Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas...

18
Crystal structure of Crystal structure of the the HrcQ HrcQ B B -C protein -C protein from from the the Type Type ΙΙΙ ΙΙΙ secretion secretion system system of of Pseudomonas syringae Pseudomonas syringae Vasiliki Fadouloglou Vasiliki Fadouloglou Nicholas M. Glykos Nicholas M. Glykos & Michael Kokkinidis & Michael Kokkinidis University of Crete & IMBB, FORTH, Heraklion Crete, University of Crete & IMBB, FORTH, Heraklion Crete, Greece Greece MBG, DUTH, Alexandroupolis, Creece MBG, DUTH, Alexandroupolis, Creece

Transcript of Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas...

Page 1: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

Crystal structure of theCrystal structure of theHrcQHrcQBB-C protein -C protein from from

the the TypeType ΙΙΙ ΙΙΙ secretion secretion

systemsystem of of Pseudomonas syringaePseudomonas syringae

Vasiliki Fadouloglou Vasiliki Fadouloglou Nicholas M. Glykos Nicholas M. Glykos ‡‡ & Michael Kokkinidis & Michael Kokkinidis

University of Crete & IMBB, FORTH, Heraklion Crete, University of Crete & IMBB, FORTH, Heraklion Crete, GreeceGreece

‡ ‡ MBG, DUTH, Alexandroupolis, Creece MBG, DUTH, Alexandroupolis, Creece

Page 2: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

The type III secretion systemThe type III secretion system::

is a multiprotein secretion machinery which is a multiprotein secretion machinery which is composed of approximately 20-25is composed of approximately 20-25 proteinsproteins. .

is found in many gram negative bacteria i.e. is found in many gram negative bacteria i.e. Salmonella, Shigella, Yersinia, Salmonella, Shigella, Yersinia,

Pseudomonas, Xanthomonas, ErwiniaPseudomonas, Xanthomonas, Erwinia. . is used to transfer pathogenic proteins from is used to transfer pathogenic proteins from

the pathogen to the cytoplasm of the host the pathogen to the cytoplasm of the host

→ → a pathogenic mechanism.a pathogenic mechanism. has morphological similarities with the has morphological similarities with the

bacterial flagellum. bacterial flagellum.

Page 3: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

The type III secretion The type III secretion systemsystem: : Morphology of the Morphology of the

secretion apparatussecretion apparatus

ShigellaShigella

Tamano et al. 2000Tamano et al. 2000

Page 4: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

The type III secretion The type III secretion systemsystem: : Morphology of the Morphology of the

secretion apparatussecretion apparatus

ShigellaShigella

FlagellumFlagellum

Tamano et al. 2000Tamano et al. 2000

Thomas et al. 1999Thomas et al. 1999 Zhao et al. 1996Zhao et al. 1996

Page 5: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

TheThe HrcQHrcQBB protein: protein: is smallis small (14 (14 kDa), hydrophilickDa), hydrophilic, , member of the member of the

type type ΙΙΙ ΙΙΙ secretion system of secretion system of Pseudomonas Pseudomonas syringaesyringae pv. phaseolicola. pv. phaseolicola.

is a highly conserved member of the secretion is a highly conserved member of the secretion machinery.machinery. It has homologues inIt has homologues in every studied every studied type III secretion system and the bacterial type III secretion system and the bacterial flagellum. flagellum.

among the HrcQamong the HrcQBB homologues homologuesthe conservation is restricted the conservation is restricted to the carboxy terminus.to the carboxy terminus.

Page 6: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

Severe Severe proteolysis proteolysis problemsproblems

Purification of the HrcQPurification of the HrcQΒΒ and and

attempts to crystallize itattempts to crystallize itStable protein was Stable protein was produced after the produced after the use of a second, Ni-use of a second, Ni-NTA agarose NTA agarose column.column.

Numerous Numerous crystallisation crystallisation attempts were attempts were unsuccesful.unsuccesful.

Page 7: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

Is there a Is there a crystallizable, crystallizable,

functional domain functional domain of the protein ?of the protein ?

Page 8: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

The conserved carboxy-terminal domain is The conserved carboxy-terminal domain is a promising candidate => HrcQa promising candidate => HrcQBB-C-C

CLUSTAL W (1.82) multiple sequence alignment

HrcQb_Psph -------MSTEDLYQEDVEMLDDYEDPS-------------TEQHWSEEDGEPSGYATAE 40HrcQb_Psto -------MSTEDLYQDDVESLEDYDDETAEQE-----HEHEHEQQWAEPDDE-SEYAEAE 47HrcQ_Erwi CTEQLQALTAGDLLIPPVSYFTPDGQGSLTVAGQRLYGELQLPHHFLLNHLESTALNSAD 240FliN_Ecol ---------MSDMNNPADDNNGAMDDLWAEAL--------------SEQKSTSS-KSAAE 36FliN_Yers ---------MSDPKFPSADGKESVDDLWAYAF--------------NEQQATEKPTATTE 37FliN_Brad ---------MSDT-----DGQVPLPDLNG------------------PMPPTGTDVGYNE 28 * . : . :

HrcQb_Psph PDD--HAAQEEQD---EPPALDSLALDLTLRCGELRLTLAELRRLDAGTILEVTGISPGH 95HrcQb_Psto PDDDEQEEQEEQQ---APSGLDSLALDLTLRCGELRLTLAELRRLDAGTILEVGGVAPGY 104HrcQ_Erwi DDALTEGSLPEYTGCEDNPQLASLPLSLEVRCDRTALTLGELQRLQAGSVVTLDNVTPGE 300FliN_Ecol TVFQQFGGGDVSGTLQDIDLIMDIPVKLTVELGRTRMTIKELLRLTQGSVVALDGLAGEP 96FliN_Yers GVFKSLEAPEGLGNLQDIDLILDIPVKLSVELGRTKMTIKELLRLSQGSVVSLDGLAGEP 97FliN_Brad DEYAAR-------AAADLEAVFDVPVQVSAVLGRSKMDVGELLKLGPGTVLELDRRVGEA 81 : .:.:.: .. : : ** :* *::: :

HrcQb_Psph ATLCHGEQVVAEGELVDVEGRLGLQITRLVTRS-------- 128HrcQb_Psto ATLCHGERVVAEGELVDVDGRLGLQITRLAAQP-------- 137HrcQ_Erwi AGLYHGDTLIARGELVDVEGHLGLQLTQLLLTSCQEVG--- 338FliN_Ecol LDILINGYLIAQGEVVVVADKYGVRITDIITPSERMRRLSR 137FliN_Yers LDILINGYLIAQGEVVVVADKYGVRITDIITSSERMRRLSR 138FliN_Brad IDIYVNNKLVARGEVVLVEDKLGVTMTEIIKTERT------ 116 : . ::*.**:* * .: *: :* :

Page 9: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

The HrcQThe HrcQBB-C :-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 The structure was solved by Multiple

Anomalous Dispersion (three data sets were Anomalous Dispersion (three data sets were collected at Hamburg Outstation (DESY)).collected at Hamburg Outstation (DESY)).

Page 10: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

The structure of the The structure of the HrcQHrcQBB-C-C

Page 11: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

The structure of the The structure of the HrcQHrcQBB-C-C

Page 12: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

The structure of the The structure of the HrcQHrcQBB-C-C

Page 13: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

The structure of the The structure of the FliN-CFliN-C

Page 14: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

Crystal structure Crystal structure comparison of the HrcQcomparison of the HrcQBB--C (1o9y.pdb) and FliN-C C (1o9y.pdb) and FliN-C (1o6a.pdb) proteins.(1o6a.pdb) proteins.

The HrcQThe HrcQBB-C and FliN-C proteins have :-C and FliN-C proteins have :(i) Similar structures, (ii) Sequence (i) Similar structures, (ii) Sequence similarities, (iii) Common subcellular location, similarities, (iii) Common subcellular location, (iv) Analogous pattern of interactions with (iv) Analogous pattern of interactions with other members of the systems. other members of the systems. (Francis et al. 1994, Thomas et al. 1999, Lux et al. 2000, Thomas (Francis et al. 1994, Thomas et al. 1999, Lux et al. 2000, Thomas et al. 2001)et al. 2001)

It has been shown that: It has been shown that: (i) FliN is the major (i) FliN is the major component of a ring-component of a ring-shaped cytoplasmic shaped cytoplasmic structure called C-ring structure called C-ring and (ii) the FliN-C is the and (ii) the FliN-C is the functionally essential part functionally essential part of the protein while the of the protein while the N-terminus is N-terminus is dispensable.dispensable.

Comparison between the Comparison between the HrcQHrcQBB-C & -C & FliN-C proteinsFliN-C proteins

Page 15: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

…thus it is reasonable to it is reasonable to suggest that the HrcQsuggest that the HrcQBB protein is the major protein is the major component of a component of a cytoplasmic structure cytoplasmic structure analogous to the flagellar analogous to the flagellar C-ring.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).

What is the oligomerization state of the structural unit which is used in the assembly of the C-ring ?

Comparison between the Comparison between the HrcQHrcQBB-C & -C & FliN-C proteinsFliN-C proteins

Page 16: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

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.

Molecular Dynamics Molecular Dynamics SimulationsSimulations

Page 17: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

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.

SummarySummary

Page 18: Crystal structure of the HrcQ B -C protein from the Type ΙΙΙ secretion system of Pseudomonas syringae Vasiliki Fadouloglou Nicholas M. Glykos ‡ & Michael.

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)

AcknowledgementsAcknowledgements