Two-Component Signal Transduction Systems

Transcription

1 Stimulus-Response coupling by Two-component Regulator systems TR act as a stimulus-response system. detection of environmental signal transduce signal to effector protein initiate response Most transcriptional regulators, but some affect protein activity Modular system conserved signal transduction strategy signal receiver and effector domains system specific exact signal transduction pathway is system specific Simple paradigm is a two protein system : 1 Two-omponent Signal Transduction Systems Basic model: Signal OM HK H Inner Membrane D ytoplasm DA RR romoter RA : 2 1

2 The signal transduction pathway Must pass the signal detected on to a domain capable of affecting transcription hosphorylation passes signal down the pathway HK+signal HK- HK- + RR RR- + HK The RR cannot remain activated RR- + HK RR + i RR- + tase RR + i At amino acid level: AT+His AD+His- His- +Asp His+Asp- Asp-+H 2 O Asp+i : 3 The TR Superfamily Amino acid sequence comparisons show large numbers of TRs (at least 300 known in bacteria) In virtually all bacteria adequately studied genome sequencing only Mycoplasma genitalium and Methanococcus jannaschii TRs not found limited regulatory systems life-style Yeast HK and RR-like Arabidopsis HK-like Mitochorondria HK-like : 4 2

3 TR sub-families Modularity Domain organisation can be used to organise into subgroups omparison based on HK or RR associated domain HK RR Signal-specific input domain Asp conserved receiver domain His conserved kinase transmitter domain Regulon-specific output domain : 5 hey SpoOF FrzZ hob ho VirG BvgA AgrA tr AlgB heb Receiver domain D RR domain comparisons D K Output domain Domain organisation of RRs. The -terminal receiver domains are conserved among RRs and contain the highly conserved amino acids D, D, and K. Based on similarity of -terminal domains, RRs can be sub-divided into sub-groups. Receiver domain Winged HtH domain HtH domain Sigma-54 interaction domain HtH domain Methylesterase domain : 6 3

4 Genetic Analysis of TRs Identification of TR systems lassical approaches: Mutants (Transposons etc) Mutant phenotype lone DA flanking Tn mutant omplementing library Sub-clone, sequence, analysis. Utilise domain conservation Southern probe consider probe and stringency RDO consider sequence conservation and codon usage Genome Sequencing BLAST comparisons of cds during annotation : 7 Genetic analysis of mechanisms Delete component ie remove HK is RR constitutively active? Over express component increase RR quench phosph of receiver? increase HK in absence of signal is it in active state? Domain deletion ie delete signal domain. Is kinase constitutively active? signal domain act negatively ie delete receiver domain -what effect on transcription? Domain disconnection ie separate input and transmitter disrupt signal pathw? Domain chimeras use domains from different systems : 8 4

5 Signal transduction mechanisms The transmitter and receiver are the common domains that define a TR onsider general ideas about mechanisms before look at detailed example /EnvZ Highly conserved amino acids in transmitter and receiver are important in accepting phosphate and in kinase activity : 9 Input signal Output signal AD i O 4 AT O 4 H G1 D/F G2 D D K Input domain Transmitter domain Receiver domain Output domain Histidine protein kinase Response regulator : 10 5

6 Histidine rotein Kinases The HK sensor (input) domain is specific EnvZ osmolarity by membrane changes? hoq binding of divalent cations see diagram The transmitter has 2 sub-domains H boxsubdomain phosphorylated His -G-D/F-G box sub domain has ATbinding activity and contains kinase activity see diagram these are conserved AA in most transmitters : 11 HKs (cont.) Specificity between cognate HK-RR systems why does HK-a interact with RR-a and not also RR-b, RR-c, RR-d? surrounding AA show conservation in subfamily members each system will have specificity in conserved areas ross-talk evidence of interaction between some systems : 12 6

7 trb VirA hoq AgrB EnvZ BvgS hea SpoIIAB H box box D box F box G box hydrophobic (TM) RR receiver domain H box like domain : 13 HK sub-domains H - b o x - b o x G 1 - b o x D /F - b o x G 2 - b o x AFQ2_STRO DMSHELRTLT VILALIGA RVRDGGIED FDRF GSGLGL ARB_EOLI TISHELRTL QILWLISA EVEDSGIGIQD FAMY ATGIGL ATOS_EOLI GVAHEVRLT QVLLILIA SIEDGGIDLS FDF GTGLGL BAES_EOLI DISHELRTLA QLFLLES TFADSAGVSDD FERF GSGLGL BASS_EOLI DVAHELRTLA MLLRLVEA AVEDEGGIDES SKAF GIGLGL BVGS_BORBR TMSHEIRTM QVLSLVGA SVSDTGGISEA FKF GTGLGL XA_EOLI DISHELRTLT SALEIVRA TVDDDGGVSE FRF GTGLGL UTS_STRLI ASHELRTLA RIALLVQA VVTTGVVAY FEF GVGLGL DEGM_BASU SIAHEVRLT QALMFVKG EIDGVGMSRQ GTAY GGIGT DIVJ_AUR MSHELRTL QIVLLVSA VVADTGVGISE GRY GTGLGL EVZ_EOLI GVSHDLRTLT RAVAMVVA QVEDDGGIAE FQF GTGLGL FIXL_AZOAL TLAHELQLS QVLVLFRA SVDTGHGISDE FTF GMGVGL KDD_MYTU AVSHDLRTLA RVLALIDA VIDEGGVRG FAF GVGLGM LUXQ_VIBHA KMSHEIRTI QILFLVSA ELTDTGIGIESD FEF GSGLGL ODV_BRAJA SIAHEITQLA QVIILVMG SVTDGVGIAE LDF GLGMGL TRB_EOLI GLAHEIKLG QVLLIVRA DVEDGGIH FYM GTGLGL HOR_BASU VSHELKTIT QVFLLVA EVADSGIGIQKE FERF GTGLGL RESE_BASU VSHELRTIS QVFTLIDA DIKDSGSGIEE FERF GTGLGL VAS_ETF YLAHDIKTLT KALSVILA SVLMGVHIDDT FIF GSGLGL VIRA_AGRTU GIAHEFILG QMLMLKA SVSDDGEGIAET FEF GTGLGL OSESUS HEXXXL QXXXXXXA DXGXG FXF GXGLGL hosphorylation of His AT binding & Kinase : 14 7

8 HKs The D/FG boxes acts as kinase bind AT HKs act as dimer intermolecular phosphorylation kinase sub domain involved in dimerisation (hea mutants) mix mutants and domain deletions: kinase- H box- kinase- wt : 15 HKs Rate of autophosphorylation determined by input signal influence dimerisation or influence dimer structure Kinase has dephosphorylation activity need to dephosphorylate RR receiver so response terminates when signal finishes Domain structure in HKs can be complicated: BvgS his kinase asp his : 16 8

9 Response Regulators Receiver domain is conserved output domain sub family & system specific hey protein interaction Transfer of requires transmitter-receiver interaction Many (not all ) RR receiver domains inhibit output domain delete receiver constitutive active output domain ½ life of Asp- affect response dephosphorylation HK + others Receiver domain output domain A.. Domain D D K Mg 2+ binding acidic pocket with Ds : 17 hey SpoOF FrzZ hob ho VirG BvgA AgrA tr AlgB heb Receiver domain D RR domain comparisons D K Output domain Domain organisation of RRs. The -terminal receiver domains are conserved among RRs and contain the highly conserved amino acids D, D, and K. Based on similarity of -terminal domains, RRs can be sub-divided into sub-groups. Receiver domain Winged HtH domain HtH domain Sigma-54 interaction domain HtH domain Methylesterase domain : 18 9

10 TR example: EnvZ/ Background response/sensitivity to high/low osmolarity in Ec. conc of small molecules salts/sugars reciprocal changes in two OM Mutant studies id 3 loci omp, ompf, ompb. conjugation mapping! omp (21 min), ompf (48min), ompb (74 min). ompb locus has two genes envz & ompr gene fusions show ompb involved transcriptional regulation EnvZ 450aa 10 copies/cell inner Memb 239aa 1000 copies/cell cytoplasmic : 19 Omp 1.08nm 1.16nm OmpF OM signal EnvZ IM whth H D omp ompf conc. porin low total porin Omp OmpF osmolarity high : 20 10

11 The signal responds to concentration of small molecules: not molecule specific Stimulus periplasmic trucated mutant (EnvZ115) not respond TnphoA study show mutant not in periplasm mutant partially compensates for EnvZ null mutant Stimulus assoc. with low osmol. sensor domain mutants result in constitutive high osmol phenotype Omp>OmpF stimulus complex unlikely specific molecules not cytoplasmic membrane/cell wall changes? : 21 HK: EnvZ EnvZ activities: autophosphorylation phosphorylation phosphatase 2 membrane spanning domains 2 domains transmitter conserved How show -domain is sensor? Gene fusions show -domain is periplasmic sequence show TM regions EnvZ115 cytoplasmic in right place mutations in sensor give constitutive F-+ phenotype : 22 11

12 EnvZ (cont.) Is -domain a sensor? cont Domain chimeras: chemotaxis sensor (respond to AA) attached to transmitter. Omp/F controlled wrt [AA] phosphate transfer H243V not autoph. vivo& vitro EnvZ- - in vitro EnvZ+- dephos in vitro balance of phos vs dephos activity? EnvZ signalling states envz null allele F+/- - not= to low or high osmolarity phenotype two active states: not simply On or Off : 23 EnvZ mutants cont. kinase vs phosphatase activity EnvZ kinase- mutants envz247 and envz250 both confer F-/- phenotype merodiploid of phosphatase- (envz473) and either kinasemutant gives constitutive low osmolarity phenotype co-dominant opposite extremes in signalling state: ph tase dominant or kinase dominant in vitro w/ mutants: low is > phosphatase activity high is > kinase activity stimulus (auto-) modulates: kinase phosphatase : 24 12

13 conc. porin OmpF Omp low osmolarity high envz 247 envz null envz 473 hos+ Kin- hos- Kin : 25 RR: transduces signal from sensor to changes in transcription reciprocal regulation of porins Omp and OmpF likely to have at least 2 states associated with low and high osmolarity Has receiver domain and DA binding domain rystal structure of DA binding domain show has winged HtH domain bind DA and interact with RA : 26 13

14 hosphorylation states of Two classess of ompr mutants: constitutive F+- (low) constitutive F-+ (high) F-+ (high) dominant to F+- thus F-+ allele stops F+- allele activating F transcription Due to simple /-? o: envz null or kinase- are F-- when not phos ot -p/- (MR) Ratio of to -? current model based on this DA protection studies show binds to large regions around OmpF/ promoters binding change with phosphorylation mutant bind different places : 27 OmpF O Low Osmolarity. Low - and high OmpF OFF High Osmolarity. High - and low : 28 14

15 Omp OFF Low Osmolarity. Low - and high Omp O High Osmolarity. High - and low : 29 interaction with RA RA α-subunit (-terminal) required for regulation rpoa mutants can suppress envz/ompr mutants rpoa mutants can affect porin expression mutants map to -terminal Mutations in ompr that affect interaction with RA map to 20 amino acid exposed loop in -terminal part of HtH domain : 30 15

16 Model based on promoter binding studies for interaction with ompf o-operative interactions between - molecules from: Huang et al. AS : : 31 conc. porin low osmolarity high total porin Omp OmpF LOW EnvZ: p tase>>kinase : >>- orin: OmpF>>Omp HIGH EnvZ: kinase >> p tase : ->> orin: Omp>>OmpF : 32 16

17 Summary TR Modular signal transduction systems Super-family/sub-family conservation of transmitter and receiver domains transduction by phosphorylation many systems regulate transcription detailed example in EnvZ/ Differences from paradigm in other TR systems : 33 17