the haemolysin system from e. coli - ldri / ucl - brussels
TRANSCRIPT
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The Haemolysin System from E. coli – a Paradigm for Type I Secretion Systems?
Lutz Schmitt
Institute of Biochemistry – Membrane Transport Group –
Heinrich Heine University Düsseldorf
Protein Secretion in Gram-negative Bacteria
CytosolInner membranePeriplasmic spaceOuter membraneExtracellular space (secretion)
Desvaux (2004) Trends Microbiol
The Haemolysin A Machinery
ABC transporter
Membrane fusionprotein
Outer membraneprotein
Substrate(RTX toxin)
What we think happens ...
TolC
HlyD
HlyB
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HlyA
Bacteriocine ABC Transporters
Dawson & Locher (2006) Nature
NukT! ! LQNSDQDCLLACYSMILSYFGKNVSINSLYKREM-IPPDGLSISYLKELNIKYELNMKVYCvaB! ! HQTETAECGLACLAMICGHFGKNIDLIYLRRKFN-LSARGATL--AGINGIAEQLGMATRClyB! ! AQGEHSECALACITMLLNYYGNQSTLVELREKYG-VPKGGLTIKN--IRTVFDEYGFDVSLagD! ! YQQDEKDCGVACIAMILKHYGTEITIQRLRELSG-TDLDGTSA--FGIKKTFEKLGFDAPLtnT! ! FQVAQTECGLCCVRTILDYFGYETTVTKLRILKE-PGRDGSSF--NDIRKLLERFGVDSKComA! ! PQVDQMDCGVASLAMVFGYYGSYYFLAHLRELAK-TTMDGTTA--LGLVKVAEEIGFETRHlyB! ! -SCHKIDYGLYALEILAQYHNVSVNPEEIKHRFD-TDGTGLGL--TSWLLAAKSLELKVKLktB! ! -SQKNTNLALQALEVLAQYHNISINPEEIKHKFD-IDGHGLNQ--TKWLLAAKSLGLKVRPaxB! ! SFKQKNDYGLHALVILAQYHNIAVSPEEIKHKFD-PEGKGIDL--VAWLLAAKSFELKAKRtxB! ! -K--TSQPALSALIILAHYHGIAANPADISHHFSGSLNNDLSE--TEWLLAAKKLELKAKApxIIIB! PFNEKIDYGLHALVILAQYHNVAVNPEEVKHKFD-LDGKGLDL--VAWLLAAKSLELKMKAqxB! ! -FNQKEDYGLYALTILAQYHNIAVNPEELKHKFD-LEGKGLDL--TAWLLAAKSLELKAK
NukT! ! RIKDKEKTFR--VISKIKKPIIVH---W-DLNHFVIVKN---VKKKHIEIVNPEI—-GKV…CvaB! ! ALSL---ELD--ELRVLKTPCILH---W-DFSHFVVLVSV---KRNRYVLHDPAR—-GIR…ClyB! ! TFKS---SFS--NYLDLPTPVISY---W-NNQHFVVIEK---IKKKKVLILDPAS--NKR…LagD! ! AFKA---GDETWQEKDIPLPLIAHIISEQKYQHYVVVYK---VKGDEIWIADPAK--GKI…LtnT! ! LYKV---KDNR-IFSTLQLPIIIY---W-KNVHFVCVER---ISKKTVIIMDPSV--GRT…ComA! ! AIKA---DMTLFDLPDLTFPFVAHVLKEGKLLHYYVVTG---QDKDSIHIADPDPGVKLT…HlyB! ! QVKK---TID--RLNFISLPALVW---REDGRHFILTKVS--KEANRYLIFDLEQ-RNPR…LktB! ! TANK---TVD--RLPFLHLPALAW---RDDGEHFILLKID--QETDRYLIFDLIQ-KNPI…PaxB! ! KVKK---SID--RLPFIHLPALIW---RDDGQHFILTKID--TQTNRYLIFDLEE-RNPK…RtxB! ! VVKQ---PIS--RLPMASLPALVW---REDGDHFLLAKIDGTGEATQYLIHDLSE-SRPI…ApxIIIB! RVKK---SIE--RLPFIHLPALIW---RDDGQHVILMKID--TQTNRYLIFDLEE-RNPK…AqxB! ! QVKK---SID--RLEFIALPALVW---RDDGQHFILTKID--SKAQKYLIFDLET-RNPR…
C39 Peptidases
C39 peptidases are cysteine proteases
Specific for ABC transporters
Cleavage occurs after a GG motif
Catalytic dyad/triade (Cys-His-Asp)
Consensus sequence – LSXXELXXIXGG (Substrate)
The C39-domain of HlyB:Consensus sequence – GGXGXDUX (RTX)Tyr instead of Cys
C39-like domain (CLD)
A Potential Role of the CLD
E. coliSE 5000
E. coliBL21/HlyB+HlyD
E. coliBL21/HlyB-C39+HlyD
Interaction Partner(s)
30
25
20
15
Unfol
ded
HlyAc
C39-d
omain
Elut
ion
Was
h
Controls ....
Was
h
Fold
ed H
lyAc
CLD Elut
ion
30
25
20
15 !"#
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!%#
CLD Was
h
Elut
ion
Unfol
ded
HlyAc –
SecS
eq
The unfolded state without the SecSeq is recognized
The NMR Structure of the CLD
The Overall Fold
The Active Site
CLD – C39: A Comparison
ComA-PEP* / CLD
*Ishii et al. (2010) JBC 285 10777- 10885
Mechanistic Implications
No thiolate (Tyr9)
His83 – Trp77 interaction
Tyr9Cys is still inactive
Cys S
NH
HN
His
R
HN R'
O
O O
Asp
Cys S
NH
HN
His
R
HN R'
O O
Asp
O
Cys S
NH
N
His
R
H2N R'
O O
Asp
O
O HH
R–GG^–R'
Interactions by NMR
Titration of HlyAc* (no SecSeq) / CLD
CLD harbors a 'continous' binding site
Interactions by NMR
Titration of HlyAc* (no SecSeq) / CLD
CLD harbors a 'continous' binding site
Mutations – CLD versus ComA
Phylogenetic Consequences
'Trp77' – 'His83' as an indicator
HasA/HasD systems
A Model of the Early Steps
TolC
HlyD
HlyB
C39 / NBD
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ReceptorEngine
Negative regulation
Recruitment of TolC
Substrate transport in the unfolded state
Text
André AbtsZainab AlKatibDiana ClausnitzerPhilipp EllingerIris GarwarzeskiRakesh GuptaNils HanekopNacera InfedMiro Kirov
Marianne KluthMichael LendersTatu MeyerRicarda MoselerSusanne PrzybylaBritta RiesUlrich SchulteSander SmitsChristian SchwarzSabrina Thomas
CollaborationsErhard Bremer (Marburg) Arnold Driessen (Groningen) Ralf Kubitz (Düsseldorf)Karl Kuchler (Vienna)Barry Holland (Paris)Bert Poolman (Groningen)Dieter Willbold (Düsseldorf)