-ring ? ►the HrcQB-C tetramer seems to be stable enough, • ►the FliN-C dimer is compatible with a HrcQB-C like tetramer.

18. Acknowledgements 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)