This article isThis article by protected copyright. All rightsreserved. 10.1111/febs.14047doi: differences to lead between the of thisversion citethisarticle and asVersion Record.Please copyediting been throughthe This article acceptedhas been for publication andundergone fullpeer review buthasnot transduc cyclase adenylate Keywords E Tel FAX: +49 72076 Tübingen, Germany, J C Paper :Regular Article type Novel title Running 72076 Tübingen, 2 Germany Tübingen, 1 Joachim E. Schultz Miriam Ziegler A of Characterization . - orrespondence denylate Max 72076 8, Morgenstelle Auf der Tübingen, Universität der Institut Pharmazeutisches mail: mail: AcceptedD 8, Morgenstelle Auf der Tübingen, Universität der Institut Pharmazeutisches E. Schultz, Article :
+49 - Cylase Planck Joachim.schultz@uni tion 7071 7071
Cyclases , - Legionella -
Transducer Institut für Entwicklungsbiologie, Abt. Proteinevolution, Spemannstr. Spemannstr. Abt.Proteinevolution, für Entwicklungsbiologie, Institut 2972475
1 292476 , JensBassler Germany
,
cyclic AMP cyclic
1* a and and
N ;
ovel - Element in A in Element Guanylate
, pagination andproofreadingtypesetting, process,may which - S tuebingen.de
(cAMP) ignal 2 , Stephanie Beltz , Stephanie T
C , receptor regulation, regulation, , receptor ransducer ransducer denylate Cyclases denylate yclases
Element I Element 1 , Anita Schultz , Anita
quorum ntrinsic ntrinsic 1 , Andrei N.Lupas , Andrei - sens to C ing lass , signal signal I II a/ b 35,
2 , - This article isThis article by protected copyright. All rightsreserved. cAMP regulation. cellular engineered AC model system. Thismay forecastapreviously unknownmechanism for rece membrane that suggest Thedata cyclases. guanylate vertebrate in and ACs IIIb cyclase distinct that established whenstar successful AutoInducer ligand to the responses orinhibitory stimulatory in exit resulting membrane of the receptor Coupling activation. N located 19 novel a identified chimera, we Usingthis LqsS. hexahelical bythe ACRv1625c mycobacterial of the anchor membrane hexahelical the we replaced thisquestion, address To unknown. yet as are that characteristics functional important possesses region thatthis suggesting via been identified inbacteria andeukaryotes. comprise III ACs Class cAMP. messenger second the produce that proteins signalling are (ACs) cyclases Adenylate Abstract Cholera Sequences of ANalysis CLuster Legionella AC, adenylate Abbreviations
Accepted Article hexahelical domain hexahelical iver domains transduce regulatory signals to the downstream catalytic domains inan domains catalytic to downstream the signals regulatory transduce iver domains
- terminally to the catalytic domain that links receptor stimulation to effector effector to receptor stimulation links that domain catalytic tothe terminally
a utoinducer - 1 (LAI cyclase; GC, guanylate cyclase; GC, guanylate cyclase;
- 1). In contrast, on the AC effector side functional coupling was only was coupling functional side ACeffector onthe In contrast, 1). ting with the cyclase the ting with - s s 1; CAI . four groups (class IIIafour groups (class In eukaryotic ACs, m ACs, In eukaryotic
- 1, - .
transducer elements are universally present in class IIIa and IIIaand class in present universally are elements transducer Legionella
to the AC was possible at several positions distal to the tothe distal positions atseveral possible to ACwas the - transducer element. Bioinformatics approaches approaches Bioinformatics element. transducer Many c Many
autoinducer
embrane anchors are well conserved, conserved, well are anchors embrane - quorum
d); of these, class IIIa and IIIb ACs have ACs and IIIb IIIa class d); these, of CTE, cyclase - lass ACs IIIa lass amino - sensing receptor from receptor sensing - 1;
- acid cyclase acid QS, quorum - transducer
are anchored to membranes membranes to anchored are - transducer element transducer - sensing; sensing; - element; LAI element; Legionella Legionella - delimited delimited CLANS,
- 1, , This article isThis article by protected copyright. All rightsreserved. and histidine CqsS LqsS in AcceptedACs IIIa class in effector cytosolic to receptor from transduction the ACs IIIa class of anchor membrane a for ligand, extracellular from receptor the replaced we Recently, protein m is it loops aliphatic as the for ligands in present receptors ACs 6TM loops connecting described been have sp transmembrane individual Articlethe that is note of feature One protein. congeners activity homolog bacterial monomeric have ACs vertebrate (6TM) domains characterized are and IIIa class to belong only bacteria in present Classes signaling are messenger (ACs) cyclases adenylate III Class Introduction
6TM AC anchors anchors AC 6TM , .
such as the mycobacterial class IIIa AC Rv1625c, a prototype of the mammalian mammalian the of prototype a Rv1625c, AC IIIa class mycobacterial the as such IIIa and IIIb are found in bacteria and eukaryot and bacteria in found are IIIb and IIIa [3, 4, 9, 10] 9, 4, [3, [4]
ost likely likely ost cAMP hydroxy
The membrane anchors of such ACs comprise approximately 40 % of the total total the of % 40 approximately comprise ACs such of anchors membrane The Vibrio Vibrio QS
n to opeetr ctltc domains catalytic complementary two and [6
- receptors from receptors [1] - Cholera - 8] Vibrio, Legionella, Burkholderia Legionella, Vibrio, and demonstrated that the receptor conferred direct regulation by the the by regulation direct conferred receptor the that demonstrated and keto [5] that . . .
that
This This They are essentially identical in identical essentially are They . hy are They
This assertion requires that the mechanism of mechanism the that requires assertion This [2] - this type of ligand ligand of type this lipids
kinases oss 6M ude wt minimal with bundles 6TM possess hexahelical hexahelical .
receptor type is represented by bacterial quorum bacterial by represented is type receptor AutoInduc etbae eoe ecd tn C sfrs n isoforms, AC ten encode genomes Vertebrate ans 8 1, 12] 11, [8,
[5] Vibrio subcategorized into four groups, groups, four into subcategorized
[5, 13] .
y Recently, membrane receptors receptors membrane Recently, er
ebae nhr f h R12c C y h QS the by AC Rv1625c the of anchor membrane lack significant extra significant lack , CqsS, , ,
- this is a proof of proof a is this . CAI 1,
.
n h asne f iebe extra sizeable of absence the In binds binds y two by
- and 1 in [5]
to and o and ue Legionella .
different different
Short of Short rtis ht prod that proteins the membrane space of the receptor the of space membrane the s e
layout layout s, whereas classes IIIc and IIId are are IIId and IIIc classes whereas s, that ther eubacteria eubacteria ther principle
- [3] membranous loops connect loops membranous eur hmdmrzto for homodimerization require
to the membrane anchors of anchors membrane the to , LqsS, , . hexahelical hexahelical the identification of a ligand ligand a of identification the -
sh egh α length h pseudo The
ares similarities similarities ares for a receptor function of function receptor a for denoted denoted
intramolecular intramolecular have been identified identified been have with known ligands ligands known with - [6, 8, 11, 12] 11, 8, [6, helices and short short and helices c te second the uce transmembrane transmembrane - - - heterodimeric heterodimeric sensing (QS) (QS) sensing membranous membranous i ls IIIa class e of ne with that that with signal signal which .
The The ing - d. -
This article isThis article by protected copyright. All rightsreserved. transduction. signal of point the that that LAI exceed not did A ( LqsS 6 the replaced we Here, is memb 6TM we Recently, the Connecting RESULTS vertebrates are Bioinformatic past. the in detection escaped had which and acids coupling functional pneumophila domain. catalytic the of start the within potential i far, So Arg218 uto
Accepted directly Article - 1 ( 1 rsn in present Rv1625c Rv1625c I , , nducer
and initially used the the used initially and the Fig. ) ly
membrane n o lnig ( linking for
stimulated 190 % by 1 µM of the ligand CAI ligand the of µM 1 by % 190 stimulated
c 0 o 0 residues 80 to 60 involved in in involved 1). The rather The 1). . rane yclase -
1 n te v65 AC Rv1625c the and have partners with partners
120 %. 120 (LAI all
anchor of the of anchor connecti QS - t ? known known
- ransducer eosrtd ht CqsS that demonstrated
- 1 receptor LqsS to Arg218 of the Rv1625c of the toArg218 LqsS receptor ee w Here (S) , Fig. - signal signal H eiie cas Ia ACs IIIa class delimited alf on
weak M ude of bundle TM - 1). AC activity activity AC 1). ls IIIa class 3 - equivalent equivalent maximal activation activation maximal
LqsS LqsS - identif e between LqsS and Rv1625c was suboptimal for suboptimal was Rv1625c and LqsS between Generating a chimera between the QS the between chimera a Generating hydroxy -
that transducti Rv1625c e , albeit albeit , lement ( lement
less efficient less bridge the distance between the membrane exit and the the and exit membrane the between distance the bridge ,
y - we n II ACs IIIb and pentadecan
statistically statistically sequence positions positions sequence n characterize and
on CTE AC Rv asked a siuae in stimulated was
r unknown are 1625c [5] ), the , . ly which is is which :
(EC
AC
W than with the CqsS receptor from receptor CqsS the with than - significant significant
, 4
Q , and and , hat are are hat AC by the the by AC 50 activity in such a CqsS a such in activity - cytosolic cytosolic on S ) - was was eetr from receptor e - 1 ((S) 1 indispensable for signal transduction transduction signal for indispensable )
. Such elements should be located located be should elements Such .
. con a also H graphically graphically in the sequence requirements for a a for requirements sequence the
analyse owever, its maximal stimulation stimulation maximal its owever,
stimulation might have indicated indicated have might stimulation LqsS LqsS
- the chimera chimera the 3 sequence sequence in QS evd emn o 19 of segment served -
hydroxy AC ligand - eetr from receptor ( - s demonstrate that CTEs that demonstrate s
Met191 receptor from receptor Vibrio, estimated to be to estimated - regulated regulated - tridecan elements elements - by the the by Rv1625c chimera chimera Rv1625c )
can replace the the replace can and in and intramolecular - 4 Legionella L Legionella GCs - Vibrio egionella on Rv1625c Rv1625c that
e 48 nM nM 48 amino amino
;
from from [5] , or , are are ) . ,
This article isThis article by protected copyright. All rightsreserved. connection significantly concentration maximal ( sequences linker longer with prevailed responses stimulatory whereas exit, membrane the to residues 8 within was T EC estimated LqsS the in achieved was % 190 of stimulation exit membrane the at observed was stimulation Significant exit. membrane facilitated was dimer AC productive a of formation chimeras latter the LqsS of exit membrane three in observed also signif C cAMP·mg nmol presumed (4.9 active themodestly at directly i.e. Tyr178, at linked chimera equivalent membrane unabridged cAMP·mg nmol 64 to 15 from ranged activities were results The unchanged. remained Arg218, cyclase, of exit cytosolic predicted the to distal positions different 11 at Rv1625c with joined was LqsS Therefore, Accepted together, aken Article icantly affected by LAI by affected icantly epne wr i te ae re o mgiue o al constructs all for magnitude of order same the in were responses
o tee constructs these for in subsequent constructs. in subsequent
affect 50 - epne curves response
was was mostly mostly rnmmrn hlx 6 helix transmembrane
receptor ,
, 48 nM LAI 48
aa A atvte wr cnieal higher considerably were activities AC basal uh s l15 Ls8, e11 n Aa9 ( Ala192 and Met191 Lys187, Gln185, as such o - , respectively , ther anchored Rv1625c AC AC Rv1625c anchored niioy epne wr osre we te on of point the when observed were responses inhibitory Fig. - - ligand interactions. interactions. ligand 1 ( 1 constructs conjoined four, six and eight amino acids distal to the the to distal acids amino eight and six four, conjoined constructs - estimated estimated
1. 2). , o shown not
s nlzd y etr blotting Western by analyzed as The
- 1 (at Met182, Gln184 and Gln186; Gln186; and Gln184 Met182, (at ·min estimated estimated
IC t Tyr178 at - ) 1 Ti i This . 50 ).
oee, w However, = 65 nM; 65 = M1 Lys187 Lys187 - - K187 1 [4] ·min iad concentration ligand ndicat with . Rv1625c
while while ees f p of Levels - 1
, comparable to the activity of the the of activity the to comparable , Fig. greater distance from the the from distance greater in LqsS LqsS in c ed onnection t ams 7 % inhibition % 70 almost ith
the 2A that R218 unexpected - terminus of TM6, was was TM6, of terminus
). on of point - V443 a ue a te on of point the as used was I the nhibition of chimeras was was chimeras of nhibition Ti may This .
oen xrsin w expression rotein
positions more distal to distal more positions dt nt shown) not (data
chimera modifications Fig. s
Fig.
connection to the the to connection eurd o half for required (
Fig. 2
A) ( 2 F i A
. Notably, in in Notably, .
ndicat ig 2 ). ( connection respective respective . A pre 2B). The 2B).
). Basal Basal ). Maximal Maximal , did not not did
e it was was it dicted . The The .
only only that that ere -
This article isThis article by protected copyright. All rightsreserved. Rv1625c of regulation the in function essential an have Lys236 to Arg218 in Rv1625c in Arg218 of downstream ( regulation and activity AC of loss progressive a in resulted Ala224) to (Ser219 residues downstream six and Arg218 of deletion Stepwise segment was stimulation that indicated This R218D. for stimulation % 206 and R218Q for % 241 R218L, for % 210 i.e. stimulation), % (190 construct parent pot ligand and protein chimeric the of stimulation the (R218L), hydrophobic a with Arg218 charged positively the replacing mutants, point three generated we role, functional ( homologs in position this at variability sequence high the to contrary LAI by stimulation LAI to unresponsive were Connection but activities, basal high in had 217 to chimeras 214 resulting (residues Arg218 to prior acids amino additional four to up serine successfully the in position corresponding Rv1625c identified be not was terminus indicated me between sequences linker N the Connecting ext
AcceptedFig. Article ,
- we
sensitive AC chimera AC sensitive 4 ; 170 pmol cAMP·mg pmol 170 ;
rather than on the physical properti on physical the than rather analyzed the the analyzed s h fso pit o ciea between chimeras for point fusion the as a
conserved segment characterized by an invariant central proline. proline. central invariant an by characterized segment conserved i Ag1 rsle i a 75 a in resulted Arg218 via fused fused
evident from evident AC s clearly as - to the 2TM chemotaxis receptor Tsr from Tsr receptor chemotaxis 2TM the to ( 1
Rv1625c to Lys187 of the the of to Lys187 Rv1625c Fig. connecting connecting
2 and and 2 a
( a Fig. [14] distinct - 1 class IIIa IIIa class polar (R218Q), or or (R218Q), polar ·min bae oan ad aayi dmis f ye Ia ACs IIIa type of domains catalytic and domains mbrane
. We . 3 Fig. , ). point on point - 1 up to up
) and a loss of regulation. This indicated that residues residues that indicated This regulation. of loss a and ) drop of sequence conservation sequence of drop ale eprmns a scesul ue Arg218 used successfully had experiments Earlier
tested LqsS tested 4) C yG from CyaG AC .
Lys236 The % drop of basal activity, but but activity, basal of drop % es es the
of th Fig. importance of Arg218 in Rv1625c seemed seemed Rv1625c in Arg218 of importance QS side of side an ,
- resulted in low basal AC activity (Lys236 (Lys236 activity AC basal low in resulted is particular particular is
- Lys receptor receptor acidic (R218D) residue. In all instances, instances, all In residue. (R218D) acidic 4 ). ency were comparable to the Arg218 Arg218 the to comparable were ency h AC the
187 The outright deletion of deletion outright The
the rhopr pla Arthrospira - Rv1625c constructs constructs Rv1625c dependent Rv1625c Rv1625c Escherichia
Lqs n CqsS and amino acid. amino S
. The AC. Inspection of the the of Inspection AC. on Fig. tensis
coli [5 the
N ihy significant highly
3 ] AC activity AC - . terminus terminus ). To clarify its its clarify To ).
,
( Similarly, the the Similarly, length of length resulting in in resulting connected at connected Arg4 Fig. 19 - It ( 1 s
residues residues iey C likely 6 was 56) Fig. 4 ). The The ). could could . W .
this this 4 of ). e a -
This article isThis article by protected copyright. All rightsreserved. domain catalytic pseudo In proline. central the lacks C the though even deviation, square mean root Å 1.5 than less to superimpose which structures, crystal aforementioned the with agreement are homodimeric the of CTEs ( catalytic ACs proline the that noted We IIIb and cyclases. IIIa class modern the that conclude pseudo 3hls GC ACs. III class to outgroup an form which cyclases, eukaryotic regulated 3 II cyclases nucleotide of sequences in presence its analyzed comprehensively we fold, cyclase the of component Asking Bioinformatic c transduction. signal intramolecular the region this termed ) membrane the including ACs, IIIb and Ia Accepted Article were CTEs .
highly highly , n o te ls II slbe C rm human from AC soluble IIIb class the of and ), from rat, where it was solved together with the upstream signaling helix (S helix signaling upstream the with together solved was it where rat, from . In the class IIIb AC10 IIIb class the In . , - whether heterodimer were solved together with the catalytic domains (pdb: 4clf (pdb: domains catalytic the with together solved were heterodimer which C1 similar for all CTEs, irrespective of the presence of presence the of irrespective CTEs, all for similar )
domain
a oiial cuh or attention our caught originally had characterization beti cass Ic n IIId and IIIc classes in absent
s the CTE is in fact a novel signaling element, rather than a constitutive constitutive a than rather element, signaling novel a fact in is CTE the
CTE is CTE contain an invariant serine three residues upstream of the central proline, proline, central the of upstream residues three serine invariant an contain
CTE sequences sequences CTE by bioinformatics by GCs , whereas it whereas
, a dis a bacterial class IIIb class bacterial
which are closely related to class IIIa ACs, but not in diguanylate diguanylate in not but ACs, IIIa class to related closely are which from ycl tinct ase
of
vertebrates is
element, which likely evolved in the common ancestor of ancestor common the in evolved likely which element, - a transducer
absent universal c universal . We found it N it found We . are are - eeoiei ACs heterodimeric o oe extent some to - bound and soluble isoforms of vertebrates of isoforms soluble and bound
in ACs ,
- this proline is conserved conserved is proline this ACs CTE - element (CTE) for its putative role in in role putative its for (CTE) element terminal CTE of the hAC the of CTE terminal . Secondary structure predictions, however, however, predictions, structure Secondary . yclase ,
CE were CTEs . s is conserved only in CTEs of class IIIa IIIa class of CTEs in only conserved is C
- of the the of rystal structures of CTEs exist from a a from exist CTEs of structures rystal terminal to terminal
( -
h transducer AC10)
diverged rm vertebrates from C2 this this domain wee oh Ts f the of CTEs both where ,
also also catalytic domains of class class of domains catalytic proline
- ( element Fig. rsn in present . It is also absent in in absent also is It . in 10
residue. T residue. 3 CTE
is divergent and divergent is ). ,
;
The invariant invariant The CTEs [15, 16] [15, s - helix) (pdb: (pdb: helix)
h ligand the of the the of his
of )
. ( is in is
Fig. first first We C1 C1 -
This article isThis article by protected copyright. All rightsreserved. domains, membrane the of presence the the between complex W AC from CTE the conjunction catalytic the to signal CTEs cognate their AC an introducing by replaceable homodimeric the in whether, co close The isoforms adenylate cyclase between elements transducer of Exchange [1, 2] originally were designations because anchors membrane and domains catalytic respective their with together ca the of map sequences their between differences conserved yet minor, to nine the from origin their to according subdivided further their C1 vertebrate ACs, along bacterial groups three into expected, as separated, sequences The ACs. IIIa class from CTEs of analysis cluster a conducted we speciation this by Prompted aspa fr sequences in asparagine invariant an by occupied is position this whereas
Accepted CTEs that propose e Article activity ( activity ragine, and probably reflect the ancestral state. ancestral the reflect andprobably ragine, .
with t with Fig. om bacterial ACs and and ACs bacterial om - talytic domains ( domains talytic evolution of CTEs with their cognate catalytic domains prompted us to ask ask to us prompted domains catalytic cognate their with CTEs of evolution AC5 either of the the of either
6 he 5 A) -
C2 was involved were involved was C2 . Nevertheless, we note we Nevertheless, . CTE into the LqsS the into CTE . This . Rv1625c CTE Rv1625c
catalytically catalytically domains, both as a homodimer and as a as and homodimer a as both domains,
are critically involved in controlling controlling in involved critically are emphasized
two based on sequence variations in the transmembrane domains transmembrane the in variations sequence on based or data not shown not data vertebrate vertebrate LqsS CTEs of the mammalian mammalian the of CTEs inactive monomers inactive ,
eukaryotic GCs eukaryotic resulting in significant stimulation. stimulation. significant in resulting - - Rv1625c chimera, the CTE might be functionally functionally be might CTE the chimera, Rv1625c
Rv1625c chimera chimera Rv1625c the
we employed we
barely or not at all all at not or barely C2 close relationship relationship close d
). that the CTE from CTE the that domains This implies that CTEs must have coevolved coevolved have must CTEs that implies This
are intermediate, having either serine or serine either having intermediate, are
. To explore whether this depende this whether explore To
(Fig. 5) (Fig. a
resulted in in resulted membrane
AC5 soluble Rv1625c construct Rv1625c soluble between catalytic domains and and domains catalytic between the responsive to LAI to responsive
. isoform ( isoform
n engineered n , The vertebrate groups were were groups vertebrate The
closely mirroring a cluster cluster a mirroring closely omto o a active an of formation AC5 C2 - a dramatic drop in in drop dramatic a delimited delimited A - C1 pass C1 ll constructs in which which in constructs ll CTEs Fig.
soito with association
6
heterodimer heterodimer ). ( isoforms, due isoforms, Fig. ed - In all cases all In
1 ( 1 the isoform the
the LAI the
Fig. 3 , )
. linked linked basal basal
CTE CTE d
6
AC AC A on - in in ). 1 ,
This article isThis article by protected copyright. All rightsreserved. the of domain receiver retinal proteins sensory diverse coiled functionally a CTE the With of Interaction catalytic ofthe the dimerization regulate specific suggested this us, To CTEs. no has derived was domain was CTE Rv1625c that functional notable, further context is it In this appreciated crystallization for and assays enzymatic for used Mostly isoform. AC single a from dimers AC active soluble obtain to past the in difficulties enormous Rv for conditions a required even that implies CTEs nmol activity constructs a by
Accepted Article 1625c n established n
- cAMP·mg , coil eue A atvt b 80 by activity AC reduced GC
dimers dimers [4] signal
catalytic catalytic , . an S an
domain eiae CTE delicate
the catalytic domains domains catalytic the h affinity The to at thetime
a seamless seamless a transducers, which convert which transducers, the other composed of C1 and C2 domains domains C2 and C1 of composed - - helix is located directly in front of the CTE the of front in directly located is helix 1 s an independent transducer element it should should it element transducer independent an s ·min
without membrane attachment, formation of an active catalyst apparently apparently catalyst active an of formation attachment, membrane without tetrade
Cyclase of homo
partly independent independent - 1 p o 50 to up ;
E. coli E. they they - uiid v65 AC Rv1625c purified , Fig.
dimer c interaction. interaction. including histidine kinases a kinases histidine including interaction of the two two the of interaction apeptide possible - Transduc
w
6 serine receptor Tsr Tsr receptor serine ere B
). Replacement of the Rv1625c CTE by the human human the by CTE Rv1625c the of Replacement ). a impaired was residue
generally not included in the in included not generally - are inl rndcn dmis Te S The domains. transducing signal 9
used 5 lhuh h His the although
n the In
er cmae to compared % mutated such that that such mutated
domains s -
Element Element fud ewe rcie ad upt domains output and receiver between found , previously in coming signals into signals coming context 204 Ti fnig may finding This . AC5 -
443 coupling of LqsS to the Rv1625c AC via the viathe AC Rv1625c the to of LqsS coupling (see (see via a HAMP domain, a CTE and an S an and CTE a domain, HAMP a via from different different from with the the with [17
for this purpose purpose this for hetero
of the Rv1625c Rv1625c the of - CTE kinase from which the LqsS receptor receptor LqsS the which from kinase nd nucleotide cyclases cyclases nucleotide nd - below 22] h parent the
s are are s . [15] hetero
dimer had high AC activity (380 (380 activity AC high had dimer S A
). ht CTEs that - a s
heli se .
In a construct construct a In vertebrate vertebrate missing, the missing,
conformational motions motions conformational - constructs oe for role association x e osbe o onc it connect to possible be
osrc (i. 6B). (Fig. construct relevant concerning concerning relevant [4, 17] [4, aayi domain catalytic - ei i a conserved a is helix prt a cyclase as operate h CTEs the AC isoforms AC . .
In these soluble soluble these In formation of the the of formation
[23, 24] [23, connecting the connecting is
eurd for required
was not not was .
s I - n the the n were were , helix helix AC5 This This
that the the
in -
This article isThis article by protected copyright. All rightsreserved. domain membrane elements. transducer other the that depend data the together, LAI constru other t in diminished control, cAMP·mg nmol 0.58 of activity AC basal a had which chimera, S the with similarity sequence no shares but helical as residues As remain interactions stimulation maximal half a for required concentration a in % inverted was Fig. the ( S t whether investigate To signal theoutput of sign the productively interacted participants all i.e. observed, to Fig. -
helix
Accepted Article of domain catalytic the a -
CTE and cyclase domain of domain cyclase and CTE 1 stimulated AC activity with with activity AC stimulated 1 7
control 7 ). In the protein with the S the with protein the In ). ). These ‘triple’ chimeras ‘triple’ These ). ,
ent we incorporated incorporated we
CTE h S the ( LAGMAAAAGMIAHELRSPLLGIKSG
n h odr f h tasue elements transducer the of order the on of the parent construct construct parent the of ,
the cts (see Western blots in in blots Western (see cts compared to the parent construct parent the to compared represents a signal transducer element capable capable element transducer signal a represents he chimeric protein, although expression of the chimera was equivalent to the to equivalent was chimera the of expression although protein, chimeric he - ei ws ins was helix length of the the of length ed unimpaired by unimpaired ed [14, 25] demonstrated - eedn mne ( manner dependent he CTE he the the AC CyaG from from CyaG AC the .
T
[ 14 he interaction appear interaction he inhibition inhibition 25 aa long long aa 25 erted posterior to the CTE. CTE. the to posterior erted S , ] - - . helix was mimicked by mimicked was helix Rv1625c receptor LqsS the of consisting helix in front of the CTE the of front in helix
an Rv1625c of
ht h efc o lgns n h otu domain output the on ligands of effect the that
identical potency as the parent construct ( construct parent the as potency identical insertion of an additional transducer element. element. transducer of anadditional insertion ( 1 nM; 21 Fig. 4 n) a smlr o h EC the to similar was nM) (48 ,
S were well expressed in expressed well were
- 7 ei o CyaG of helix
) w Fig. . Fig.
) A. platensis A. ould functionally functionally ould , i.e. LAI i.e. , On the basis of the attenuated basal activity activity basal attenuated the of basis the On
in front of the CTE the of front in
7 s ) Ti means This 7).
). The The ).
[14, 25] . to be to
Similarly, Similarly, a respective respective a - ,
1 inhibited AC inhibited 1
the sign of the receptor stimulation stimulation receptor the of sign the -
helix. This led to an unregulated unregulated an to led This helix. , independent of the nature of the of nature the of independent
. no h LqsS the into estimated estimated robust regulation by serine was was serine by regulation robust
Removal of the S the of Removal AC activity was considerably considerably was activity AC - , the S the , of functional interaction functional of 1 interact interact h dt suppor data the ·min E. coli E. .
ht h ligand the that extension of LqsS by LqsS of extension This stretch stretch This - 1 -
helix from helix ( ligand concentration concentration ligand activity by almost 50 almost by activity Fig. with a with
50 (see Western blots blots Western (see - Rv1625c Rv1625c
for half maximal maximal half for
7 ). - Fig.
helix heterologous As a second second a As t the notion notion the t is predicted predicted is CyaG
7 -
). receptor receptor inverted inverted chimera T
, aken
was was with with and
25
This article isThis article by protected copyright. All rightsreserved. AcceptedC the into disturbances minor only with extends helices terminal coiled a in end that extend AC10 approximately of bend central a during have we analogy, this on Based to ability our GCs such of presence widespread linker upstream the different of capable Articleiso nearly between transitions between transitions conformations involve asymmetric to and shown symmetric been has activity, kinase versus phosphatase domains output input domains, transduction kinases, histidine phosphotransfer the of and CTE element the transducer between parallels analogous conspicuous the by guided were CTE considerations how wondered we Next, A s tructural tructural output domains output .
[23, 26, 27] 26, [23, The
region region t signal tran signal e N he in conformational inputs conformational to
signaling signaling functional functional
conveying opposite direction opposite - generate m termi r known are odel for CTE whose , . domain sduction
Both a hlcs om sot coiled short a form helices nal . - domains
coil, notably HAMP and and HAMP notably coil, Polar linkers appear to be a be to appear linkers Polar analogies analogies
signals to signals chimeras between between chimeras such as as such
elements
to (DHp . ee o atvt te set they activity of level
The severely severely s to the the to s - ignal
. . oh r feunl cnetd t hi N their at connected frequently are Both ). linkers energetic states for states energetic HAMP, GAF, PAS GAF, HAMP, into into
available available in signaling signaling in Th both
invariably connect directly at their C their at directly connect invariably 45 us th us s defined
osdrd possible considered transducer elements transducer ° t
ransduction DHp impair impair mig 1, 16] [15, N e CTE is is CTE e - terminal to the CTEs of CTEs the to terminal class IIIa class structures structures t mechanistically ht
and CTE and
structural between CTEs and DHp provide a rationale for rationale a provide DHp and CTEs between
inln (e.g. signaling the S the . 2, 28] [26,
n h pseudo the In
, or S or , directly key key
-
ACs - helix the ol fo wih h C the which from coil, of CTEs of , . In DHp, setting of the output, i.e. i.e. output, the of setting DHp, In . it appears that that appears it
change feature
- .
helices, helices,
CTE and .
We The hydrophobic register of the N the of register hydrophobic The compatible [26, 28] [26, conformational changes changes conformational the . His [14] show show
Given the diversity of domains domains of diversity the Given s - niae similar envisage in this this in heterodimer
rndc te signal. the transduce
that from which they they which from - kinases. ) dimerization and histidine histidine and dimerization terminal helices terminal
. . type IIIa and IIIb ACs IIIb and IIIa type
Accordingly two In DHp, In determine
the process with upstre with - termini to the catalytic catalytic the to termini helices, separated by by separated helices, se
ic CTE of human human of CTE ic eff - - m termini to signal signal to termini terminal helices helices terminal
iciently iciently utations in utations connecting , the activity of activity the
ly w am receive ,
suggesting suggesting e concerted concerted of note domains domains convert convert
CTEs
their their
and and O this the the ur -
This article isThis article by protected copyright. All rightsreserved. domains membrane harveyi by receptor remained has of years manner specific isoform [9] of sequencing initial the isoforms The Discussion o thedecoding to enable have diverged hetero domains for explanation attractive state. dimer active might segment domains N tuberculosis pH the for reported was transition structural analogous An be might swi could CTE the that - emnl o h ctltc oan osiue a aty non α unwound partly a constitutes domain catalytic the to terminal Accepted. Article
At present, we know that that know we present, At hexahelical
dimers affect The pairwise adaptation adaptation pairwise The CqsS, ,
, evolution
(Fig amenable amenable are free to self to free are forms have remained remained have
sgetn ta the that suggesting , [29] the conformational flexibility nee flexibility conformational the biochemical biochemical s
are particularly striking particularly are
.
elusive 3 a rigid a and the the and
, , membrane anchors of class IIIa ACs IIIa class of anchors membrane
which
5 [5]
to experimental testing testing to experimental and S5/2 and supported .
. tch b tch However helix , ot r Most -
v65 AC Rv1625c being a assemble into assemble from sort of sort characterization of a chimera between the the between chimera a of characterization
mammalian AC mammalian e
h co the that t ). ween bent and straight straight and bent ween the membrane anchors in eukaryotic ACs are ACs eukaryotic in anchors membrane the
ecently, coelacanth coelacanth a In this context, the context, this In the proposed receptor function receptor proposed the ,
a biochemical mystery for the past 27 years. years. 27 past the for mystery biochemical a holds class IIIc class a defined a required for such conformational switching switching conformational such for required
straight conformation of CTE correlates with the inactive inactive the with correlates CTE of conformation straight - evolution , A . because
the
f we a more
n bioinformatic
AC , to inactive monomers monomers inactive by introduction of of by introduction
active dimer active role beyond beyond role have have a transporter or channel function was proposed was function channel or transporter a
ded by the catalytic domains to domains catalytic the by ded , of man
complex, complex,
lacks a CTE a lacks they
ebae domains membrane provided evidence for a role as as role a for evidence provided , differences between the CTEs of CTEs the between differences
eaae by separated suggest that, suggest conformations ( conformations , in particular of particular in , analysis . In the inactive state the N the state inactive the In . possibly possibly that of a passive a of that
.
In the active state, the the state, active the In appropriate mutations appropriate -
esn Rv1264 sensing apart [5]
of shared properties properties shared of asymmetric signals. asymmetric .
approximately 400 400 approximately in vertebrate in F
or Fig. - [29] helix helix , a receptor to transmit transmit to receptor a QS the nine nine the CTE
. 8)
membrane membrane Similarly, the CTE CTE the Similarly, - . In the future this this future the In . and the catalytic catalytic the and eetr from receptor
conserved conserved s
At the time of time the At assemble the assemble n catalytic and s, AC provides mammalian
CTEs .
- segment segment a pseudo from terminal terminal
anchor anchor million million ligand of the the of in an in may an M. V - .
This article isThis article by protected copyright. All rightsreserved. % 90 to up by drop activities specific CTE without proteins Rv1625c soluble conservation for reasons the rationalize to difficult mechanisms catalytic identical because possibility remote rather catalysis the of between transmission purpose the for other each to tuned” “finely allosteric the O specificity respective to trio domains HAMP through signaling bacterial the between receiver of composed ACs cytosolic membrane to restricted not is probably function that claim the for termed identified E study. this entity transducer converter or extracellular an xamination Acceptedn Article the structur is the other hand, on hand, other the CTE
tuned tuned sensory cyclase
and the effector output domain output effector the and CTEs . a
e
enzymatic output domain output enzymatic conserved segment of about 19 about of segment conserved
to
, of the and
connection connection for a shared AC effector domain domain effector AC shared a for CTEs are signaling devices signaling are CTEs
which is apparent in in apparent is which
each other each
- probably transducer signal to signal domain
present present function. The The function. connecting connecting e may may e
are are are with with a
in class IIIa and IIIb ACs IIIb and IIIa class in such that that such ligand - cytosolic effector effector cytosolic element, CTE element,
functionally functionally scaled scaled a ask ask points between points
in functional sensor domain and that the catalytic domain catalytic the that and domain sensor - binding whether the the whether all sequence and structural information indicates indicates information structural and sequence t . Such mechanisms mechanisms Such . and he to trigger trigger to class III ACs III class CTEs from from CTEs small energetic differences differences energetic small
significant significant
communicate in a in communicate recep effector domains domains effector h hg dge of degree high the
.
involved in involved interaction between between interaction Probably, a Probably,
residues present in class IIIa class in present residues 3, 31] [30, domain
the LqsS LqsS the tor CTE h cnomtoa cags required changes conformational the i s and etbae AC vertebrate
-
(Fig eiie As u my el prt in operate well may but ACs delimited [18, 29, 32, 32, 29, [18, iey o e required be to likely s has been identified and characterized and identified been has ,
.
regulation of regulation could could a the ot likely, Most
. central
suitable suitable receptor and and receptor 6
A provide a high degree of signaling of degree high a provide catalytic d catalytic [2, 13] [2, ). This concerted not
only ly located ly CTE isoform play a rather passive role in in role passive rather a play 33] molecular signal transducer transducer signal molecular s .
, but not allosteric allosteric not but , indicates indicates
The sequence sequence The class IIIa and and IIIa class the ( . caused by ligand by caused Fig. Fig.
imer and In ad In
the AC the manner reminiscent of reminiscent manner - receptor/ specific specific
. . We consider this a this consider We . the 5). proline proline
Such a molecular molecular a Such dition, it would be be would it dition, and IIIb and that thereceptor
s eakby i Remarkably, S effector domain domain effector
[4] - and CTEs are are CTEs and helix CTE evolutionary
is important important is b ACs b whereas whereas differences differences essentially essentially
ACs.
/effector /effector - bolsters bolsters binding in signal signal .
This This
It is is It the
in in n ,
This article isThis article by protected copyright. All rightsreserved. P9WQ35 (Germany). Munich LMU, Hilbi, H. from His LqsS the containing plasmid A Materials PROCEDURES EXPERIMENTAL cone dominant autosomal mutants plausible expression activity anchors myokymia facial by new entirely signal regulatory direct implies this be to out provide data the summary, In ofCTEs. capacity transducing signal CTE the between interaction to inability single a from domains catalytic µ absent mammalian soluble Accepted Articlemol recent reports recent ·m ,
in in HEK293 HEK293 in .
aa A atvt i lw bt yegsial siuae by stimulated synergistically but low, is activity AC basal
and ) - primary signal signal primary
1
A that · and mutant clones with point mutations in catalytic residues were available in the the in available were residues catalytic in mutations point with clones mutant and
mg properly properly
that next to the well the to next that r rti folding protein or similarly located mutation mutation located similarly
regulatory pathway pathway regulatory h rsetv CTEs respective the - n s e un yet as an 1 ; . The point mutations mutations point The .
of [34] cells cells
two mutations two mutations interact in the absence of the respective CTEs. Finally, the functional functional the Finally, CTEs. respective the of absence the in interact ) AC .
s oee, t However, receivers transfected with with transfected -
5 - impinge on the AC membrane domains. domains. membrane AC the on impinge rod dystrophy dystrophy rod C1: characterized characterized and the established S established the and
AC are are a - characterized indirect regulation by G by regulation indirect characterized vertebrate stringent argument that membrane that argument stringent of - . 2
in the human AC type 5 type AC human the in un For - e failu he C2 kinase receptor DNA ( DNA receptor kinase [35 cAMP
impaired
the nine the - 37] heterodimeric heterodimeric is [38, 39] wildtype or respective AC5 respective or wildtype are are
mode of AC regulation is is regulation AC of mode . biosynthesis e to re also
isoform may actually actually may isoform T . tuberculosis M. hese . located between the hexahelical hexahelical the between located
Along the model proposed here, it appears appears it here, proposed model the Along membrane . known
functionally combine combine functionally - single single helix helix constructs
.
in the human retinal retinal human the in Th ( Fig.
- Uni on mutation point that cause familial dyskinesia and and dyskinesia familial cause that delimited is argument argument is
p
7 C v65 ( Rv1625c AC rot )
, strongly implies an intrinsic intrinsic an implies strongly
entry Q5ZRY7 entry suggest suggest in which the CTEs are are CTEs the which in
- isoforms mutants delimited ACs delimited - dysfunctional T proteins his his forskolin/ soluble soluble is further supported further is s
do not not do a conformational conformational a will ,
of vertebrates, vertebrates, of indicating , Uniprot entry entry Uniprot
GC yet unknown unknown yet establish C1 and C2 C2 and C1 Gs ) was a gift gift a was ) membrane membrane , affect
α in these these in will turn turn will causing causing
~ 10 (~
that
AC an
This article isThis article by protected copyright. All rightsreserved. s AC IIIa class mycobacterial a and mammalian a in used successfully been has linker This S301T). Rv1625c AC mammalian Rv1625c pETDuet introducing thus pQE30, of that by replaced was MCS N an carried Constructs cloning. facilitate to introduced were sites restriction silent necessary, 3´ the at and EcoRI or request on available ( manipulations DNA for used were methods biology molecular Standard Plasmid fromthe respectively; (Q used were corresponding the Glu237) to (Arg218 platensis A Arthrospira were usually curves response to LAI Roth. and Merck Diagnostics, Roche Sigma, from purchased were laboratory equencing by GATC, Konstanz, Germany. Konstanz, by GATC, equencing 431 - terminal terminal Accepted Article[12] LENTNRELEQRVLERTAA
and dissolved in DMSO. Because the ligand has has ligand the Because DMSO. in dissolved and 204 - 4 17] [4, construction 3 carried a C a carried 3 were fitted with with fitted were [4] MRGS - 443 .
(N3 Radiochemicals were from Hartmann Analytik and Perkin Elmer. All enzymes enzymes All Elmer. Perkin and Analytik Hartmann from were Radiochemicals 427 . -
1 n AC and C1 from New England Biolabs or Roche Diagnostics. Other chemicals were were chemicals Other Diagnostics. Roche or Biolabs England New from QERLLLSVLPRHVAMEMKA 72T, - h fdlt o al osrcs a cnimd y double by confirmed was constructs all of fidelity The His ). 6 Uniprot entry O95622 entry Uniprot DNA fragments and vectors were restricted at their 5´ their at restricted were vectors and fragments DNA - - -
ends by HindIII sites and inserted into inserted and sites HindIII by ends terminal S terminal tag for detection in Western blots. In the pETDuet the In blots. Western in detection for tag R376H)
catalytic residues mutated according to those those to according mutated residues catalytic - C2 domains. The linked linked The domains. C2 L LQEKE run - - TRAAGGPP AC tag for Western blot detection. The linked hetero linked The detection. blot Western for tag
up 455 0 aa 20 to . o te T rpaeet i te v65 AC Rv1625c the in replacements CTE the For ).
). 1 CyaG CyaG
μM
446 emns rm ua AC5 human from segments
AAGGR
and LAI - - 1 was synthesized in synthesized was 1 1 Y . surfactant properties surfactant
Uirt nr K1VRL5 entry (Uniprot
1035 S the The The d - Rv1625c mrc osrc ws His was construct imeric NRRLLHNILPKDVAA His pQE80 5 aa 25 6 - tag. The second MCS in in MCS second The tag. 204
(Δ S - 443 - XhoI; ΔNcoI). When ΔNcoI). XhoI; helix was from the the from was helix (D256S, D300S, D300S, (D256S, a list of list a - - - 3 vector the first first the vector 3 house according according house 1 n AC5 and C1 - - , ends by BamHI BamHI by ends
stranded DNA DNA stranded concentration present present
p HFLA dimers of of dimers rimer 6 - tag - s is is s 1054 C2 C2 in - - ; , This article isThis article by protected copyright. All rightsreserved. kinas creatine µg 230 and phosphate Tris mM 50 protein, µg 1.5 contained 10 for determined was activity cyclase Adenylate cyclaseassay Adenylate P β mM against overnight dialyzed were eluates The imidazole. mM 150 10 Tris mM 50 C: buffer Wash imidazole. Tris mM 50 8 Tris mM 50 A: buffer Wash wash). per ml (2 washed and column a into poured was resin the ice, on hrs 3 for rocking gentle After supernatant. the to added (Qiagen) slurry NTA debris cell proteins mM 1.6 ( g x 100,000 at press g, x (4.300 debris French cell of removal After p.s.i.). (1100 by disintegrated and 8) pH NaCl, mM 50 thioglycerol, mM 2 Tris/HCl, mM at stored Tris mM 50 with once washed centrifugation, by harvested were (pET A an At 37°C. at grown and preculture a of ml 5 with inoculated was antibiotic) (with medium L in overnight with Vectors expression Protein , 10 mM β mM 10 , urified proteins were stored in di stored in were proteins urified 600
Accepted Article mM NaCl, 15 mM imidazole. Protein imidazole. mM 15 NaCl, mM
of 0.3 the temperature was lowered to 22°C and expression was started with 500 μM μM 500 with started was expression and 22°C to lowered was temperature the 0.3 of v - ectors) ectors) ecpotao, 0 M MgCl mM 10 mercaptoethanol, thioglycerol, 20 % glycerol) and assayed for AC activity. For preparation of cytosolic cytosolic of preparation For activity. AC for assayed and glycerol) % 20 thioglycerol, - 80°C. For preparation of cell membranes cells were suspended in lysis buffer ( buffer lysis in suspended were cells membranes cell of preparation For 80°C. - - mercaptoethanol C p 8 1 m β mM 10 8, pH HCl DNA constructs were transformed into into transformed were constructs DNA B or 100 µM isopr µM 100 or
medium ( medium 1
h and membranes membranes and
at 4°C). Membranes were suspended in buffer (40 mM Tris/HC mM (40 buffer in suspended were Membranes 4°C). at 20
g , 2 mM MgCl mM 2 ,
opyl β opyl LB broth/l) at 30°C containing 100 μg/ml ampicillin. 200 ml LB LB ml 200 ampicillin. μg/ml 100 containing 30°C at broth/l) LB alysis buffer at4°C. buffer alysis - ecpotao, m MgCl mM 2 mercaptoethanol, - w D - HCl pH 8, 22 8, pH HCl ere - - , 5 M [α μM 75 e, HCl pH 8, 10 mM β mM 10 8, pH HCl 2 thio s were eluted with 0.5 0.5 with eluted were s
and 20 % glycerol and assayed for AC activity. activity. AC for assayed and glycerol % 20 and
2 removed (48,000 x g, 30 min, 4°C) and 200 µl Ni µl 200 and 4°C) min, 30 g, x (48,000 removed , 400 mM NaCl, 5 mM imidazole. Wash buffer B: B: buffer Wash imidazole. mM 5 NaCl, mM 400 , galacto pyranosid
- 30 % glycerol, 3 mM MnCl mM 3 glycerol, % 32 min in 100 μl at 37°C. The reactions reactions The 37°C. at μl 100 in min E. coli coli E.
P]
min -
T, n 2 M [2,8 mM 2 and ATP, , 4°C) membranes were collected collected were membranes 4°C) , - mercaptoethanol, 10 mM MgCl mM 10 mercaptoethanol, BL21(DE3). Strains were grown grown were Strains BL21(DE3).
(pQE v (pQE to1 - HCl, 1mM EDTA, pH 8 and and 8 pH EDTA, 1mM HCl,
50 mM Tris mM 50 2
, 400 mM NaCl, 15 mM mM 15 NaCl, mM 400 , ml of buffer C containing containing C buffer of ml ectors) for 3 for ectors) 2 , 6 mM creatine creatine mM 6 , - HCl pH 7.5, 2 7.5, pH HCl - 3 - H] 4 hrs. Cells Cells hrs. 4 - cAMP to to cAMP - l HCl , pH 8, pH , pH 50 2 - ,
This article isThis article by protected copyright. All rightsreserved. Accepted[46] HHblits tool sensitive highly the using units C2 vertebrate and C1, vertebrate bacteria, of ( database at available uniprot20 the searched we CTEs, of distribution taxonomic [44] software CLANS the using N segments as CTEs, and domains EMBL (E search 3 HMMer a in identified were cyclases Nucleotide Sequenc Analysis Sequence methods Bioinformatic Healthcare). (GE Imager DIGE Article Ettan observed. not was products expressed of Proteolysis the with out carried was the Detection of Healthcare). dilution 1:2500 a with subsequently fluoroph and Systems) R&D (Novagen antibody RGS an with incubated membrane, PVDF a onto blotted SDS to applied and proteins membrane of µg 1 to added was of integrity The analysis blot Western throughout. out carried Ligand purification cAMP during yield monitor .
ae o te epcie cluster respective the on based
SMART’s cycc family alignment alignment family cycc SMART’s was r cnuae scnay nioy y (C Pe goat Plex (ECL Cy3 antibody secondary conjugated ore es were taken from the Uniprot Reference Proteomes databank (release 2015_04). 2015_04). (release databank Proteomes Reference Uniprot the from taken were es
http://toolkit.tuebingen.mpg.de; de i DS nt exceeding not DMSO in added all xrse poen ws oioe b Wsen ltig Sml buffer Sample blotting. Western by monitored was proteins expressed
[43] .
Sequence Logos were created using the WebLogo 3 server server 3 WebLogo the using created were Logos Sequence - terminal to the former, and analyzed them respectively respectively them analyzed and former, the to terminal s from the cluster analysis. For the analysis of the the of analysis the For analysis. cluster the from s [4, 40] [4, [42] [45]
. .
1
Subs )
We extracted the sequences of catalytic catalytic of sequences the extracted We l ovn/ue Rsetv cnrl were controls Respective solvent/tube. µl with alignments built from CTE sequences sequences CTE from built alignments with trate conversion was kept below 10 below kept was conversion trate
- His - - 4 au ctf 1E cutoff value PAGE (12 %). Proteins were were Proteins %). (12 PAGE - nioy Qae) r S or (Qiagen) antibody - α - os IgG mouse version 2016_2; 2016_2; version - 5; [41] - Cy3, GE GE Cy3,
using using -
tag tag %. %.
This article isThis article by protected copyright. All rightsreserved. Accepted manuscript. the versionof final the approved and results the reviewed authors All data. analyzed and assistance cloning provided AS 8. in data analyzed and designed desig Fig in shown experiments analyzed and performed designed, SB and MZ coauthors. all with together paper the wrote and study the coordinated and conceived JES Author contributi The authors thatdeclare no conflict of interestexists. interest of Conflict Article concentration respective from derived were figures. the of legends the in are experiments of ± means as presented are Data analysis Statistical segment. this for constraints structural using Bst2 the S the and for 2x7a) (pdb: 4clf), Ectodomain (pdb: rat from AC 10 type soluble a 4p2f), (pdb: Rv1625c Stru Models Structure ctural models were created using Modeller (version 9.16; (version Modeller using created were models ctural ned, performed and analyzed analyzed and performed ned, on
s
F ig.’s ig.’s SEM - helix. The straight CTE was modelled without a template template a without modelled was CTE straight The helix. 3
experiments shown in in shown experiments
when applicable. Student’s t Student’s applicable. when and 5. JB and AL designed and analyzed data in in data analyzed and designed AL and JB 5. and - response curves. response
h etmtd EC estimated The
[47] F ig . ) ’s
- from crystal structures of structures crystal from test was used. was test
4 50 6, , /IC
50 . B n JES and JB 7. .’s
concentrations concentrations
1
n 2 and -
Thetherin Thetherin Numbers Numbers . MZ MZ . F ig . This article isThis article by protected copyright. All rightsreserved. Prob (2010) B. L. & Bassler, N.S. Wingreen, M.F., Koch, M.,Semmelhack, T., Long, J., J., L. Cong, Y.,Perez, Wei, W. L., Ng, 12. microbiology. Molecular S (2011) B.L. M.F.&Bassler, Y.,Kraml,C., Semmelhack, Wei, L.J., Perez, W. L., Ng, 11. cyclases, adenylyl mammalian of anddiversity Complexity G.(1996) A. C. Gilman, W. & R. K.,Dessauer, Sunahara, 10. channel possible sequence: acid amino cyclase Adenylyl A.G.(1989) R. &Gilman, R. C.,Reed, Slaughter, P. G., Orth,K., Feinstein, J., W. A., Tang, H. Bakalyar, F., Coussen, J., Krupinski, 9. Vibrio, and 8. Tiaden, A. & Hilbi, H.(2012) alpha Chem. Biol J Legionella autoinducer synthase LqsAproduces an alpha A J. C., Vorholt, P.,Buchrieser, A., Kiefer, T.,Tiaden, Spirig, 7. ofgenetics. review quorum Bacterial B.L.(2009) &Bassler, L. Ng, W. 6. e13098. t for function areceptor indicates Vibrio from sensor quorum by the Regulation J. E.(2016) J. & Schultz, S., Bassler, Beltz, 5. EMBO J. cyclases, adenylyl of mammalian progenitor a tuberculosis: Mycobacterium of Rv1625c E.(2001 J. J.U.&Schultz, Kurz, U., Linder, T., Seebacher, L., Guo, Y. 4. cyclases, III nucleotidyl class of evolution and regulation mechanism, Structures, R.(2006) S. S.C.&Sprang, Sinha, 3. modules, signalling 2. Linder, J. U. & Schultz, J.E. (2003) Theclass III adenylyl cyclases:multi enzymes, O.& Barzu, 1. References theMax from funds supported Acknowledgements:
Accepted quorum CqsA/CqsS inVibrio specificity detection and production ignal Article
20
Prog Nucleic Acid Res Mol Biol. MolBiol. Res Acid Nucleic Prog in part in Sensors. Sensors. , 3667
283
Danchin, A. (1994) Adenylyl cyclases: a heterogeneous class of ATP class aheterogeneous cyclases: Adenylyl (1994) A. Danchin, -
, 18113 by the Deutsche Forschungsgemeinschaft (SFB 766) and institutional institutional and (SFB766) Forschungsgemeinschaft Deutsche by the
or transporter - 3675. - 43 Cell Signal. Cell Signal. Planck 12 , 197
We thankU ,
79 2899 -
23. , 1407 - - 222. Society.
Rev Physiol Biochem Pharmacol. Biochem Physiol Pharmacol. Rev - 919. Annu Rev Pharmacol Toxicol. Toxicol. Annu Pharmacol Rev - 15
like structure, structure, like he membrane anchors of adenylate cyclases, cyclases, of adenylate anchors he membrane - 17. rsula ,
1081
- Hydroxyketone synthesis and sensing by Legionella by Legionella and sensing synthesis Hydroxyketone
Kurz for expert technical assistance. forexpert technical Kurz – 1089. 49 ing bacterial transmembrane histidine kinase kinase histidine transmembrane bacterial ing , 241 Science. Science.
- 83. -
sensing network architectures, architectures, network sensing 244
- hydroxyketone signaling molecule, signaling hydroxyketone , 1558 36 . & Hilbi, H. (2008) The The H.(2008) . &Hilbi, 157 , 461 - 64. , 105 - 80. - sensing systems, sensing -
40. ) Adenylyl cyclase cyclase ) Adenylyl - purpose
This work was This Elife. - 5 utilizing utilizing Annual Annual , This article isThis article by protected copyright. All rightsreserved. newstructures, from insights kinases: histidine K. S. M. P.,Molnar, Bhate, 26. act, balancing a HAMP domains: by transduction signal Intraprotein M. (2015) K.&Ziegler, Kanchan, E., J. Schultz, 25. for r coiled within the Interactions (2001) B. J. Hurley, D.M.& Hunt, V., E.,Daggett, S. Wilkie, C., V.,Tucker, Ramamurthy, 24. f helix:common a signaling The (2006) L. S. &Aravind, Balaji, V., Anantharaman, 23. cyclase, on typeVadenylyl site binding Gialpha a of Identification A.G.(1998) S.R.&Gilman, J.,J. Sprang, Tesmer, C. W., Dessauer, 22. 17 studies, overexpression and from knockout insights isoforms: cyclase adenylyl Gprotein for roles Physiological (2009) C.W. R.&Dessauer, Sadana, 21. 272 of P analysis kinetic and binding Equilibrium cyclase. adenylyl ofmammalian mechanism catalytic The G. (1997) A. & Gilman, C. W. Dessauer, 20. Chem. cycl adenylyl mammalian of domains cytoplasmic (1996)Two J. W. H.&Tang, Z. Huang, D., Hahn, S.Z., Yan, 19. 278 Gsalpha.GTPgammaS, acomplex in with cyclase adenylyl of domains catalytic the 18. Tesmer, J.J., Sunahara, R. K., Gilman,G. A. & forskolin, and by Gsalpha 17. Tang, W. J.& Gilman, A. G. (1995) Construction ofasoluble adenylyl cyclaseactivated o of Sciences Academy National bicarbonate, through its of and activation catalysis of mechanisms reveal adenylyl cyclase soluble human of structures Crystal C. (2014) &Steegborn, J. Buck, S. A.,Moniot, S.,Diaz, Kleinboelting, 16. biology. coiled helix the of signaling structure Crystal (2010) F. Akker, van den X., A.& Ma,Beuve, 15. reporter, cyclase receptor/adenylyl 14. Winkler, K., Schultz, A. & Schultz, J.E. (2012) Thes inproteins, modular signaling intraprotein (2013)Regula J. E.&Natarajan, J. Schultz, 13. America. of States the of United of Sciences Academy National receptor unctional theme in diverse signaling proteins, proteins, signaling theme diverse in unctional
Accepted, 5 Article , 1907 , 27787 egulation rather thanfor highaffinity, - 22. - coil domain of the beta1 subunit of the soluble guanylyl cyclase, cyclase, guanylyl soluble of the subunit beta1 the domain of coil 271 - 10
ligand interactions with natural and synthetic molecules, molecules, synthetic and natural with interactions ligand - 16. , 2. , 10941 , - 95.
- 5.
ase form aGsalpha form ase Science. Science. , Goulian, M. & DeGrado, W. F. (2015) Signal transduction in in transduction Signal (2015) F. W. M.&DeGrado, , Goulian, f the United States of America. of America. States f theUnited Int J Med Microbiol. Int MedMicrobiol. J J Biol Chem Biol J - 268 coil domain ofRetGC domain coil , 1769 , van den Heuvel, J., Weyand, M., Levin, L. R., L. M.,Levin, Weyand, J., Heuvel, , vanden J Biol Chem.Biol J Trends Biochem Sci. Sci. BiochemTrends - -
Biology direct. direct. Biology 72. and forskolin and Structure. Structure. . ted unfolding: a basic principle of ted unfolding: abasic J Biol Chem.Biol J 287
Sprang, S. R. (1997) Crystal structure of structure Crystal (1997) S.R. Sprang, , 15479 305 276
- , 243 helix determines the signal in a tsr in tsr a signal the determines helix 23 - , 26218 , optimized are cyclase guanylyl 1 - - , 981 activated enzyme in vitro, vitro, enzymein activated 88. 1 - 273 - , 25. inhibition, site 51. 38
111 107 - Proceedings of the of the Proceedings , 25831 , 538 94. -
Proceedings of the of Proceedings 29. , 3727 , 5575
- BMC structural BMC structural 45. - 9. - - 32. 80. -
regulated regulated
J Bio J
Neurosignals. Neurosignals. l Chem. Science. Science. J Biol Biol J This article isThis article by protected copyright. All rightsreserved. Neurology. ADCY5 H. (2015) W. Bird, & Raskind, E.,T.D. Roze, A., E. E.,Torkamani, M., Eichler, O., Weiss, M. M.,Dorschner, O., L. Amendola, S.,Trouillard, M.,Winesett, Vidailhet, C., D., Tranchant, D., Hi D., Gras, Grabli, B., N.,Degos, Perriere, M.Y.,Damon Davis, S., M., Bernes, C.,Anheim, D.,Mignot, Doummar, H.A., Stessman, E. S., Bonkowski, A., O.,Gad, R.,Korvatska, J. A.,Friedman, H., Meneret, D. Chen, 37. 75 myokymia, facial with dyskinesia familial in ADCY5mutations function Gain A.(2014) & Torkamani, W. H. Raskind, N.J., D.,Schork, T. Bird, P.D., Swanson, G. K.,Zhang, J. Lee, E.R., Scott, E.S., H.,Bonkowski, Pham, P. A.R., Carson, S.E., Topol, M., F. Hisama, C.S., G.C.,Bloss, D.H.,Chan, R.,Chen, J. Friedman, Z., Y. Chen, 36. 5, cyclase adenylyl in mutation exome sequencin single myokymia: facial and dyskinesia familial dominant H. (2012) Autosomal W. & Raskind, T.D. D. A.,Bird, Nickerson, E.E., H.,Eichler, Lipe, F., S.,Antonacci, M.,Girirajan, P.,Rieder, Robertson, M. M., Matsushita, Z., Y. Chen, 35. cyclase, adenylyl mammalian form of of a soluble andcharacterization Purification G. (1996) A. & Gilman, C. W. Dessauer, 34. st from thecrystal inhibition cyclase foradenylyl mechanism Anovel (2005) H. R. &Wu, Levin, L. J., R.,Buck, A. B.,Taussig, Capper, K.C., N.,Hess, T. C.,Litvin, Steegborn, 33. J. cyclases adenylyl in asymmetry of Origin J. U. (2005) E. J. & Linder, Schultz, S.R., M., Sprang, S. C.,Wetterer, Sinha, 32. signaling, transmembrane in rotation E., J. Schultz, J., A.,Martin, Schultz, V., U.,Truffault, J. M.,Linder, Gruber, F., M., Berndt, Hulko, 31. Chem. aReporter, as Cyclase Adenylyl Mycobacterial a Probedwith Transduction Signal A.&Schu A.,Schultz, L. G.,Lupas, Mondejar, 30. 1023. ofapH structure 29. Tews, Findeisen,F., Sinning,I., Schultz, A., Schultz, J.E. & Linder,J. U. (2005) The Struc kinases, histidine ofreceptor ofregulation Mechanism M.(2012) A.N.&Coles, K., Lupas, 28. Ferris, H. U., Dunin 1156 Signa Transmembrane for Implications Dunin 27. ructure of its complex with catechol estrogen, estrogen, withcatechol its complex of ructure , Oliveira, G., Xu, Scott G., J., , Oliveira, 24 Accepted, 542 Article - ture. 663
1174. 287 - - related dyskinesia: dyskinesia: related 9. - 673.
- 20 , 1022 , Horkawicz, S. & Lupas, A. N. (2010) Comprehensive Analysis of HAMP Domains: Domains: HAMP of Analysis Comprehensive (2010) A.N. S. &Lupas, Horkawicz,
85 , 56
, 2026 helix implies structure domain HAMP The M.(2006) A. N. &Coles, Lupas, - - - sensing mycobacterial adenylyl cyclase holoenzyme, holoenzyme, cyclase adenylyl mycobacterial sensing 66. 31. -
35.
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Horkawicz, S., Hornig, N., Hulko, M., Martin, J., Schultz, J. E., J. Zeth, Schultz, J., M.,Martin, N.,Hulko, S., Hornig, Horkawicz, Broader spectrum and genotype and spectrum Broader - Van Zeeland, A. A., Chen, Q., Levy, S., Topol, E. J., Storm, E.J., D., Q.,Levy,Topol, Chen, S., A.A., Zeeland, Van Arch Arch Neurol. : structures of Mycobacterium tuberculosis Rv1900c, Rv1900c, tuberculosis of Mycobacterium : structures Cell. Cell. J Biol Chem.Biol J l Transduction, l Transduction, 126 69 , 929 sama, F. M., Mackenzie, K. M., Swanson, P. P. K. M.,Swanson, Mackenzie, sama, M., F. , 630 J Biol Chem. J Biol ltz, J. E. (2012) HAMP Domain HAMP E.(2012) J. ltz, 271 - 940. - 5. Journal of molecular biology. biology. molecular of Journal
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308 - mediated mediated J Biol Biol J 397 , 1020 EMBO - , of - - – This article isThis article by protected copyright. All rightsreserved. Accepted S1 Table Supplementary Information Supporting Bioinformatics. Curr Protoc 47. Webb, B. & Sali, A. (2014) Comparative Protein Structure Modeling UsingMODELLER, byHMM searching sequence protein lightning HHblits: (2012) J. A. &Soding, Hauser, A., M.,Biegert, Remmert, 46. Res. an integrative platform for advanced proteinsequence andstructure analysis, Toolki MPIbioinformatics The (2016) A.N. J. &Lupas, Soding, V.,Nam,S.Z., Alva, 45. generator, logo asequence WebLogo: (2004) S.E. M.&Brenner, J. G., Chandonia, G.E.,Hon, Crooks, 44. similarit pairwise on based Article families protein visualizing for application Java CLANS:a A.(2004) & Lupas, T. Frickey, 43. America. of States oftheUnited of Sciences Academy too research architecture modular simple a SMART, C.P.(1998) P.&Ponting, Bork, F., Milpetz, J., Schultz, 42. e1002195. HMMSearches, Profile Accelerated R.(2011) S. Eddy, 41. Adenylate Y. (1979) Salomon, 40. Genet. cone dominant with patients in gene (GUCY2D) cyclase guanylate theretinal in mutations of frequency and Clustering D.M.(2001) S.&Hunt, Bhattacharya, S.,Bi Johnson, S.M., A.G.,Downes, A.M.,Morris, Payne, 39. Genet. cone dominant with autosomal inpatients severity disease with chara Functional M. (2000) D. &Hunt, M.J. Warren, S.S., B.,Bhattacharya, J. Hurley, 38. Wilkie, S. E., Newbold, R. J.,Deery, E., C. Walker, E., Stinton, I., Ramamurthy,V., 44 cterization of missense mutations at codon 838 in retinal guanylate cyclase correlates correlates cyclase guanylate retinal in 838 codon at mutations missense of cterization 38 9 , W410 , 3065 , 611
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This article isThis article by protected copyright. All rightsreserved. Accepted 4clf) (pdb: domains catalytic the with together solved pseudo the of CTEs both where (AC10), human from AC soluble IIIb class (S helix signaling upstream the with together cyclase 8. Fig. Article Top : CTEs in available crystal structures. (A) structures. crystal available in CTEs :
Structure of a CTE from a rat guanylate guanylate rat a from CTE a of Structure - [16] helix) (pdb: 3hls). 3hls). (pdb: helix)
. (C) Superposition of the CTEs in in CTEs the of Superposition (B) - heterodimer were were heterodimer
Structure of the the of Structure
This article isThis article by protected copyright. All rightsreserved. Accepted Article C the coiled the extending interact,thereby helices terminal in residues hydrophobic The conformation. straight hypothetical the adopt CTEs other the while conformation straight hypothetical a adopted has bent a in are orange) and conformation. blue in (highlighted CTEs The (B). and (A) in structures the conformation. straight a or bent a adopt can that hinge a as operates CTE the that proposed is It CTEs. Å. 1.5 than less of deviation sq mean root a at superimpose and similar highly structurally are CTEs The (B). and (A) (E) (D)
The transition progresses through an asymmetric state in which one CTE CTE one which in state asymmetric an through progresses transition The cmuainl oe o te S the of model computational A
Bottom :
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