solution structure and dynamics of crh, the bacillus subtilis catabolite repression hpr

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Solution Structure and Dynamics of Crh, the Bacillus subtilis Catabolite Repression HPr Adrien Favier 1 , Bernhard Brutscher 1 , Martin Blackledge 1 Anne Galinier 2 , Josef Deutscher 3 , Franc ¸ois Penin 4 and Dominique Marion 1 * 1 Institut de Biologie Structurale, Jean-Pierre Ebel C.N.R.S.-C.E.A., 41, rue Jules Horowitz, 38027 Grenoble Cedex, France 2 Laboratoire de Chimie Bacte ´rienne, C.N.R.S UPR 9043, 31, chemin Joseph Aiguier, 13402 Marseille Cedex 20, France 3 Laboratoire de Ge ´ne ´tique des Microorganismes, I.N.R.A.- C.N.R.S. URA 1925 78850 Thiverval-Grignon France 4 Institut de Biologie et Chimie des Prote ´ines, C.N.R.S UMR 5086, 7, passage du Vercors 69367 Lyon Cedex 07, France The solution structure and dynamics of the Bacillus subtilis HPr-like pro- tein, Crh, have been investigated using NMR spectroscopy. Crh exhibits high sequence identity (45 %) to the histidine-containing protein (HPr), a phospho-carrier protein of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system, but contains no catalytic His15, the site of PEP-dependent phosphorylation in HPr. Crh also forms a mixture of monomers and dimers in solution whereas HPr is known to be mono- meric. Complete backbone and side-chain assignments were obtained for the monomeric form, and 60 % of the dimer backbone resonances; allow- ing the identification of the Crh dimer interface from chemical-shift map- ping. The conformation of Crh was determined to a precision of 0.46(0.06) A ˚ for the backbone atoms, and 1.01(0.08) A ˚ for the heavy atoms. The monomer structure is similar to that of known HPr 2.67(0.22) A ˚ (C a rmsd), but has a few notable differences, including a change in the orientation of one of the helices (B), and a two-residue shift in b-sheet pairing of the N-terminal strand with the b4 strand. This shift results in a shortening of the surface loop present in HPr and conse- quently provides a flatter surface in the region of dimerisation contact, which may be related to the different oligomeric nature of these two pro- teins. A binding site of phospho-serine(P-Ser)-Crh with catabolite control protein A (CcpA) is proposed on the basis of highly conserved surface side-chains between Crh and HPr. This binding site is consistent with the model of a dimer-dimer interaction between P-Ser-Crh and CcpA. 15 N relaxation measured in the monomeric form also identified differential local mobility in the helix B which is located in the vicinity of this site. # 2002 Elsevier Science Ltd. Keywords: phosphotransferase system; carbon catabolite repression; Crh; HPr; solution structure *Corresponding author Introduction Bacteria are highly adaptive organisms capable of growing under a great variety of environmental conditions. The key to adaptability is the large number of catabolic genes whose expression can be switched on and off in response to the compo- sition of the environment. In several bacteria, the histidine-containing protein (HPr), a phosphocar- rier protein of the phosphoenolpyruvate(PEP):car- bohydrate phosphotransferase system (PTS), plays a central catalytic and regulatory role in the uptake and utilization of carbohydrates. It is phosphory- lated by the PEP-dependent protein kinase enzyme I at His15 and transfers the phosphoryl group to several enzymes II, each specific for a particular carbohydrate (reviewed by Postma et al. 1 ). In low-GC Gram-positive bacteria, HPr is also phos- phorylated by the ATP-dependent HPr kinase at Ser46. 2–4 P-Ser-HPr functions as corepressor in E-mail address of the corresponding author: [email protected] Abbreviations used: Crh, catabolite repression HPr; HPr, histidine containing protein; PEP, phosphoenolpyruvate; PTS, phosphoenolpyruvate:carbohydrate phosphotransferase system; CcpA, carbon control protein A; Cre, catabolite response element; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; r.m.s., root mean square. doi:10.1006/jmbi.2001.5397 available online at http://www.idealibrary.com on J. Mol. Biol. (2002) 317, 131–144 0022-2836/02/010131–14 $35.00/0 # 2002 Elsevier Science Ltd.

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doi:10.1006/jmbi.2001.5397 available online at http://www.idealibrary.com on J. Mol. Biol. (2002) 317, 131±144

Solution Structure and Dynamics of Crh, the Bacillussubtilis Catabolite Repression HPr

Adrien Favier1, Bernhard Brutscher1, Martin Blackledge1

Anne Galinier2, Josef Deutscher3, FrancËois Penin4

and Dominique Marion1*

1Institut de BiologieStructurale, Jean-Pierre EbelC.N.R.S.-C.E.A., 41, rue JulesHorowitz, 38027 GrenobleCedex, France2Laboratoire de ChimieBacteÂrienne, C.N.R.S UPR9043, 31, chemin JosephAiguier, 13402 Marseille Cedex20, France3Laboratoire de GeÂneÂtique desMicroorganismes, I.N.R.A.-C.N.R.S. URA 192578850 Thiverval-GrignonFrance4Institut de Biologie et Chimiedes ProteÂines, C.N.R.S UMR5086, 7, passage du Vercors69367 Lyon Cedex 07, France

E-mail address of the [email protected]

Abbreviations used: Crh, cataboliHPr, histidine containing protein; Pphosphoenolpyruvate; PTS,phosphoenolpyruvate:carbohydratesystem; CcpA, carbon control proteiresponse element; TOCSY, total corrspectroscopy; NOE, nuclear Overhanuclear Overhauser effect spectroscomean square.

0022-2836/02/010131±14 $35.00/0

The solution structure and dynamics of the Bacillus subtilis HPr-like pro-tein, Crh, have been investigated using NMR spectroscopy. Crh exhibitshigh sequence identity (45 %) to the histidine-containing protein (HPr), aphospho-carrier protein of the phosphoenolpyruvate (PEP):carbohydratephosphotransferase system, but contains no catalytic His15, the site ofPEP-dependent phosphorylation in HPr. Crh also forms a mixture ofmonomers and dimers in solution whereas HPr is known to be mono-meric. Complete backbone and side-chain assignments were obtained forthe monomeric form, and 60 % of the dimer backbone resonances; allow-ing the identi®cation of the Crh dimer interface from chemical-shift map-ping. The conformation of Crh was determined to a precision of0.46(�0.06) AÊ for the backbone atoms, and 1.01(�0.08) AÊ for the heavyatoms. The monomer structure is similar to that of known HPr2.67(�0.22) AÊ (Ca rmsd), but has a few notable differences, including achange in the orientation of one of the helices (B), and a two-residue shiftin b-sheet pairing of the N-terminal strand with the b4 strand. This shiftresults in a shortening of the surface loop present in HPr and conse-quently provides a ¯atter surface in the region of dimerisation contact,which may be related to the different oligomeric nature of these two pro-teins. A binding site of phospho-serine(P-Ser)-Crh with catabolite controlprotein A (CcpA) is proposed on the basis of highly conserved surfaceside-chains between Crh and HPr. This binding site is consistent with themodel of a dimer-dimer interaction between P-Ser-Crh and CcpA. 15Nrelaxation measured in the monomeric form also identi®ed differentiallocal mobility in the helix B which is located in the vicinity of this site.

# 2002 Elsevier Science Ltd.

Keywords: phosphotransferase system; carbon catabolite repression; Crh;HPr; solution structure

*Corresponding author

Introduction

Bacteria are highly adaptive organisms capableof growing under a great variety of environmental

ng author:

te repression HPr;EP,

phosphotransferasen A; Cre, cataboliteelationuser effect; NOESY,py; r.m.s., root

conditions. The key to adaptability is the largenumber of catabolic genes whose expression canbe switched on and off in response to the compo-sition of the environment. In several bacteria, thehistidine-containing protein (HPr), a phosphocar-rier protein of the phosphoenolpyruvate(PEP):car-bohydrate phosphotransferase system (PTS), playsa central catalytic and regulatory role in the uptakeand utilization of carbohydrates. It is phosphory-lated by the PEP-dependent protein kinase enzymeI at His15 and transfers the phosphoryl group toseveral enzymes II, each speci®c for a particularcarbohydrate (reviewed by Postma et al.1). Inlow-GC Gram-positive bacteria, HPr is also phos-phorylated by the ATP-dependent HPr kinase atSer46.2 ± 4 P-Ser-HPr functions as corepressor in

# 2002 Elsevier Science Ltd.

132 3D NMR Structure of Crh

carbon catabolite regulation (CCR) by interactingwith the catabolite control protein A (CcpA).5,6 Thecomplex between P-Ser-HPr and CcpA binds to thecatabolite response element (cre),7 an operator-likesequence present in most catabolite-repressedgenes and operons.8 For Bacillus subtilis, about 200cres were predicted9, 10 and the expression of about10 % of the genome seems to be regulated by theCcpA-dependent CCR mechanism.11

Sequencing of the B. subtilis genome12 revealed agene encoding HPr-like protein named Crh (forcatabolite repression HPr), which was furtherdemonstrated to be involved in catabolite regu-lation of several genes and operons.13 Crh is com-posed of 85 amino acid residues and exhibits 45 %sequence identity when compared to HPr(Figure 1). Since the catalytic His15 of HPr isreplaced with a glutamine in Crh, it cannot bephosphorylated with PEP and enzyme I13 andtherefore plays no role in PTS-catalyzed carbo-hydrate transport and phosphorylation. However,when Gln15 is replaced with a histidine, the result-ing mutant Crh can be phosphorylated by PEP andenzyme I and carry out some of the catalytic andregulatory functions of P-His-HPr,14,15 suggesting aclose structural similarity between the two pro-teins. In addition, like HPr, Crh is phosphorylatedby the ATP-dependent HPr kinase at Ser463,13 andsimilar to P-Ser-HPr, P-Ser-Crh was found to allowbinding of CcpA to several cre sequences.16 ± 18 Infact, Crh regulates the expression of some of thecatabolic genes and operons controlled by P-Ser-HPr.10

Crh seems to be restricted to Bacilli, as Crh-encoding genes have so far been found only inB. subtilis, B. stearothermophilus, B. anthracis andB. halodurans14. It is therefore tempting to assumethat Crh carries out a Bacillus-speci®c function.Although the in vitro results suggest a role for P-Ser-Crh in CCR similar to that of P-Ser-HPr, noeffect on catabolite regulation was observed when

Figure 1. Sequence comparison of Crhs of B. subtilis, B. sB. subtilis, and E. coli. The secondary structure of B. subtilis Cabove the corresponding primary sequence were calculated1HDN,31 respectively, by using the program promotif v.2.5

B. anthracis Crhs that are identical to the B. subtilis Crh residthe HPrs is colored in red. The regulatory site Ser46 is colorE. coli HPr whose side-chains participate in analogous hydcomplex and HPr/enzyme IIAGlc complex, and sharing similsponding residues of B. subtilis HPr and Crh are shaded inHPr selectively broadened during the titration of 15N labeleNMR chemical shift mapping6 are boxed.

B. subtilis Crh was inactivated.13 By contrast, repla-cing Ser46 of HPr with an alanine led to partial orcomplete relief from glucose repression for manycatabolite-repressed genes and operons,10 demon-strating that P-Ser-Crh insuf®ciently replaces P-Ser-HPr. These results further supported the idea thatthe true function of Crh might be to regulate oper-ons carrying out Bacillus-speci®c functions, whosecres might not be recognized by P-Ser-HPr/CcpA.

An intriguing difference between HPr and Crhhas recently been reported, which might partlyaccount for the different af®nities and speci®citiesof their CcpA complexes. Whereas HPr is mono-meric in solution, Crh was observed as a mixtureof monomers and dimers in a slowly exchangingequilibrium in hours time scale.19 This dimerizationof Crh suggests that in contrast to the P-Ser-HPr/CcpA complex, a dimer/dimer interaction couldoccur in P-Ser-Crh/CcpA and may be importantfor the speci®c functional characteristics of the twoprotein complexes. To obtain more detailed infor-mation about the Crh structure, we carried outNMR spectroscopy using Crh solutions containingdimers and monomers in slow exchange at equili-brium. This allowed us to determine the completestructure of the Crh monomer, to assign about60 % of the resonances of the dimer backbone andto identify the dimer interface. Comparison of Crhwith HPr shows some signi®cant structural differ-ences as well as a high degree of conservation atthe binding surface with CcpA. The present struc-tural data constitute an important step towards abetter understanding of the function of Crh.

Results and Discussion

NMR resonance assignment

NMR assignment of Crh was obtained at asample temperature of 25 �C, at pH 7.5 and a pro-tein concentration of 0.5 mM. These conditions

tearothermophilus, B. halodurans, B. anthracis and HPrs ofrh (this study), B. subtilis HPr, and E. coli HPr displayed

from the NMR structure ensembles of 1K1C, 2HID40 and0 The residues of B. stearothermophilus, B. halodurans andues are represented with dashes. The active site His15 ofed in yellow in Crh and HPr sequences. The residues ofrophobic and electrostatic interactions in the HPr/EINar hydrophobic or electrostatic properties with the corre-green and blue, respectively. The residues of B. subtilis

d B. subtilis P-Ser-HPr with B. magaterium CcpA-C using

3D NMR Structure of Crh 133

were optimized to enhance the stability of thesample, though more complex spectra areobtained. As illustrated in Figure 2(a), the 1H-15NHSQC spectrum showed the presence of a mixtureof Crh monomer (70-80 %) and dimer (20-30 %).Due to the large number of resonances and the loweffective concentration of monomeric Crh (0.35-0.4 mM) assignment of this molecule was dif®cultto achieve. As reported previously,19 the Crhmonomer/dimer exchange is temperature-depen-dent. At temperature higher than 25 �C, the dimericfraction of Crh decreases likely due to aggregationof the dimer, which led to a decrease in samplelifetime (a few days at 35 �C). However, in order toget a starting point for the NMR assignment at25 �C we used additional 3D-HNCA, 3D-NOESY-

Figure 2. (a) 1H-15N HSQC spectrum of Crh at 25 � Crecorded at 800 MHz 1H frequency. All signals arisingfrom Crh monomers are colored in black and labeledwith the corresponding residue number. All signals aris-ing from Crh dimers are colored in red. The mostshifted dimer peaks are indicated by arrows pointingfrom the assigned monomer peak towards the corre-sponding dimer correlation peak. * Unassigned His resi-due contained in the poly(His)C-terminal extension.(b)Backbone amide chemical shift differences between themonomeric and dimeric form of Crh. Filled circles at 0ppm indicate that �d could not be calculated becausethe corresponding residues could not be assigned in Crhdimer. The weighted average chemical shift differenceswere calculated as described by Garrett et al.51 i.e.{[(�H)2 � (�N/5)2/2}1/2. Secondary structure elements(b-strands and a-helix, this study) are shown schemati-cally above the histogram.

HSQC and 3D-TOCSY-HSQC spectra recorded at35 �C to clearly distinguish monomer from dimercorrelation peaks. This strategy allowed completebackbone and side-chain assignment of the mono-meric form of Crh at 25 �C, for essentially all of the1H, 13C and 15N resonances using standard three-dimensional experiments as detailed in Materialsand Methods. No signal was detected for theamide 15N and 1H of Val2 due to the absence ofresidue Met1, which was mainly removed duringexpression in Escherichia coli as evidenced by massspectrometry. In addition, no NMR signal could bedetected for the side-chain resonances of Val2 andthe backbone amide group of Gln3 indicating prob-ably a high degree of ¯exibility for these two N-terminal residues. In addition, the amide protonand nitrogen resonances of Ala16 and Arg17 arevery weak in the 1H-15N HSQC spectrum and theside-chain resonances of Arg17 could not beobserved in the 3D HCCH-TOCSY suggesting con-formational exchange in this region of the protein.A close examination of the assigned HSQC spec-trum of the monomeric form of Crh (Figure 2(a))shows a high similarity with HSQC spectra ofB. subtilis and E. coli HPrs20,21 and suggests a highstructural homology between these proteins.

Partial 1H-15N assignment of the Crh dimerpeaks was obtained by comparing strips extractedfrom a 3D CBCA(CO)NH spectrum for the unas-signed 1H-15N correlation peaks with the Ca, Cb

resonance assignments of monomeric Crh. CarbonCa and Cb chemical shifts are known to be stronglyrelated to the amino acid type and the local back-bone structure.22 It has been shown previously bynear UV circular dichroõÈsm19 that the overall per-centage of the backbone structure of the mono-meric and dimeric forms of Crh is very similar.The differences in Ca and Cb shifts between themonomer and the dimer were therefore expectedto be small. Despite the low intensity of the dimerpeaks (three to four times weaker than the corre-sponding monomer peaks) we could unambigu-ously assign 60 % of the 1H and 15N resonances ofthe Crh dimer. Figure 2(b) showing the chemicalshift variations between monomer and dimer forassigned residues is detailed below.

Secondary structure determination

The secondary structural elements of Crh sum-marized in Figures 1 and 3 were determined onthe basis of sequential and medium range NOEconnectivities involving HN, Ha and Hb protons23

and the chemical shift index (CSI).22 The two lar-gest a-helical regions from residues Arg17 toArg28 (helix A) and from Glu70 to Glu83 (helix C)were easily distinguishable due to the presence ofstrong dN,N(i,i � 1) and weak da,N(i,i � 1) connectiv-ities. The dN,N(i,i � 1) pattern is however broken atthe N terminus of helix A owing to residue Pro18.Strong sequential da,N(i,i � 1) connectivities wereobserved for four b-strands comprising amino acidresidues Gln4 to Arg9, Asp32 to Lys37, Lys40 to

Figure 3. Secondary structure and topology compari-son of B. subtilis Crh and B. subtilis HPr highlighting thebase-pairing difference between b1 and b4 strands inboth proteins. Strong NOE connectivities de®ning the b-sheet pairing of the four b-strands of Crh are rep-resented with arrows.

134 3D NMR Structure of Crh

Asn43 and Glu60 to Gln66. Eight strong Ha-Ha andeight strong HN-HN inter-strand NOE peaks wereidenti®ed, unambiguously de®ning the b-sheet

pairing of the four strands (Figure 3). The second-ary structural elements were con®rmed by the CSIcalculated from the CO, Ca and Ha chemical shifts.In addition, the CSI revealed the presence of athird small helical region from residue 47 to 50(helix B).

Experimental restraints andstructure calculation

All distance restraints used for the structure cal-culation of Crh were derived from 13C and 15N edi-ted 1H-1H NOESY experiments. Because of the lowconcentration and the higher tumbling correlationtime of the dimer leading to broader lines for thesepeaks, NOE cross-peaks in these spectra werecaused predominantly by the Crh monomer. Aninitial set of unambiguous distance constraints wascompiled and used with 19 empirical distancesimposed on the atoms involved in the H-bondswithin the a-helices A and C, deduced from theCSI data and backbone NOEs, to de®ne the globalfold of the protein using a structure calculationfrom randomized initial coordinates. Subsequently,remaining NOEs were added using a standarditerative procedure and the empirical distanceswere removed. A total of 1048 unambiguous NOE-derived distance restraints (244 intra- and 804inter-residue constraints) were used in the ®nal cal-culation. Among the 244 intra-residue distancerestraints, 56 % were found structurally relevantusing the program AQUA.24 An additional set of327 NOEs were treated as ambiguous restraints.The distribution of the ®nal NOE-derived con-straint set along the protein sequence is shown inFigure 4(a). A set of 51 f dihedral angle restraintswas derived from the measured 3JHNHa and 1JCaHacoupling constants and added to the constraint list.Following the ®nal structure calculation, 24 struc-tures were selected from a set of 170 calculatedstructures on the basis of the experimental targetfunction (E < 21 kcal molÿ1) to form the ensembleEI, and these structures were all re®ned to form the®nal re®ned ensemble EII. The statistical and ener-getic details of the ensembles EI and EII are sum-marized in Table 1. There are no individualviolations greater than 0.20 AÊ in the selected struc-tures of EI and no violations greater than 0.30 AÊ inthe ®nal re®ned structure ensemble of Crh. Anal-ysis of the ensemble with PROCHECK24 showsthat 71.9 % and 27.5 % of the residues in the 24structure ensemble EII have backbone conformationin favorable and allowed regions of the Ramachan-dran plot, respectively. The backbone and heavyatom r.m.s. deviation per residue with respect tothe mean structure shown in Figure 4(b) re¯ects, ingeneral, the distribution of the number of con-straints per residue. The structure has an overallr.m.s. deviation of 0.46(�0.06) AÊ for the backboneatoms and 1.01(�0.08) AÊ for the heavy atoms withrespect to the mean coordinates. In summary, thecalculated structures fully satis®ed the experimen-

Table 1. Statistics of the 24 ®nal simulated annealing structures of Crh

Energy (kcal molÿ1)EI EII

Structural statisticsBond 4.0 � 0.5 10.0 � 0.7Angle 61.3 � 4.7 96 � 6Dihedral 77 � 5 113 � 6Out of plane 0 0Hbond ÿ25.3 � 3 ÿ44 � 2VDW ÿ279 � 14 ÿ406 � 9Electrostatic ÿ845 � 13 ÿ1190 � 17Total ÿ1005 � 27 ÿ1414 � 21

Experimental statisticsNumber of distance violations >0.2 AÊ 0 0.9 � 0.1Number of distance violations >0.1 AÊ 1.0 � 0.8 16 � 1Number of distance violations >0.05 AÊ 12.4 � 3.3 56 � 1Violation energy 7.9 � 0.9 17.21 � 1.9

Convergence statisticsBackbone (AÊ ) 0.69 � 0.14 0.46 � 0.06Heavy (AÊ ) 1.11 � 0.09 1.01 � 0.08

Ramachandran plot (PROCHECK)Residues in most favorable regions 60.0 % 71.9 %Residues in additional allowed regions 34.2 % 25.8 %Residues in generously allowed regions 5.7 % 1.7 %No. of bad contacts 0.1 % 0.6 %

3D NMR Structure of Crh 135

tal NMR data and yielded a highly de®ned Crhstructure.

Structure comparison of B. subtilis Crhand HPr

Figure 3 shows that the overall topology ofB. subtilis Crh and HPr is nearly identical. Thebabbaba secondary structural elements form anopen-faced b-sandwich. However, despite the highsequence identity (Figure 1) and the similar overall3D fold (Figure 5 (a) and (b) for B. subtilis Crh andB. subtilis HPr, respectively), the structure ofB. subtilis Crh exhibits some major differences withrespect to previously determined structures of HPr.The superposition of secondary structure elementsof B. subtilis Crh and HPr from several organismsrevealed strong similarities between the variousHPr with a mean value of the r.m.s. deviation ofthe trace being 1.25(�0.45) AÊ , whereas the simi-larity between Crh and any of the HPrs was some-what weaker with a mean value of the r.m.s.deviation of 2.67(�0.22) AÊ (the structure ofStaphylococus aureus HPr was not included in thesecalculations, since it exhibits unusually large devi-ations of the r.m.s. values when compared to otherHPrs). The principal differences which distinguishCrh from the HPr family are discussed in the fol-lowing section.

The backbone structures of B. subtilis Crh andB. subtilis HPr are presented in Figure 5 (a) and (b),respectively. The most obvious difference in thefold of these proteins concerns the orientation ofthe a-helices (color-coded in red) relative to theb-sheet scaffold of the molecule. The short a-helixB is oriented quite differently in HPr and Crh,

while the C-terminal half of this helix is replacedby a 310-like turn in B. subtilis Crh. In view of thefunctional importance of this region, we have paidparticular attention to the potential causes of thesedifferences. As no localized restraint violations orforce ®eld strain were observed in the NMRensemble, we suppose that this conformation isrepresentative of the solution structure. The signi®-cant bend of helix C observed in B. subtilis HPr isnot found in Crh. This correlates with the C-term-inal stabilization in HPr, involving backbonehydrogen bonding between the N-terminal b-strand 4 and the extended three-amino acid loopfollowing helix C, which is absent in Crh. In con-trast, the conformation and orientation of helix Cin Crh and E. coli HPr are very similar.

A further important difference between the twoprotein structures lies in the H-bond network con-necting the strands b1 and b4 of the b-sheet (color-coded in green in Figure 5), resulting in an appar-ent shift in the alignment of the b-strands. In Crh,the amide proton of Gln4 forms a hydrogen bondwith the CO of Ala65, whereas the correspondingamide proton is H-bonded with the CO of Leu63in all HPr structures reported so far (see alsoFigure 3). As a consequence, the N-terminal resi-dues of Crh appear more disordered than that ofHPr which are partially involved in the b-strandH-bond network. This different arrangement of theb1 strand is clearly responsible for the confor-mational differences of loop L1 connecting b1 withhelix A (color-coded in blue in Figure 5) which isshortened by two residues in Crh. Moreover, theL1 loop of Crh is folded onto the core of the pro-tein, yielding a ¯at surface, whereas this loop pro-trudes at the surface of HPr and is mostly exposed

Figure 4. Structural characterisation of Crh monomer.(a) Number of NOE-derived distance constraints used inthe structure calculation. The stacked bars represent thenumber of intra-residue, unambiguous inter-residue andinter-residue NOEs whose contributions arise from sev-eral proton pairs (shaded, white, and black, respect-ively). (b) Average atomic r.m.s. deviation between theindividual structures in the ensemble and the meancoordinates calculated by superposing the backbone (C0,Ca and N) atoms of Crh. The broken line represents ther.m.s.d. calculated from the backbone atoms and thecontinuous line the r.m.s. deviation taking into accountall heavy atoms of the residues. The high r.m.s. devi-ation values observed for residues marked with a starare discussed in the text.

Figure 5. Structure comparison of B. subtilis Crh andB. subtilis HPr. (a) Stereoview of the ribbon represen-tation of the backbone of B. subtilis Crh. (PDB entry:1K1C) (b) Stereoview of the ribbon representation of thebackbone of B. subtilis HPr (PDB entry: 2HID40). In (a)and (b), the a-helices, the b-strands and the loop L1 con-necting the a1 helix and the b1 strand are colored inred, green, and blue, respectively. The phosphorylableregulatory residue Ser46 is represented in yellow. (c)Stereoview of the ribbon representation of the back-bones of Crh (in black) and of the known HPr 3D struc-tures (in light grey; PDB entry: 1FU0,52 1SPH,53 1CM3,54

1QFR,55 2HID,40 1HDN,31 1JEM,40 1PFH,33 2HPR,53

1QR5,56 1PTF,57 1POH,58 1PCH59 and 1OPD60). S. aureusHPr structure is not reported in this HPr ensemble,because its comparison to the other HPr structuresrevealed a relatively large r.m.s. deviation. Note how-ever that the L1 loop of S. aureus HPr is close to that ofthe other HPr proteins but not to that of Crh. All struc-tures have been superposed on all secondary structureelements except the b1 strand, which is shifted in Crhwhen compared to that of HPr (see the text).

136 3D NMR Structure of Crh

to the solvent. The different conformation of the L1loop in B. subtilis HPr and Crh also implies adifferent orientation of helix A (tilted by 16 �) inthe two proteins. The structural differences for thisloop region and the shift in the b-strand are high-lighted in Figure 5(c), where the secondary struc-ture elements from b1 to a-helix A of B. subtilisCrh and various HPr have been superposed. Thesedifferences are also correlated with changes in theconformation of loop L2 between helix B and b-strand 4 of Crh when compared to the correspond-ing loop in HPr (see details below).

15N relaxation and backbone dynamics

15N spin relaxation data contain information onthe time-dependent ¯uctuation of the individual

backbone N-H bond vectors. To analyze themeasured relaxation data of monomeric Crh wehave used the Lipari-Szabo model-free

3D NMR Structure of Crh 137

approach25,26 in combination with the descriptionof rotational diffusion anisotropy formulated byWoessner.27 Residues with overlapping 1H-15N cor-relation peaks or weak cross-peak intensities dueto chemical exchange were removed from the anal-ysis. In addition, all residues for which the 1H-15Ncorrelation peak of the dimer was not assignedwere also left out, as one cannot exclude that themonomer and dimer resonances may overlap forthese residues. The generalized order parametersS2 obtained for the 36 remaining residues are dis-played in Figure 6. The color scheme re¯ects differ-ent order parameters, i.e. degrees of local ¯exibilityalong the protein backbone of Crh. Internal mobi-lity is restricted (S2 > 0.8) for residues located inhelices A and C and in the b-sheet. The turn con-necting the strands b2 and b3 exhibits a muchhigher ¯exibility (S2 � 0.2-0.7). Interestingly theregion including helix B (residues 48-54) alsoundergoes some signi®cant ¯exibility. The timescale of these dynamic processes includes contri-butions on the nanosecond timescale, as well as onthe millisecond timescale as evidenced by thechemical shift exchange contribution (Rex) presentthroughout this region. It has been noted thatunder conditions of anisotropic rotational diffu-sion, NH sites in helical regions can exhibit ®ctiveRex contributions due to the orientation of the helixrelative to the axes of the rotational diffusiontensor,28,29 if an isotropic model is used. For this

Figure 6. Ribbon representation of the backbone ofB. subtilis Crh colored according to the S2 values derivedfrom the Lipari-Szabo analysis of the R1, R2 and NOErelaxation data assuming anisotropic rotational diffu-sion. The residues colored in gray could not be inte-grated into this approach (see the text). The decreasingS2 order parameter color-coded from yellow to bluere¯ects local increasing ¯exibility. The relative dimen-sions of the rotational diffusion tensor axes determinedusing the program TENSOR247 are graphically dis-played using the grid representation around the ribbondiagram. The details of this analysis can be found in theSupplementary Material.

reason we used the rotational diffusion tensordetermined from relaxation data from helices Aand C and from the b-sheet region, to describe theoverall reorientational characteristics of the mol-ecule in the analysis of local motion. Despite thereduced number of sites available for this analysis(23 residues), the tensor geometry (which wasfound to have approximately axial symmetry) iscompatible with the shape of the monomer(Figure 6). We can therefore exclude the possibilitythat the differential mobility observed in this helixis caused by rotational diffusion anisotropy of theprotein. The increased ¯exibility of the Crh poly-peptide chain observed near the regulatory site,also observed in Hpr,30,31 may be important forbinding in the active site of HPr kinase and/or theinteraction with CcpA.

Putative interaction surface of Crh withprotein partners

In Gram-positive and Gram-negative bacteria, P-His-HPr is known to interact with a variety of pro-teins of unrelated structure including the N-term-inal part of enzyme I,32,33 enzyme IIA34 ± 36 andtranscriptional regulators containing a PTS regu-lation domain (reviewed by StuÈ lke et al.37). InGram-positive bacteria, P-His-HPr has also beenshown to phosphorylate glycerol kinase.38 Numer-ous NMR chemical shift mapping studies haveshown that P-His-HPr binds to its different proteinpartners by using the same surface area encom-passing helices A and B.35 By carrying out NMRchemical shift mapping, this surface region of HPrhas also been identi®ed as the binding interface inthe interaction of P-Ser-HPr with a C-terminal frag-ment of CcpA.6 These observations suggest thatsome key residues are crucial for the interaction ofHPr with its protein partners. Two recentlyresolved high-resolution structures of the HPr-EINand HPr-EIIAGlc complexes con®rm thishypothesis.32,39 Although the secondary structuresof the binding surface of N-terminal enzyme I,mainly composed of a helices, and enzyme IIAGlc,mainly formed by b sheets, are quite different, thebinding surfaces for these two enzymes with HPrshare 17 residues in common, out of 18 that inter-act with enzyme IIAGlc and 23 with N-terminalenzyme I.39 It was therefore interesting to seewhether these residues are also found at the corre-sponding surface region of Crh and if their confor-mations are similar to those found in HPr.

The residues of E. coli HPr involved in intermo-lecular interactions in the HPr/enzyme I and HPr/enzyme IIAGlc complexes and conserved in B. subti-lis HPr are boxed in Figure 1 and colored inFigure 7(a) and (b). The majority of the contactsbetween HPr and enzyme I or enzyme IIA arehydrophobic (colored in green). The residues of theB. subtilis and E. coli HPrs involved in these hydro-phobic interactions have very similar conformationin both proteins. The corresponding hydrophobicpatch is also found in B. subtilis Crh despite a

Figure 7. Comparison of bindingsurface for enzyme I and enzymeIIAGlc on E. coli HPr to the corre-sponding surface of B. subtilis HPrand Crh. The PDB entries usedfor E. coli HPr, B. subtilis HPr andCrh are 1HDN,31 2HID40 and1K1C, respectively. (a) The residuesof helices A and B, involved inanalogous hydrophobic and/orelectrostatic interactions in the(HPr-EIN)E. coli and (HPr-enzymeIIAGlc)E. coli complexes and con-served in B. subtilis HPr are high-lighted on the HPr surfaces. Theresidues, whose side-chains partici-pate in hydrophobic and electro-static interactions, are colored ingreen and blue, respectively. ThePTS active site residue His15is indicated in red and the CCRregulation site residue Ser46 inB. subtilis is indicated in yellow.The corresponding residues ofB. subtilis Crh are colored in thesame way but Gln15 of Crh iscolored in magenta. Leu50 of Crh,which is part of the central hydro-phobic core, is colored in darkgreen. (b) Stick representation ofthe residues colored in (a). (c) Samepicture as in (a) but containingalso residues situated around thehydrophobic core involved inhydrophobic and/or electrostatic

interactions in the (HPr-EIN)E. coli and (HPr-enzyme IIAGlc)E. coli complexes, but which are only partly conserved inB. subtilis HPr and Crh.

138 3D NMR Structure of Crh

different position of helix B in Crh (Figure 7(b)).Indeed, Leu47 of E. coli HPr and Ile47 of B. subtilisHPr are positioned between helix A and helix Bbridging the hydrophobic patches formed by side-chains of these two helices. In B. subtilis Crh, Ile47is shifted away from helix A and Leu50 of helix Btakes over its function in bridging the two hydro-phobic patches. By contrast, Leu50 in E. coli HPrand Val50 in B. subtilis HPr are buried. This showsthat Crh possesses the appropriate structural fea-tures to bind enzyme I and enzyme IIAGlc andexplains the previous observation that replacingGln15 of Crh with a histidine allowed its PEP-dependent phosphorylation and that P-His-Crh(Q15H) could carry out several of the catalyticand regulatory functions of HPr.14 In view of theabsence of the key residue His15 required for thereaction with enzyme I, it is conceivable that Crhmight act as a decoy and indirectly regulate HPractivity by interacting with enzymes of the PTS ina non-productive manner.

When extending the conserved binding regionshown in Figure 7(a) by including the surface-exposed residues surrounding it, we obtain the

pattern shown in Figure 7(c). In the three proteins,the homologous residues in positions 48 (Met orPhe) and 52 (Thr or Ser) as well as Lys45 enlargethe conserved binding surface. In contrast, lysineresidues 24, 27, and 49 surrounding the hydro-phobic patch in E. coli HPr are observed neither forHPr nor Crh from B. subtilis. This differentenvironment around Ser46 between HPr fromE. coli and Crh or HPr from B. subtilis mightexplain why no phosphorylation at Ser46 could beobserved for E. coli HPr. In addition, this compari-son shows that the surface properties of helices Aand B of B. subtilis Crh and HPr are very similar interms of hydrophobicity and relative position ofnumerous conserved residues. We therefore pro-pose that this region represents the binding inter-face in the P-Ser-Crh/CcpA complex, as reportedfor the analogous site in the P-Ser-HPr6 complex. Atwo-step mechanism could be envisaged, wherethe long-range recognition of the partners isachieved through electrostatic interaction by meansof the peripheral polar residues (Arg17, Gln24,Lys45) while the precise docking involves the cen-tral hydrophobic core.

Figure 8. Surface (on the left) and ribbon (on theright) representations of Crh illustrating regionsinvolved in the dimeric interface. The surface high-lighted by the NMR chemical shift mapping of dimerand the CCR regulatory site Ser46 are colored in redand yellow, respectively. On the ribbon representationof Crh, the loop L1 is colored in blue. (a) Side view ofCrh similar to that in Figure 7(c). (b) Top view obtainedafter a 90 � rotation of the protein represented in (a).

3D NMR Structure of Crh 139

Crh dimer formation and dimer interface

The functional differences between HPr and Crhduring catabolic regulation might be related to thedifferent oligomerisation state of the two proteins.The current NMR study was performed under con-ditions of slow exchange on the NMR time scale(tex4one second), allowing the simultaneouscharacterization of structural features of bothmonomeric and dimeric forms of Crh. Althoughthe NMR signals arising from the Crh dimer weretoo weak to calculate a complete three-dimensionalmodel, the assignment of the majority of reson-ances from the dimer allowed us to identify thesurface region of Crh involved in the dimer inter-face. The chemical shift variations for amide 1Hand 15N are plotted in Figure 2(b) as a function ofthe primary sequence. The residues experiencingthe largest chemical shift variations between themonomer and the dimer are most likely located atthe dimer interface. These residues are shown inFigure 8, where the most strongly shifted residuesare color-coded in red. The predicted dimer inter-face comprises mainly the loop region labeled L2(residues 53-59), and the C-terminal part of helix B.Interestingly, loop L2 lies adjacent to loop L1,whose conformation is different in Crh and HPr(see above). Nevertheless, the contacts betweenthese two loops are quite similar in both proteins.In the structure ensemble of B. subtilis HPr (PDBnumber: 2HID40), two hydrogen bonds are foundlinking loop L1 to loop L2 in most of the confor-mers. The ®rst hydrogen bond involves HN ofVal8 and CO of Ala59 and the second CO of Val8and HN of Gly58. The hydrophobic interactioninvolving Ile14 of loop L1 and Val50, Leu53 andIle55 of loop L2 stabilizes the two loops. In ourstructure ensemble of Crh, a hydrogen bond con-nects the HN of Leu10 to the CO of Thr59 in allconformers. The hydrophobic patch linking bothloops in Crh is found at the same location as inHPr. The residues involved are Leu14, Leu50 andAla54. The shift of two residues in the b-sheet pair-ing pattern observed in Crh when compared toHPr is propagated into this loop, such that Val8hydrogen bonds to Ala59 in HPr whereas Leu10hydrogen bonds to Thr59 in Crh. This shift hashowever no longer an effect on the location of thehydrophobic interaction involving more or lesssequence-aligned residues. This interaction net-work implies that loop L1 of Crh is two residuesshorter due to a shift of two residues between Val8and Ile14. As a result, the loop L1 in Crh is packedinto the protein core and gives rise to a ¯at surfaceof the corresponding region as illustrated inFigure 8. Such ¯at surface favors a close contactbetween two Crh molecules which involve bothpolar and hydrophobic residues. In contrast, the L1loop in HPr is protruding and may sterically pre-vent dimer formation.

On the basis of these observations and takinginto account the information given by the dynamicanalysis, two models can be constructed for the

Crh dimer (Figure 9). In the ®rst one (Figure 9(a))the two Crh molecules are directly attached via theL2 loop region by hydrophobic and/or polar inter-actions in a head to head conformation. In thismodel, the L1 loop should also be involved in thedimer interface, an interaction unfortunately notevidenced by experimental data as the amide res-onances of the L1 loop in the Crh dimer are notassigned. This lack of assignment may beexplained either by peak superpositions of theweak dimer peaks with monomer peaks, or by asigni®cant conformational change of the L1 loopupon dimerisation which may signi®cantly changethe Ca and Cb chemical shifts, making assignmentdif®cult. Assuming a conformational rearrange-ment of the L1 loop, a second model of the dimercan be made which involves a swapping of the b1strand from one monomer to the other(Figure 9b)).41 This second model is more compati-ble with the high energetic barrier between themonomeric and the dimeric form of Crh suggestedby the slow exchange between the two forms,19

although no further experimental evidence hasbeen obtained to support this model. In bothmodels, two positively charged residues, Arg9 and

Figure 9. Diagram showing the two proposed modelsfor Crh dimer topology. (a) direct association via the L1and L2 loops. (b) Association via the swapping of the b1strand.

140 3D NMR Structure of Crh

Lys40, are located close to the dimer interface,which could theoretically destabilize the dimericform of Crh due to electrostatic repulsion. Interest-ingly, these two residues marked with a star inFigure 4(b), explore a large conformational spacein the NMR ensemble of the monomeric form ofCrh, suggesting that the ¯exibility of both residuescould allow the minimization of the electrostaticrepulsion during the dimer formation. A similararm exchange in dimers has been reported for sev-eral proteins42 ± 44 where the occurrence of bothmonomeric and dimeric species in the NMR spec-trum depends on the kinetic and thermodynamicproperties. Bergdoll et al.44 have reported an extre-mely frequent occurrence of proline residues in thevicinity of the linker between the protein and theexchanged arm. As a matter of fact, this trend isvalid in the case of Crh with Pro18. While the con-formation of this proline is clearly trans in themonomeric form, we cannot exclude that a cis-transisomerization may play a key role in the dimerisa-tion process.

Conclusions

Crh shares structural and functional similaritieswith HPr, a protein that plays a central role in bac-terial metabolism. Although Crh and HPr exhibitvery similar overall folds, some signi®cant localconformational deviations were found whichwould have been dif®cult to predict on the basis ofsequence homology. The most interesting structur-al difference is the different pairing of strands b1and b4, which are shifted by two residues betweenCrh and HPr, yielding a distinctive conformationin the L1 loop region, which no longer protrudesaway from the protein core as in HPr. In addition,loop L2, which is in close contact with loop L1,forms part of the dimer interface of Crh. Whereas

the pairing of the two strands b1 and b4 is strictlyconserved for all known HPr structures, theirdifferent arrangement in Crh may be functionallyrelevant for its interaction with CcpA. The packedconformation of loop L1 in Crh might favor thedimer formation of Crh whereas the moreextended and protruding loop L1 conformation inHPr may prevent the interaction of monomers. Theputative binding site of Crh for CcpA identi®ed bystructural and functional comparison with HPrborders the dimer interface of Crh. We proposetwo possible models for the overall architecture ofthe Crh dimer, although the model assuming theb1 swapping better explains the slow exchangebetween monomeric and dimeric form of Crh.Nevertheless, in both models the two Ser46 resi-dues are suf®ciently far away from the dimer inter-face to allow the phosphorylation of Crh and theinteraction with a CcpA dimer to form a functionalregulatory complex. Further investigation on thedimeric form would be necessary to delineate thepossible role of Pro18 in the dimerisation of Crhprior to its interaction with its partner. Titrationexperiments with P-Ser-Crh and CcpA followed byNMR spectroscopy will show whether the mono-meric or the dimeric form is involved in the inter-action, whose localisation on the surface of Crhcould be identi®ed using NMR chemical shiftmapping.

Materials and Methods

Sample preparation

Crh (residues 2-85) was overproduced with a C-term-inal LQHHHHHH extension and puri®ed as describedpreviously.19 Uniformly 15N and 13C/15N-labelled Crhwas obtained by growing bacteria in Silantes growthmedia. Mass spectroscopy showed that Met1 had beenremoved during expression in E. coli. NMR sampleswere prepared at a concentration of 0.5 mM in 20 mMsodium phosphate buffer (90 % H2O, 10 % 2H2O), pH 7.5,50 mM NaCl, 0.05 % sodium azide.

NMR spectroscopy

All NMR experiments were performed on VarianINOVA 600 and INOVA 800 spectrometers, bothequipped with a triple-resonance (1H, 15N, 13C) probeand shielded z-gradients. For sequential backboneassignment, 3D 15N-edited NOESY-HSQC and TOCSY-HSQC spectra were recorded on an 15N-labelled sampleof Crh at 800 MHz 1H frequency. The NOESY andTOCSY mixing times were set to 100 ms and 50 ms,respectively. Additional HNCA and HNCO triple-reson-ance experiments were performed on the 13C/15N-labelled sample at 800 MHz 1H frequency for unambigu-ous sequential backbone assignment, and for the assign-ment of the backbone 13C resonances. All experimentsfor backbone resonance assignment were carried out atboth 25 �C and 35 �C. 1H and 13C aliphatic side-chainassignments were accomplished using a set of four 3Dtriple-resonance experiments recorded at 600 MHz 1Hfrequency and a sample temperature of 25 �C: CBCA(CO)NH, H(C)C(CO)NH-TOCSY, (H)C(CO)NH-TOCSY,and H(C)CH-TOCSY. Data processing and peak picking

3D NMR Structure of Crh 141

were performed using FELIX program version 2000 (MSITechnologies). Inter-proton distance restraints werederived from the 3D 15N-edited NOESY and 13C-editedNOESY spectra. 3JHNa scalar coupling constants weremeasured from a 3D HNHA spectrum.45 The sign of thef torsion angles was derived from the analysis of 1JCaHascalar coupling constants measured in a 3D HN(CO)CAspectrum where no 1H decoupling was applied in the Ca

dimension.45

NMR relaxation measurements

15N R1 and R1r relaxation rates and heteronuclearNOE were measured using pulse sequences similar tothose proposed by Farrow et al.46 The relaxation decaywas sampled at 11 different time points for R1: 0.02, 0.06,0.12, 0.18, 0.26, 0.34, 0.42, 0.52, 0.60, 0.70, and 1.00seconds, and at ten different time points for R1r: 0.08,0.012, 0.016, 0.025, 0.032, 0.048, 0.064, 0.080, 0.104, and0.132 seconds. For both series one of the measured pointsof the relaxation decay curve was recorded twice toallow for error estimation of the relaxation rates. Off-res-onance 1H irradiation was applied during the recycledelay to ensure a constant temperature for individualexperiments of a relaxation series. In the R1r exper-iments, a spin lock ®eld of jgNB1j/2p � 2.5 kHz wasapplied on the 15N spins to suppress chemical shift andscalar JNH coupling evolution. Transverse relaxation rateconstants R2 were then calculated from the measured R1rrate constants, taking into account the frequency offset�n, using the equation:

R1r � cos2�y�R2 � sin2�y�R1

with

y � tanÿ1�2p�n=gNB1�For the heteronuclear NOE measurements, two spectrawith and without proton saturation, were acquired in aninterleaved manner. 1H saturation was achieved using aWALTZ-16 sequence of three seconds.

Relaxation data analysis

The 15N heteronuclear relaxation rates were inter-preted using the program TENSOR2, which uses thedescription of the molecular diffusion derived by Woess-ner, in combination with the Lipari-Szabo model-freeanalysis of local ¯exibility.25 In the model-free approach,internal mobility is characterized using an order par-ameter S2, which may be interpreted as the amplitude ofthe motion and a correlation time ti the characteristictime constant of this motion. The physical nature of themotion is not constrained, but the internal and globalmotions are assumed to be independent. The programTENSOR2 has been described elsewhere29,47 and all dataanalysis was performed as described in these references.Brie¯y the analysis uses the following approach:

Rotational diffusion of the molecule is characterizedusing the ratio R2/R1 measured in regions of the mol-ecule where contributions to this ratio from internalmotion are expected to be negligible. In this particularcase, sites whose monomeric form had been assignedfrom the b-sheet region and helices A and C were usedfor this analysis. Once the diffusion tensor has beencharacterised, the contribution to R1, R2 and the hetero-nuclear NOE from the global motion is derived from thisanalysis and the remaining contribution is ®tted to the

local motional parameters (S2 and ti). In the case ofinsuf®cient reproduction of the experimental data usingthese parameters, an additional chemical shift exchangecontribution to R2 (Rex), or an extended motion compris-ing two independent internal motions may be evoked.R1, R2 and the heteronuclear NOE are expressed in termsof the angular spectral density function of the inter-nuclear vector at different transition frequencies as pre-viously described. The inter-nuclear distance rNH wasassumed to average to 1.01 AÊ , and the chemical shift ani-sotropy of the 15N nucleus (sk-s?) was approximated toÿ170 ppm.

Five models of internal motion are iteratively testedstarting with the simplest model and invoking morecomplex models until the proposed model could giverise to the measured relaxation rates within 95 % con®-dence limits: (1) S2; (2) S2, ti; (3) S2, Rex; (4) S2, ti, Rex;and (5) Sf

2, Ss2, ti. In model (1), motions on the fast time-

scale are too fast (<20 ps) to be characterized and affectR1 and R2 in a similar way. In model (2), internal motionis relaxation active. In models (3) and (4) Rex contributesto R2. Model (5) refers to an extended motion comprisingtwo independent internal motions.

Structure calculations

NOESY peak volumes were calibrated using severalknown distances in a-helices and b-sheets.23 An upperbound uncertainty of 25 % was applied to NOErestraints, the lower distance limit being effectivelyequivalent to the sum of the van der Waals radii of thetwo protons. Hydrogen bonding restraints were deducedfrom backbone NOEs and backbone chemical shiftsusing standard criteria. Structural information concern-ing the backbone of Crh was derived from the measure-ment of 3JHNHa scalar coupling constants, which arerelated to the backbone dihedral angle f by the semi-empirical Karplus equation. The Karplus equation gener-ally yields four different f angles for a measured 3JHNacoupling constant. Two of them can be discarded by theadditional measurement of the 1JCaHa coupling constantwhich provides information on the sign of the f angles.The program TALOS48 was used to solve the remainingambiguity when possible. TALOS performs a databasecomparison for residue triplets in protein structures withknown NMR shift assignments. It provides a distributionof (f,c) angles for the tri-peptides with the lowest valueof the target function. If the (f,c) value ensemble pro-posed by TALOS was well de®ned we used a single fangle with a lower and upper bound of �20 �. If not, thef angle was restrained to a lower bond of (f1 ÿ 20 �)and an upper bond of (f2 � 20 �), with f1 and f2, thetwo experimentally determined solutions. Structure cal-culation, visualization and analysis were carried outusing the Discover programs (ACCELRYS). The struc-ture determination protocol used a simulated annealingcalculation (ensemble EI) starting from randomized Car-tesian coordinates to explore the conformational spaceand a restrained molecular dynamic calculation (ensem-ble EII) to re®ne each structure as described elsewhere.49

The o dihedral angles were forced to be 180 � for all resi-dues except for Pro18, which was allowed to evolvefreely during the simulated annealing calculation. Sincein all structures of EI Pro18 was found to be in the transconformation, the o dihedral angles of all residues wererestrained in the trans conformation during the re®ne-ment calculation.

142 3D NMR Structure of Crh

Accession numbers

The coordinates of the 24 re®ned structures of themonomer of Crh and the NMR restraints have beendeposited with the Brookhaven Protein Data Bank underthe accession codes 1K1C. The 1H, 13C and 15N chemicalshifts of Crh monomer residues have been depositedwith the BioMagResBank (BMRB) under the accessionnumber 4972.

Acknowledgments

We thank Roland Montserret for excellent technicalassistance, Nico van Nuland and Pierre Gans for helpfuldiscussions. We thank ACCELRYS for continuing collab-oration. This work has been supported by the CentreNational de la Recherche Scienti®que and the Commis-sariat aÁ l'Energie Atomique. A. F. was recipient of aMESR fellowship.

References

1. Postma, P. W., Lengeler, J. W. & Jacobson, G. R.(1993). Phosphoenolpyruvate:carbohydrate phospho-transferase systems of bacteria. Microbiol. Rev. 57,543-594.

2. Deutscher, J. & Saier, M. H., Jr (1983). ATP-depen-dent protein kinase-catalyzed phosphorylation ofa seryl residue in HPr, a phosphate carrier proteinof the phosphotransferase system in Streptococcuspyogenes. Proc. Natl Acad. Sci. USA, 80, 6790-6794.

3. Galinier, A., Kravanja, M., Engelmann, R.,Hengstenberg, W., Kilhoffer, M. C., Deutscher, J. &Haiech, J. (1998). New protein kinase and proteinphosphatase families mediate signal transduction inbacterial catabolite repression. Proc. Natl Acad. Sci.USA, 95, 1823-1828.

4. Reizer, J., Hoischen, C., Titgemeyer, F., Rivolta, C.,Rabus, R., StuÈ lke, J. et al. (1998). A novel proteinkinase that controls carbon catabolite repression inbacteria. Mol. Microbiol. 27, 1157-1169.

5. Deutscher, J., Kuster, E., Bergstedt, U., Charrier, V.& Hillen, W. (1995). Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carboncatabolite repression in Gram-positive bacteria. Mol.Microbiol. 15, 1049-1053.

6. Jones, B. E., Dossonnet, V., Kuster, E., Hillen, W.,Deutscher, J. & Klevit, R. E. (1997). Binding of thecatabolite repressor protein CcpA to its DNA targetis regulated by phosphorylation of its corepressorHPr. J. Biol. Chem. 272, 26530-26535.

7. Fujita, Y., Miwa, Y., Galinier, A. & Deutscher, J.(1995). Speci®c recognition of the Bacillus subtilis gntcis-acting catabolite-responsive element by a proteincomplex formed between CcpA and seryl-phos-phorylated HPr. Mol. Microbiol. 17, 953-960.

8. Henkin, T. M., Grundy, F. J., Nicholson, W. L. &Chambliss, G. H. (1991). Catabolite repression ofalpha-amylase gene expression in Bacillus subtilisinvolves a trans-acting gene product homologous tothe Escherichia coli lacl and galR repressors. Mol.Microbiol. 5, 575-584.

9. Miwa, Y., Nakata, A., Ogiwara, A., Yamamoto, M.& Fujita, Y. (2000). Evaluation and characterizationof catabolite-responsive elements (cre) of Bacillus sub-tilis. Nucl. Acids Res. 28, 1206-1210.

10. Deutscher, J., Galinier, A. & Martin-Verstraete, I.(2001). Carbohydrate uptake and metabolism. InBacillus subtilis and its Closest Relatives: From Genes toCells (Sonenshein, A. L., Hoch, J. A. & Losick, R.,eds), pp. 129-150, American Society forMicrobiology, Washington, DC.

11. Moreno, M. S., Schneider, B. L., Maile, R. R.,Weyler, W. & Saier, M. H. (2001). Catabolite repres-sion mediated by the CcpA protein in Bacillus subti-lis: novel modes of regulation revealed by whole-genome analyses. Mol. Microbiol. 39, 1366-1381.

12. Kunst, F., Ogasawara, N., Moszer, I., Albertini,A. M., Alloni, G., Azevedo, V. et al. (1997). The com-plete genome sequence of the gram-positive bacter-ium Bacillus subtilis. Nature, 390, 249-256.

13. Galinier, A., Haiech, J., Kilhoffer, M. C., Jaquinod,M., StuÈ lke, J., Deutscher, J. & Martin-Verstraete, I.(1997). The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression.Proc. Natl Acad. Sci. USA, 94, 8439-8444.

14. Darbon, E., Galinier, A., Le Coq, D. & Deutscher, J.(2001). Phosphotransfer functions mutated Bacillussubtilis HPr-like protein Crh carrying a histidine inthe active site. J. Mol. Microbiol. Biotechnol. 3, 439-444.

15. Martin-Verstraete, I., Galinier, A., Darbon, E.,Quentin, Y., Kilhoffer, M. C., Charrier, V. et al.(1999). The Q15H mutation enables Crh, a Bacillussubtilis HPr-like protein, to carry out some regulat-ory HPr functions, but does not make it an effectivephosphocarrier for sugar transport. Microbiology,145, 3195-3204.

16. Galinier, A., Deutscher, J. & Martin-Verstraete, I.(1999). Phosphorylation of either crh or HPr med-iates binding of CcpA to the bacillus subtilis xyn creand catabolite repression of the xyn operon. J. Mol.Biol. 286, 307-314.

17. Martin-Verstraete, I., Deutscher, J. & Galinier, A.(1999). Phosphorylation of HPr and Crh by HprK,early steps in the catabolite repression signallingpathway for the Bacillus subtilis levanase operon.J. Bacteriol. 181, 2966-2996.

18. Presecan-Siedel, E., Galinier, A., Longin, R.,Deutscher, J., Danchin, A., Glaser, P. & Martin-Verstraete, I. (1999). Catabolite regulation of the ptagene as part of carbon ¯ow pathways in Bacillussubtilis. J. Bacteriol. 181, 6889-6897.

19. Penin, F., Favier, A., Montserret, R., Brutscher, B.,Deutscher, J., Marion, D. & Galinier, D. (2001). Evi-dence for a dimerisation state of the Bacillus subtiliscatabolite repression HPr-like protein, Crh. J. Mol.Microbiol. Biotechnol. 3, 429-432.

20. Wittekind, M., Rajagopal, P., Branchini, B. R.,Reizer, J., Saier, M. H., Jr & Klevit, R. E. (1992).Solution structure of the phosphocarrier protein HPrfrom Bacillus subtilis by two-dimensional NMR spec-troscopy. Protein Sci. 1, 1363-1376.

21. van Nuland, N. A., Grotzinger, J., Dijkstra, K.,Scheek, R. M. & Robillard, G. T. (1992). Determi-nation of the three-dimensional solution structure ofthe histidine-containing phosphocarrier protein HPrfrom Escherichia coli using multidimensional NMRspectroscopy. Eur. J. Biochem. 210, 881-891.

22. Wishart, D. S. & Sykes, B. D. (1994). The 13C chemi-cal-shift index: a simple method for the identi®-cation of protein secondary structure using 13Cchemical-shift data. J. Biomol. NMR, 4, 171-180.

23. WuÈ thrich, K. (1986). NMR of Proteins and NucleicAcids, Wiley, New York.

3D NMR Structure of Crh 143

24. Laskowski, R. A., Rullmannn, J. A., MacArthur,M. W., Kaptein, R. & Thornton, J. M. (1996). AQUAand PROCHECK-NMR: programs for checking thequality of protein structures solved by NMR.J. Biomol. NMR, 8, 477-486.

25. Lipari, G. & Szabo, A. (1982). Model free approachto the interpretation of nuclear magnetic resonancerelaxation in macromolecules: 1. Theory and rangeof validity. J. Am. Chem. Soc. 104, 4546-4559.

26. Lipari, G. & Szabo, A. (1982). Model free approachto the interpretation of nuclear magnetic resonancerelaxation in macromolecules: 2. Analysis of exper-imental results. J. Am. Chem. Soc. 104, 4559-4570.

27. Woessner, D. E. (1962). Nuclear spin relaxation inellipsoids undergoing rotational Brownian motion.J. Chem. Phys. 37, 647-654.

28. Tillett, M. L., Blackledge, M. J., Derrick, J. P., Lian,L. Y. & Norwood, T. J. (2000). Overall rotational dif-fusion and internal mobility in domain II of proteinG from Streptococcus determined from 15N relaxationdata. Protein Sci. 9, 1210-1216.

29. Tsan, P., Hus, J. C., Caffrey, M., Marion, D. &Blackledge, M. (2000). Rotational diffusion aniso-tropy and local backbone dynamics of carbon mon-oxide-bound Rhodobacter capsulatus cytochrome c0.J. Am. Chem. Soc. 121, 2311-2312.

30. Pullen, K., Rajagopal, P., Branchini, B. R., Huf®ne,M. E., Reizer, J., Saier, M. H., Jr, et al. (1995). Phos-phorylation of serine-46 in HPr, a key regulatoryprotein in bacteria, results in stabilization of its sol-ution structure. Protein Sci. 4, 2478-2486.

31. van Nuland, N. A., Hangyi, I. W., van Schaik, R. C.,Berendsen, H. J., van Gunsteren, W. F., Scheek, R. M.& Robillard, G. T. (1994). The high-resolution struc-ture of the histidine-containing phosphocarrier pro-tein HPr from Escherichia coli determined byrestrained molecular dynamics from nuclear mag-netic resonance nuclear Overhauser effect data.J. Mol. Biol. 237, 544-559.

32. Garrett, D. S., Seok, Y. J., Peterkofsky, A.,Gronenborn, A. M. & Clore, G. M. (1999). Solutionstructure of the 40,000 Mr phosphoryl transfer com-plex between the N-terminal domain of enzyme Iand HPr. Nature Struct. Biol. 6, 166-173.

33. van Nuland, N. A., Boelens, R., Scheek, R. M. &Robillard, G. T. (1995). High-resolution structure ofthe phosphorylated form of the histidine-containingphosphocarrier protein HPr from Escherichia colidetermined by restrained molecular dynamics fromNMR-NOE data. J. Mol. Biol. 246, 180-193.

34. Chen, Y., Reizer, J., Saier, M. H., Jr, Fairbrother, W. J.& Wright, P. E. (1993). Mapping of the bindinginterfaces of the proteins of the bacterial phospho-transferase system, HPr and IIAglc. Biochemistry, 32,32-37.

35. Wang, G., Sondej, M., Garrett, D. S., Peterkofsky, A.& Clore, G. M. (2000). A common interface onhistidine-containing phosphocarrier protein for inter-action with its partner proteins. J. Biol. Chem. 275,16401-16403.

36. van Nuland, N. A., Kroon, G. J., Dijkstra, K.,Wolters, G. K., Scheek, R. M. & Robillard, G. T.(1993). The NMR determination of the IIA(mtl) bind-ing site on HPr of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.FEBS. Letters, 315, 11-15.

37. StuÈ lke, J., Arnaud, M., Rapoport, G. & Martin-Verstraete, I. (1998). PRD-a protein domain involvedin PTS-dependent induction and carbon catabolite

repression of catabolic operons in bacteria. Mol.Microbiol. 28, 865-874.

38. Charrier, V., Buckley, E., Parsonage, D., Galinier, A.,Darbon, E., Jaquinod, M. et al. (1997). Cloning andsequencing of two enterococcal glpK genes and regu-lation of the encoded glycerol kinases by phosphoe-nolpyruvate-dependent, phosphotransferase system-catalyzed phosphorylation of a single histidyl resi-due. J. Biol. Chem. 272, 14166-14174.

39. Wang, G., Louis, J. M., Sondej, M., Seok, Y. J.,Peterkofsky, A. & Clore, G. M. (2000). Solutionstructure of the phosphoryl transfer complexbetween the signal transducing proteins HPr andIIA(glucose) of the Escherichia coli phosphoenolpyru-vate:sugar phosphotransferase system. EMBO J. 19,5635-5649.

40. Jones, B. E., Rajagopal, P. & Klevit, R. E. (1997).Phosphorylation on histidine is accompanied bylocalized structural changes in the phosphocarrierprotein, HPr from Bacillus subtilis. Protein Sci. 6,2107-2119.

41. Bennett, M. J., Schlunegger, M. P. & Eisenberg, D.(1995). 3D domain swapping: a mechanism foroligomer assembly. Protein Sci. 4, 2455-2468.

42. Ekiel, I., Abrahamson, M., Fulton, D. B., Lindahl, P.,Storer, A. C., Levadoux, W. et al. (1997). NMR struc-tural studies of human cystatin C dimers and mono-mers. J. Mol. Biol. 271, 266-277.

43. Janowski, R., Kozak, M., Jankowska, E., Grzonka,Z., Grubb, A., Abrahamson, M. & Jaskolski, M.(2001). Human cystatin C, an amyloidogenic protein,dimerizes through three-dimensional domain swap-ping. Nature Struct. Biol. 8, 316-320.

44. Bergdoll, M., Remy, M. H., Cagnon, C., Masson,J. M. & Dumas, P. (1997). Proline-dependent oligo-merization with arm exchange. Structure, 5, 391-401.

45. Vuister, G. W. & Bax, A. (1993). Quantitative J corre-lation: a new approach for measuring homonuclearthree-bond J(H. NHa) coupling constants in15N-enriched proteins. J. Am. Chem. Soc. 7772-7777.

46. Farrow, N. A., Muhandiram, R., Singer, A. U.,Pascal, S. M., Kay, C. M., Gish, G. et al. (1994). Back-bone dynamics of a free and phosphopeptide-com-plexed Src homology 2 domain studied by 15NNMR relaxation. Biochemistry, 33, 5984-6003.

47. Dosset, P., Hus, J. C., Blackledge, M. & Marion, D.(2000). Ef®cient analysis of macromolecularrotational diffusion from heteronuclear relaxationdata. J. Biomol. NMR, 16, 23-28.

48. Cornilescu, G., Delaglio, F. & Bax, A. (1999). Proteinbackbone angle restraints from searching a databasefor chemical shift and sequence homology. J. Biomol.NMR, 13, 289-302.

49. Cordier, F., Caffrey, M., Brutscher, B., Cusanovich,M. A., Marion, D. & Blackledge, M. (1998). Solutionstructure, rotational diffusion anisotropy and localbackbone dynamics of Rhodobacter capsulatus cyto-chrome c2. J. Mol. Biol. 281, 341-361.

50. Hutchinson, E. G. & Thornton, J. M. (1996).PROMOTIF: a program to identify and analyzestructural motifs in proteins. Protein Sci. 5, 212-220.

51. Garrett, D. S., Seok, Y. J., Peterkofsky, A., Clore,G. M. & Gronenborn, A. M. (1997). Identi®cation byNMR of the binding surface for the histidine-con-taining phosphocarrier protein HPr on the N-term-inal domain of enzyme I of the Escherichia coliphosphotransferase system. Biochemistry, 36, 4393-4398.

144 3D NMR Structure of Crh

52. Audette, G. F., Engelmann, R., Hengstenberg, W.,Deutscher, J., Hayakawa, K., Quail, J. W. &Delbaere, L. T. (2000). The 1.9 AÊ resolution structureof phospho-serine 46 HPr from Enterococcus faecalis.J. Mol. Biol. 303, 545-553.

53. Liao, D. I. & Herzberg, O. (1994). Re®ned structuresof the active Ser83! Cys and impairedSer46! Asp histidine-containing phosphocarrierproteins. Structure, 2, 1203-1216.

54. Napper, S., Delbaere, L. T. & Waygood, E. B. (1999).The aspartyl replacement of the active site histidinein histidine-containing protein, HPr, of the Escheri-chia coli phosphoenolpyruvate:sugar phosphotrans-ferase system can accept and donate a phosphorylgroup. Spontaneous dephosphorylation of acyl-phosphate autocatalyzes an internal cyclization.J. Biol. Chem. 274, 21776-21782.

55. Maurer, T., Doker, R., Gorler, A., Hengstenberg, W.& Kalbitzer, H. R. (2001). Three-dimensional struc-ture of the histidine-containing phosphocarrier pro-tein (HPr) from Enterococcus faecalis in solution. Eur.J. Biochem. 268, 635-644.

56. Kalbitzer, H. R., Gorler, A., Li, H., Dubovskii, P. V.,Hengstenberg, W., Kowolik, C. et al. (2000). 15Nand 1H NMR study of histidine containing protein(HPr) from Staphylococcus carnosus at high pressure.Protein Sci. 9, 693-703.

57. Jia, Z., Vandonselaar, M., Hengstenberg, W., Quail,J. W. & Delbaere, L. T. (1994). The 1.6 AÊ structure ofhistidine-containing phosphotransfer protein HPrfrom Streptococcus faecalis. J. Mol. Biol. 236, 1341-55.

58. Jia, Z., Quail, J. W., Waygood, E. B. & Delbaere, L. T.(1993). The 2.0-AÊ resolution structure of Escherichia

coli histidine-containing phosphocarrier protein HPr.A redetermination. J. Biol. Chem. 268, 22490-501.

59. Pieper, U., Kapadia, G., Zhu, P. P., Peterkofsky, A.& Herzberg, O. (1995). Structural evidence for theevolutionary divergence of mycoplasma from gram-positive bacteria: the histidine-containing phospho-carrier protein. Structure, 3, 781-790.

60. Napper, S., Anderson, J. W., Georges, F., Quail,J. W., Delbaere, L. T. & Waygood, E. B. (1996).Mutation of serine-46 to aspartate in the histidine-containing protein of Escherichia coli mimics the inac-tivation by phosphorylation of serine-46 in HPrsfrom gram-positive bacteria. Biochemistry, 35, 11260-11267.

Edited by M. F. Summers

(Received 10 October 2001; received in revised form 2January 2002; accepted 2 January 2002)

http://www.academicpress.com/jmb

Supplementary Material comprising four Figuresand one Table is available at IDEAL