small-angle x-ray scattering reveals the solution structure of a bacteriophytochrome in the...

doi:10.1016/j.jmb.2006.09.045 J. Mol. Biol. (2006) 364, 655–666

Small-Angle X-ray Scattering Reveals the SolutionStructure of a Bacteriophytochrome in the CatalyticallyActive Pr State

Katie Evans, J. Günter Grossmann, Anthony P. Fordham-Skeltonand Miroslav Z. Papiz⁎

CCLRC Daresbury Laboratory,Keckwick Lane, Warrington,Cheshire, WA4 4AD, UK

Abbreviations used: Bph, bacteriocatalytic; CBD-DR, CBD X-ray diffraD. radiodurans; CBD, chromophore bcytoplasmic part of a histidine kinasdimerization domain; PAS, Per/AmGMP/adenylyl cyclase/FhIA domakinase/adenylyl cyclase/methyl binphytochrome; NSD, normalized sparesponse regulator; SAXS, small-angE-mail address of the correspondi

[email protected]

0022-2836/$ - see front matter © 2006 P

Phytochromes are light-sensing macromolecules that are part of a twocomponent phosphorelay system controlling gene expression. Photocon-version between the Pr and Pfr forms facilitates autophosphorylation of ahistidine in the dimerization domain (DHp). We report the low-resolutionstructure of a bacteriophytochrome (Bph) in the catalytic (CA) Pr form insolution determined by small-angle X-ray scattering (SAXS). Ab initiomodeling reveals, for the first time, the domain organization in a typicalbacteriophytochrome, comprising an chromophore binding and phyto-chrome (PHY) N terminal domain followed by a C terminal histidine kinasedomain. Homologous high-resolution structures of the light-sensingchromophore binding domain (CBD) and the cytoplasmic part of a histidinekinase sensor allows us to model 75% of the structure with the remaindercomprising the phytochrome domain which has no 3D representative in thestructural database. The SAXS data reveal a dimeric Y shapedmacromoleculeand the relative positions of the chromophores (biliverdin), autophosphor-ylating histidine residues and the ATPmolecules in the kinase domain. SAXSdata were collected from a sample in the autophosphorylating Pr form andreveal alternate conformational states for the kinase domain that can bemodeled in an open (no-catalytic) and closed (catalytic) state. This modelsuggests how light-induced signal transduction can stimulate autopho-sphorylation followed by phosphotransfer to a response regulator (RR) in thetwo-component system.

© 2006 Published by Elsevier Ltd.

Keywords: bacteriophytochrome; SAXS; two-component; photosynthesis;modeling
*Corresponding author
Introduction

Adapting to changes in environmental light con-ditions is important to many organisms. One of the

phytochrome; CA,ction structure frominding domain; CHK,e sensor; DHp,t/Sim; GAF, cyclicin; HAMP, histidineding proteins; PHY,tial discrepancies; RR,le X-ray scattering.ng author:

ublished by Elsevier Ltd.

most intensively studied class of photoreceptorsinvolved in these responses is the superfamily ofphytochromes,1,2 found in both prokaryotes andeukaryotes, that utilize a linear tetrapyrrole chromo-phore (bilin) as the light sensor. Phytochromes are ofgreat importance in higher plants where they controldiverse functions such as shade avoidance, germina-tion and flowering. The family of phytochromes hasrecently been greatly extended to include cyanobac-teria, proteobacteria, actinobacteria, fungi and slimemoulds.3 Several bacteriophytochromes (Bphs) havebeen characterized and represent the most ancientbranch of the family.4–10 Bphs photoconvert betweena red absorbing Pr and far-red absorbing Pfr formand, in most cases, act by initiating an autopho-sphorylation of a histidine and phosphotransfer to acognate response regulator (RR) that controls gene

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http://dx.doi.org/10.1016/j.jmb.2006.09.045

656 SAXS structure of a Bacteriophytochrome

expression. Bphs utilize biliverdin as the chromo-phore covalently bound to anN terminal Cys residuewhich is found within the first 10 to 25 aminoresidues.11,12 The chromophore is joined to the Cysresidue through a vinyl group on pyrrole A by theintrinsic auto-lyase activity of the apo-protein. Anisomerization is believed to occur between the D andC pyrrole rings stimulated by red and far-red lightillumination. The biological functions controlled bythese light responses in bacteria are less wellunderstood than in plants; however, recently, it hasbeen shown that two contiguous Bphs in thephotosynthetic bacterium Rps. palustris are responsi-ble for the control of a unique peripheral light-harvesting complex 4 (LH4) expressed under low-light conditions.13,14 Rps. palustris is unusual inhaving six Bphs, one more than the model plantspecies Arabidopsis thaliana, and also has severalperipheral light-harvesting complex genes. Of thefive peripheral LH complex genes three encode forLH2 complexes, absorbing at 800 and 850 nm, anunusual LH4 complex that absorbs only at 800 nmand a pseudo-gene that is presumed to be nonefunctional.15–17 The complete genome sequence17

reveals that four of the Bph genes are found close tophotosynthetic genes: Bph rpa1537 controls theexpression of the major cluster of photosyntheticgenes under the influence of far-red light and isinvolved in initiating photosynthesis in the presenceof light and low oxygen tension,10 Bph rpa1490 isnear pucC and genes coding for LH2 αβ-e peptidesand Bphs rpa3015 and rpa3016 are a pair genesadjacent to genes coding for LH4 αβ-d peptides. Ithas been proposed that as the bacteria evolved andadapted to watery environments, it utilized theattenuation properties of water, in the red to near-infra red part of the EM spectrum, to detect waterdepth and light quality/intensity based on the Bphred/far-red light absorption properties, to achieve aspecific expression profile of light-harvesting com-plexes for optimal photosynthesis.13 This bacteriumtherefore invests heavily in photosynthesis and theassociated machinery for sensing light. We namegene products RPA3015 and RPA3016, Bph4 andBph5, as they are the 4th and 5th Bphs relative to theorigin of the genome numbering scheme. Bph4 andBph5 have been shown to autophosphorylate andphosphotransfer efficiently to a cognate responseregulator (RR) RPA3017 in their Pr forms.14 The Prform is created by far-red light >740 nm or byincubation in the dark which causes the Pfr form torelax to the stable Pr state. Conversely, red light∼700 nm creates the Pfr form and has been shown tostimulate LH4 peptide expression which implies thatthe de-phosphorylated form of the cognate RRswitches on LH4 production. The domain structureof Bph4, as predicted by Pfam,18 is typical of mostBphs comprising an N terminal chromophore bind-ing domain (CBD) which is composed of a PAS (Per/Amt/Sim) domain, found in many signaling pro-teins, and a GAF (cyclic GMP/adenylyl cyclase/FhIA domain) motif named for its presence in cGMP-regulated cyclic nucleotide phosphodiesterases, cer-

tain adenylyl cyclases and the bacterial transcriptionfactor FhlA but it is also found inmanyphoto-sensingproteins. This is followed by the phytochrome (PHY)domain region and is specific to the family ofphytochrome proteins that sense red/far-red light.DHp is a dimerization and a histidyl phosphoac-ceptor domain found in histidine kinase sensorswhich also contain the catalytic (CA) kinase domainC terminal to the DHp domain. The CA domain issimilar to those found in DNA gyrase B and HSP90and is responsible, in class 1 histidine kinases, forthe autophosphorylation of a His residue on DHp.In the SMART and Pfam databases CA is namedHTAPase_c and DHp is named HisKA. For brevityand consistency with the nomenclature employedin describing the first atomic structure of acytoplasmic histidine kinase,24 we use the shorterabbreviation. The CA domain is characterized by aseries of sequence motifs named H, N, G1, F and G2boxes19 and a recently defined G3 box.20 The atomicstructure of a Bph CBD from Deinococcus radio-durans has recently been determined in the Pr stateand reveals a novel light-sensing knot that stabilizesthe PAS and GAF domains that form the biliverdinbinding pocket.21 The important steps of a func-tioning Bph are therefore: the initial light sensing inthe CBD, the transduction of this change throughthe PHY domain, the autophosphorylation of thehistidyl group by the ATP bound in the CA domainfollowed by phosphotransfer to an aspartate on theRR. In an attempt to define the domain organiza-tion and to shed light on some of the componentsteps that go towards determining the function inBphs, we have elucidated the small angle X-rayscattering (SAXS) solution structure of Bph4 fromRps. palustris in the dark stable and catalyticallyactive Pr form.

Results

Spectroscopic characterization of the Pr/Pfrstates in Bph4

Spectra show (Figure 1(a)) recombinant Bph4undergoing photoconversion by illumination withred (690 nm) or far-red light (750 nm): Bph4converts to the Pfr state with red light or convertsin the dark or on far-red illumination, to the Prform. Spectral overlap between Pr and Pfr statesresults in an incomplete photoconversion to the Pfrform with an apparent far-red spectrum composedof 35% Pr and 65% Pfr states. However, a 100% Prstate can be obtained easily by incubation in thedark or by far-red illumination. In the presence ofATP, the Pr state autophosphorylates most effi-ciently although the Pfr state is the actual func-tionally active state (i.e., releases the RR repression)which facilitates the expression of LH4 complexpeptides under red light and/or low-lightconditions.13 The Pr form is therefore the importantstate to investigate autophosphorylation and

Figure 1. Bph4 spectra showing Pr and Pfr photo-conversion. Pr form after illumination with 750 nm lightand Pfr after illumination with 690 nm light. Chromo-phore spectrum taken before and after a 45-min exposureto X-rays equivalent in dose to that experienced during aSAXS measurement.

657SAXS structure of a Bacteriophytochrome

subsequent phosphate transfer to the RR. Thedomain organization of the Pr state is describedhere using small angle X-ray scattering (SAXS) todetermine the shape of the macromolecule.

SAXS and ab initio modeling

The intensity scattered by Bphy4, in the Pr state,was collected over the range of scattering vectors0.0075<q<0.45 Å−1 corresponding to a real distancerange of 837> r>14 Å. The radius of gyration (Rg)obtained from the Guinier approximation, wasdetermined to be 52.5 Å and the derived maximumparticle diameter (Dmax) as 155 Å. Assuming a dimermolecular mass of 167 kDa, the Rg and Dmax valuesare consistent with a structure that has anisotropicaxial dimensions. Low-resolution ab initio models

were generated using the program GASBOR whichrepresents the structure as clustered sphericalresidues compatible with a chain model constrainedto a 3.8 Å inter-residue spacing. A resolution cut-offof q=0.31 Å was chosen giving the most stableconvergence for the final set of model structures. Atypical fit to X-ray scattering intensity and the pairdistribution function ρ(r) (Figure 2(b)) is shown. Thecorresponding ab initio model (Figure 2(a)) shows aflattened extended structure which, in shape,resembles the letter Y. Earlier electron microscopyimages and SAXS data22 of pea phytochrome showa similar shape although plant phytochromes haveadditional PAS domains in the dimerization regionmaking them approximately 30% larger. The largestvariation between models is found at the base of theY where the two symmetry-related domains D havea tendency tomove out of the oblate plane. To obtaina representative model, 10 structures were super-imposed and averaged (Figure 2(c)) using a suite ofprograms included in the release of DAMAVERversion 3.1. The average of normalized spatialdiscrepancies (NSD) between pairs of models was1.49 implying an absolute model discrepancy of5.6 Å which is well within the resolution limitsimposed by the data.

Domain structure

A search with the program SAM-T02,23 using theprimary sequence of the Bph4 CBD domain as atemplate, identified a CBD domain of a homologousBph from D. radiodurans exhibiting 30% sequenceidentity. The E-value of 3.1×E−51 suggests a highdegree of 3D structural similaritywith theCBDX-raydiffraction structure (Figure 3(a)) fromD. radiodurans(CBD-DR). The structure corresponds to residues 5–321 of Bph-DR and spans the whole CBD-DR apartfrom 2 short loops which presumably could not belocated in the electron density map and are missingin the PDB data. A number of structures were foundcontaining DHp and CA domains; however, themost significant in E-value (5.0×E−28), by severalorders of magnitude, was the cytoplasmic part of ahistidine kinase sensor (CHK) from the thermophileThermotoga maritima. The X-ray structure has beendetermined24 in complex with an ATP analogue andis, to date, the only example of a complete 3Dstructure of a class 1 histidine kinase showing bothDHp and CA domains in a dimeric state (Figure 3(b)). As expected, no homologous structures could befound of the PHY domain in the PDB database. Thestructures of the CBD-DR and CHK thereforerepresent 75% of the Bph structure and can be usedto explore the 3D domain organization in Bph4. It isassumed that the position and shape of the PHYdomain can be inferred from the remaining SAXSmodel after fitting CBD-DR and CHK.

Fitting domains into the SAXS model of Bph4

The dimerization of Bphs is believed to occur inthe C terminal half of the structure at the DHp

Figure 2. Small Angle X-ray Scattering of Bph4 in solution. (a) A single model form the program GASBOR. The arrowrepresents the 2-fold symmetry axis. (b) The corresponding fitted scattering data calculated from the GASBOR model.Axes are the logorithm of scattering intensity and scattering vector q. Inset is the pair distribution function ρ(r). (c)DAMAVER averaged model of ten GASBORmodels. Labeled to show the features discussed in the text which are: (A) thearms, (B) the dimerization axis passing through the center of the DHp domain, (C) the body of the structure and lobe D.


domain. This domain forms dimers through theparallel association of two domains creating a 4-helix bundle. CHK is an example of this dimeriza-tion motif as is the class 2 histidine kinase structureof CheA.25 The structure of CHK includes part of aHAMP (histidine kinase/adenylyl cyclase/methylbinding proteins) domain26,27 and comprises thefirst 22 amino acids emerging from the membranesurface into the cytoplasm formed into a coiled-coilstructure. Sequence alignment of CHK with anumber of Bphs shows the common region ofalignment with the PHY domain and the linkeramino region connecting to DHp. None of thematching regions were removed from the CHKmodel to give the canonical form of DHp and CA.For the purposes of building CHK into the Bph4SAXSmodel, the 4 helix bundle can be positioned onthe 2-fold symmetry axis in two ways. Namely thepeptide can enter from the direction nearest to thearms of the Yat A or from the base of the Y nearest tothe lobe D (Figure 2(c)). The DHp domain isconnected to the CA domain by a flexible linkerregion which can greatly affect the relative positionof the two domains. For example, in CheA25 the 4-helix bundle of the DHp domain is related by strict2-fold symmetry while the CA domain is displacedby 25°. In CHK, the ATP analogue bound to the CAdomain is 25 Å from the conserved His residue to bephosphorylated and the authors have proposed acatalytic model that requires a movement of the CAdomain facilitated by the linker region. The CAdomain is therefore relatively mobile due to theflexibility in the linker region and appears to be

essential for the functioning of the kinase. Position-ing of the CA domain in the SAXS model wasexplored by detaching the domain from the linkerandmoving to various parts of the SAXSmodel thenre-establishing connection with the linker followedby peptide torsional angle regularization. With the4-helix bundle positioned with the linker close to A,the CA domain can fill only the bottom part of thearm (A) and not very convincingly leaving theupper portion unreachable by the other domains.Fitting into the body of the structure at C results inparts of the CA structure positioned outside theSAXS model causing A to be cut-off from a directroute to D apart from a longer excursion throughdensity at the front face of the SAXSmodel. In such astructure, the PHY domain would be fragmentedinto three separate domains. The CA domain can bepositioned on the front surface of the SAXS modelbut the remaining density in the C and D lobesappears to be more than is necessary for the PHYdomain (Figure 4(a)–(c)). The alternative arrange-ment of placing the linker region at the end nearestto lobe D and reorienting CA into lobe D providesa more satisfying alternative in that the domain fitsthe shape of D well and provides un-interrupteddensity that can accommodate both the CBD andPHY domains. Superficially, in this region, theSAXS density resembles closely the shapes andrelative arrangements expected of the DHp and CAdomains, as observed in the CHK and CheAstructures, apart from a lowering of the CA domainby approximately 20 Å to accommodate one end ofthe PHY domain. The proposed repositioning is no

Figure 3. X-ray structures of homologous CBD and histidine kinase sensor. (a) CBD from the Bph ofD. radiodurans. TheCBD is formed fromaPASandGAFdomain. The biliverdin chromophore BVis in theGAFdomain and is covalentlybound toCys24which is in a region that precedes the PAS domain. The eightmissing residues are highlighted in pink. (b) Cytoplasmicdomain of histidine kinase sensor showing the dimerization helicesα1 and a2 of DHp domain and the CAdomain helicesα3,α4 and α5. The portions of structure with missing amino acids in the PDB data are highlighted by pink ellipses.


Figure 4. X-ray structures of CBD-DR and CHK fitted into the SAXS envelope. Model of CBD, DHp and CA domainsfitted into the SAXS ab initiomodel (a) top, (b) front and (c) side view. CBD in green, DHp and CA in blue. The conservedphosphorylated His residue is shown red, ADPβN in orange and biliverdin in purple.


larger than suggested for the mechanism of CHK orthat observed due to domain distortions in CheA.SAXS models have been proposed28 of the sensorhistidine kinase PrrB from Mycobacterium tuberculo-sis. These suggest some flexibility in the CA domainswith envelops larger than are required although it isdifficult to say if there is more than one CA domainconformation. Low-resolution X-ray (4.2 Å) andSAXS structures have been reported,29 of a histidinekinase in complex with a response regulator (RR),that show the RR attached to the catalytic His. Theoverall shape, of the SAXS ab initio model, is verysimilar to the C terminal half of the model proposedhere, with the difference that a CA domain takes theplace of the RR. Similar reasoning requires placingCBD-DR at the end of arm A. The orientation of thedomain is helped because of the asymmetric shape ofthe SAXS model in this region which resembles apeanut and matches well with the shape of the CBD-DR domain obtained from X-ray crystallography.

The possibility that the SAXS ab initio modelrepresents a mixture of Pr and Pfr states can beexcluded as the absorption spectra (Figure 1(b)),taken before and after the SAXSmeasurement, showthe Pr state of the sample unaffected by exposure toX-radiation.

Discussion

Description of the Bph4 model

The CBD-DR domain is a peanut shaped moleculeformed from the PAS and GAF domains (Figure3(a)). An unusual trefoil knot is formed as 35 Nterminal residues pass through a loop formed byGAF domain residues 225 to 257.21 The knot helps tostabilize this region and is an insertion loop notfound in other GAF domains. The biliverdin pocket


is lined entirely with GAF residues and of the 15 allare identical between CBD-DR and CBD of Rps.palustris apart from Ser272 which is Thr267 in CDB-RP and Phe198 which is replaced by Tyr193. Thesulfur of Cys24 in CBD-DR covalently binds to thevinyl group of biliverdin ring A and is equivalent toCys15 in CBD of Rps. palustris. In the present SAXSBph4 model (Figure 4(a)–(c)), biliverdin is situatedin the lower part of the arm while the long Cterminal α helix which terminates CBD-DR is nearthe upper part of the arm. Interestingly, secondarystructure predictions suggest that this α helixcontinues through the GAF region and into thePHY domain. This would double the length of thishelix bringing it to the base of the arm. The CBD-DRoccupies around 80% of the volume defined by Awhile the PHY domain appears to occupy the regiondesignated C which abuts the DHp domain andterminates at lobe D. Entry from the PHY into theDHp domain appears to be close to or within lobe D.In the model, the CA domain has been built into thegreater part of lobe D, although there is some roomto accommodate a small part of the PHY C terminalthat connects to DHp. It is likely that there are asignificant number of contacts, not only betweenPHYand DHp but also between PHYand CA. The 4-helix bundle spans the region that is defined by theSAXS volume at the diad axes but appears to becompletely surrounded by additional electron den-sity that is not part of the DHp. This unaccountedscattering density adds a volume that is more than isrequired by the 190 residues of the PHY domain andlinker region and appears to bury the His residuemaking it inaccessible for autophosphorylation.Irrespectively of which particular GASBOR modelis considered, there is always extra density sur-rounding the DHp domain and this differs markedlyfrom the CHK structure where the His is exposedand readily available for catalysis. The implicationof this will be discussed in the next section. The CAdomain fits the overall shape of the D lobe wellalthough there is some density unfilled. Theremaybe at least three reason for this additionaldensity: (1) nine residues in the CA domain ofCHK between 432 and 442 are absent in the PDBdata, (2) there may be some mobility of this domaindue to the flexible connection to the DHp domainand (3) it has already been noted that the PHYdomain enters the DHp domain in or around theregion of lobe D and so it maybe the case that itforms part of the lobe. The domains that have beenfitted contain the three important components thatdefine the function of Bphs: the biliverdin, thephosphorylating His (His532 in Bph4) and the ATPanalogue bound to the CA domain (Figure 4(a)–(c)).The inter biliverdin distances are 112–117 Å, biliver-din-His532 is 70–75 Å and the analogue ATP toHis532 on the opposite protomer is 30–35 Å. TheATP anologue to His distance shows, as in the CHKstructure,24 that the ATP is not in a position tocatalyze the phosphorylation reaction. In the CHKstructure, the analogue ATP molecule (AMPPNP)was hydrolyzed to ADPβN and a sulfate occupied

the position that would have been occupied by theγ-P after phosphorylation. Although hydrolysismay be one of the reasons why a locked state wasnot observed between AMPPNP and His260, a moreimportant reason is probably due to the favorablelattice contacts formed between CA domains in thecrystal structure that stabilized this open conforma-tion. There can be no such constraints in solutionand because Bph4 is in the Pr state, it would bereasonable to suggest that, at least, some of themacromolecular population has CA in contact withDHp in the region of His532 and that the additionaldensity surrounding DHp is a second conforma-tional state of CA in the catalytic conformation.

Structural model of catalysis

The model proposed for the catalytic action ofCHK has the CA repositioned through 25 Å so thatthe ATP molecule, bound to CA, is within reactiondistance of theHis residue in the DHp domain.24 TheCHK structure incorporates a sulfate ion with the O12.6 Å from theHis Nε atom and appears to mimic theinteraction of a phosphate with the histidine. Therequirement for the model is that the His residue, aswell as other parts of the DHp domain, is exposed forinteraction with the CA domain. As has been stated,all SAXS models display additional scattering den-sity surrounding the DHp domain, suggesting analternative “closed” state of the CA domain confor-mation in a catalytic orientation. The SAXS structurerepresents the Pr state of Bph4 which is the readilyautophosphorylating state and it may be expectedthat there exists a population of states showinginteractions between DHp and CA domains, even inthe absence of ATP or its analogue, as the surface areacontacts between domains are large (ca 2250 Å2 inCHK).Modeling the Bph4CAdomain, in its catalyticconformation, was achieved by aligning to the SAXSenvelopewhile constrainingOβ1 of the ADPβP to bewithin bonding distance of the sulfate ion and at adistance to NE2(His260) appropriate for the kinasetransition state, of 4.0–5.0 Å.24 The reaction is knownto proceed via phosphorylation of the histidine on theopposite protomer of the homodimer30 and somainly these sets of structures were explored in ourmodeling. The orientation of CA can be, in principle,either (1) the CA helices parallel or (2) anti-parallelwith the DHp helices. For case (1) and ADPβP in thebinding position, the majority of the CA and thelinker region, between DHp and CA, lie outside theSAXS model. An anti-parallel arrangement allowsthe linker sequence to lie between the α1 helix of oneDHp protomer and the α2 helix of the other DHpprotomer whilst fulfilling the ATP binding con-straints and SAXS model alignment requirements.Themodel (Figure 5) is therefore consistentwith boththe SAXS model electron density and chemicalconstraints, indicating that it is possible to accom-modate two conformers of the CA domain in theresting (open) and catalytic (closed) states. Animportant feature of the model is that the putativePHY domain can form extensive but different

Figure 5. CA domain in the catalytic conformation fitted to SAXS envelope. Models superimposed on the SAXSmodel showing one of the CA domains in the catalytic position. (a) Front view; (b) bottom view. Biliverdin (BV, purple),ADPβN (orange), phosphorylating histidine (His, red). DHp and CA domains of the two protomers are colored blue andcyan. The CA of one protomer (cyan) is in the position to phosphorylate the histidine.


interactions with the CA domain in the open andclosed forms. In the open case, the provisionallyassignedC terminal of PHYmustmake contactswithα3 and, possibly, α2 helices of CAwhile in the closedstate the flanking α1 and α3 helices make contact

with the C terminal half of the PHY domain. Theremaining volume after fitting the SAXS model withthe CBD, DHp and two CA domains is approximate-ly 20% bigger than required to accommodate thePHY domain. The excess is probably reasonable if


there are other conformers represented in the modelthat are in the process of changing from the open toclosed forms. The SAXS model was obtained whileimposing 2-fold symmetry; however, there is evi-dence to suggest that autophosphorylation is anasymmetric process31 implying that only a single CAdomain is attached to DHp at any one time. If this istrue there will be a population of Bph4 macromole-cules that conform to 2-fold symmetry over themajority but not all of the homodimer volume. Themodeling procedure may therefore reproduce thefeatures of the closed form but will also imposeadditional symmetry that is not there. Ideally amodel should be obtained without twofold con-straints; however, for BPh4, this produced unstablemodels due to its highly anisometric shape. Withtwofold constraints, the indications are that theposition of the CA domain varies significantlybetween GASBOR models, suggesting a relativemovement of CA out of the oblate plain and this isconsistent with the model proposed for catalyticaction.

Implication for phosphorylation and signaltransduction

In our model, the PHY domain forms a structuralbrace between the CBD and CAwhile abutting DHp.The direction taken by the CBD domains suggeststhat the protomers cross one another with theirrespective CAdomains finishing on opposite sides ofthe 2-fold axes. The contacts made by CA domainwithDHp and PHYare very different in the open andclosed states and suggest some general features forthe signal transduction mechanism initiating autop-hosphorylation. The Bph4 Pr and Pfr states arecharacterized by their spectra and the greaterefficiency for autophosphorylation in the Pr state.We propose that, in the Pr state, interactions of CAwith PHY are destabilized in the open form andstabilized in the closed form. The latter is likely toinvolve extensive tripartite interactions between thethree domains. The 30 Å movement of the CAdomain is likely to be relatively slow as the 12residues in the linker region, between DHp and CA,explore conformational space. These conformationswill include outcomes that take CA back to the openstate as well as towards the closed state hence itmaybe expected that both open and closed states arepresent in the Pr state. It is known that Bph4-Princubated with ATP is∼100% phosphorylated whileautophosphorylation is at least an order of magni-tude less efficient in the Pfr state. In addition,phosphotransfer efficiency from Bph4-Pr to theresponse regulator (RR) RPA3017 is also ∼100%efficient.14 As suggested by SAXS data, the open andclosed states can coexist in the Pr form, an essentialfeature of a model for autophosphorylation/phos-photransfer if CA and RR are to have equal access tothe DHp histidine. The implication of the model isthat phosphotransfer to a RR is only limited by therate of creation of a histidyl-phosphate residue in theDHp domain. The efficiency of phosphorylation is

likely to involve a significant number of contactsbetween the three domains that bring together andstabilize interactions of γP of ATP and Nε of His532.The CA domain covers a large area that includesboth the DHp and the PHY domains helping tostabilize the interaction. The detailed nature of theconformational changes that facilitate this can onlybe guessed at in the absence of detailed atomicstructure but two models have been proposed26,32

for sensor histidine kinases that involve the read-justment of the HAMP domain helices on signaltransduction that results in a change of the relativeorientations of the helices in the DHp 4-helix bundle.The reorientation may involve a change in the helixcrossing over angles as well as a twist applied aboutthe super-helix axis causing the His to be partiallyburied and in a none optimal binding position.Evidence exists from SAXS experiments on the Prand Pfr forms of PhyA from pea that there is arearrangement of structure.33 The model of the Pfrstate has the arms pushed further apart combinedwith movements in the C terminal domain. Howev-er, this structure has the 6 kDa N terminal domainmissing and there is no detailed interpretation of thedomain structure which differs in plant phyto-chromes. Nevertheless, the indications are thatthere maybe a movement of the arms changing therelative orientation of helices within the 4-helixbundle consistent with some of the models pro-posed. In the case of Bph4 (and by analogy otherphytochromes), the role of the HAMP domain isreplaced by the PHY domain but which, unlike theHAMPdomain, has extensive contactswith the DHpdomain andmay therefore have amore complex rolein affecting the catalytic reaction. Secondary struc-ture predications indicate the presence of two longhelices one forming a transition between the GAFand PHY domain and the other between PHY andDHpdomains. TheC terminal helix of the CBD-DR istherefore predicted to be longer by five turns andextending from the arm into the body of thestructure. This is also true of the second helixwhich will occupy a similar position as the coiled-coil of CHK. There is sufficient density in the Bph4SAXS model to accommodate this. The PHY domainis therefore flanked by α helices that provide arelatively rigid lever arm for transducing the signalfrom the CBD. The Bphs studied so far autopho-sphorylate most efficiently in either the Pr14,34,35 orthe Pfr forms5,8 and these constitute two distinctclasses of Bphs. There are also differences independence on light of the autophosphorylationand phosphotransfer steps. For example, CphA andCphB of cyanobacterium Calothrix sp. PCC7601show: (a) light-dependent autophosphorylation ofthe sensor kinase and (b) light-dependent interactionbetween kinase and response regulators RcpA andRcpB that depends on an altered access of RcpA tothe phospho-histidine and is not due to modulationof the autophosphorylation reaction.34 Bphs forwhich there are data on both the autophosphoryla-tion and phosphotransfer step do so in the sameform, either Pr or Pfr. This finding is a little surprising


as it would be expected that more biological controlcan be exerted if phosphotransfer proceeded throughopposite states rather than, as it appears, through thesame state. This suggests that phosphotransferdepends mainly on the presence of a phospho-histidine. These experiments are consistent with themodel proposed here, since the controlling feature ofthe model is the release of the CA domain from theopen state and this can be, in principle, achieved bysmall photoinduced changes. The same mechanismthen could be evolutionarily fine tuned to release theCA domain more efficiently, from the open state, ineither the Pr or Pfr forms and may explain how thetwo types of Bphs can come about. Once releasedfrom the open state, autophosphorylation can takeplace followed by phosphotransfer to the RR as theCA is freed to hop between the states. At physiolog-ical concentrations of ADP, autophosphorylation isreduced,31 indicating less affinity of ADP bound CAfor DHp that may be an additional factor facilitatingaccess of the RR to the phospho-histidine. Themodeltherefore explains how the Pr or Pfr forms can be theautophosphorylating state and why the autopho-sphorylating and phosphotransfer steps must belinked to the same state.

†http://www.soe.ucsc.edu/research/compbio/sam.html‡http://pymol.sourceforge.net

Materials and Methods

Cloning, expression and protein purification

The Bph4 (rpa3015) coding sequence was amplifiedfrom genomic DNA isolated from Rps. palustris strainCGA009. The gene was cloned, over-expressed and therecombinant protein ligated to biliverdin was purified asdescribed elsewhere.13

SAXS measurements

Synchrotron small-angle X-ray scattering (SAXS) datawere collected on the CCLRC SRS facility beamline 2.1.36

Two camera lengths (1 and 4.25 m) were used covering therange of scattering vectors 0.0075<q<0.183 Å−1 and0.052<q<0.450 Å−1 (q=4πsinθ/λ, where 2θ is the scatter-ing angle and wavelength λ=1.54 Å). Samples of Bph4were prepared in the same buffer used for spectroscopymeasurements at 5 and 13 mg ml−1 for the long and shortcamera length data, respectively. Data were collectedusing a multi-wire area detector and normalized toincident radiation intensity and then the two data setswere merged. The radius of gyration (Rg) was evaluatedusing the Guinier approximation (I(q)= I(0)exp(−q2Rg2/3)for qRg<1.3) and also from the entire scattering curve withthe indirect Fourier-transform program Gnom.37 Gnomalso provided the maximum dimension Dmax defined asthe distance r at which the distribution function p(r)approaches its lowest positive value.

Ab initio modeling of SAXS data

Low-resolution models were obtained by the ab initioSAXS modeling program GASBOR version 1.8.38 Thesearch model was constrained to the diameter Dmax<D<Dmax+15 Å compatible with a chain model con-

strained to a 3.8 Å inter-residue spacing. A simulatedannealing procedure minimized a global function com-prising the weighted χ2 fit calculated to observedintensities and a geometric constraint. The latter constraintimposes an expected histogram distribution of numbers ofnearest neighbors, a nearest residue separation of 3.8 Åand a mass center of gravity close to the origin. A 2-foldsymmetry restraint was imposed on the model through-out: initially no shape constraint was used but after severalrounds of ab initio modeling, an oblate shape constraintwas imposed. To check the results of ab initiomodeling, theprogram DAMMIN was also used.39 These models weresimilar to those obtained with GASBOR but at a lowerresolution due to the algorithm employed which uses asmaller number of atoms at larger regularly spaceddistances. To obtain a representative model, ten GASBORstructures were superimposed and averaged with thesuite of programs included in the release of DAMAVERversion 3.1.40,41 Little change in shape was observed onaveraging further models. The output DAMAVER modelis represented by a 3D matrix of hexagonal close packedatoms separated by 9 Å. To quantify the accuracy of theaveraged model normalized, spatial discrepancies (NSD)between pairs of models were calculated and averaged.During this procedure, no models were rejected based onthe default rejection cut-off criterion of NSD>NSDaverage+2σNSD. The meaning of NSD is that for each Cα atom inone structure, there is a Cα atom in the other that is withinthe distance NSD×3.8 Å.

Bioinformatics and domain fitting

The program SAM-T02 was used to search for homo-logues 3D structures based on primary sequence searchesusing linear hidden Markov model algorithms†. Theprimary sequence of Bph4, or domain subsets, was usedas templates for SAM-T02 searches. 3D models of a CBDdomain and a cytoplasmic portion of a sensor histidinekinase containing DHp and CA domains were found andused to fit the SAXS model. The modeling program O42

was used to manipulate and re-build the models into theSAXS model and COOT43 was used to regularize the bondand angle geometries of rebuilt models to ideal values.The graphics molecular drawing program PYMOL‡ wasused to investigate the relationships between parts of themodel and to produce the figures.

Protein Data Bank accession numbers

The coordinates and structure factors for CBD from theBph of D. radiodurans have been deposited in the ProteinData Bank with accession number 1ZTU and thecytoplasmic portion of a sensor histidine kinase containingDHp and CA domains with accession number 2C2A.

Acknowledgements

Kate Evans was supported by a DaresburyLaboratory/John Moores University (Liverpool,

http://www.soe.ucsc.edu/research/compbio/sam.html



http://pymol.sourceforge.net

http://pymol.sourceforge.net


UK) PhD scholarship. The authors thank Dr TinaGeraki for help in making some measurements andacknowledge CCLRC for the use of synchrotronfacilities.

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Edited by R. Huber

(Received 30 March 2006; received in revised form 29 August 2006; accepted 5 September 2006)Available online 23 September 2006

small-angle x-ray scattering reveals the solution structure of a bacteriophytochrome in the...

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