High-Resolution Solution Structureof Bacillus subtilis IIAglc

Yuan Chen,1 David A. Case,1 Jonathan Reizer,2 Milton H. Saier, Jr.,2 and Peter E. Wright1*1Department of Molecular Biology, The Scripps Research Institute, La Jolla, California2Department of Biology, University of California at San Diego, La Jolla, California

ABSTRACT The high-resolution solutionstructure of the phosphocarrier protein IIAglc

from Bacillus subtilis is determined using 3Dand 4D heteronuclear NMR methods. B. subti-lis IIAglc contains 162 amino acid residues andis one of the larger proteins for which high-resolution solution structure has been deter-mined by NMR methods. The structures havebeen calculated from a total of 2,232 conforma-tional constraints. Comparison with the X-raycrystal structure indicates that the overall foldis the same in solution and in crystalline envi-ronments, although some local structural dif-ferences are observed. These occur largely inturns and loops, and mostly correspond toregions with high-temperature factors in thecrystal structure. The N-terminus of IIAglc isdisordered in solution. The active site is lo-cated in a concave region of the protein sur-face. The histidine, which accepts the phospho-ryl group (His 83), interacts with a neighboringhistidine (His 68) and is surrounded by hydro-phobic residues. Proteins 31:258–270, 1998.r 1998 Wiley-Liss, Inc.

Key words: IIAglc; NMR; protein phosphoryla-tion; PTS

INTRODUCTION

The bacterial phosphoenolpyruvate:sugar phos-photransferase system (PTS) mediates the concomi-tant transmembrane transport and phosphorylationof a number of simple carbohydrates.1–3 The PTSconsists of two nonsugar-specific proteins, enzyme Iand HPr, and a sugar-specific permease complexknown as enzyme II. The enzyme II complex consistsof two hydrophilic domains, IIA and IIB, and one ortwo transmembrane domains. In the translocationand phosphorylation of sugars, a phosphoryl group istransferred sequentially from phosphoenolpyruvateto enzyme I, HPr, the IIA domain, the IIB domain,and finally to a sugar.

The glucose IIA domain (IIAglc, also known asIIIglc) is a central regulatory protein of the PTS inEscherichia coli. IIAglc functions not only in theuptake and phosphorylation of glucose, but also inregulation of the uptake and phosphorylation ofother PTS and non-PTS sugars by transcriptional

and posttranscriptional mechanisms.4 Bacillus sub-tilis IIAglc consists of 162 residues.5 It has been foundthat phosphorylation occurs at His83, and the phos-phorylation of the corresponding residue in the E.coli protein6,7 controls their regulatory interac-tions.8–14 Also, His 68 has been found to be importantfor phosphoryl transfer from IIAglc to IIBglc.8 The E.coli and B. subtilis PTSs are similar in mechanismand structure, although the involvement of thesesystems in regulation is entirely different.

The three-dimensional structure of B. subtilisIIAglc has been studied by both X-ray crystallogra-phy15 and two- and three-dimensional heteronuclearNMR spectroscopy.16–18 In addition, information aboutbackbone dynamics of the protein has been obtainedby 15N relaxation measurements.19 The three-dimen-sional structure of a homologous protein, E. coliIIAglc, has also been investigated by X-ray crystallog-raphy20 and NMR spectroscopy.21–23 In the X-raycrystallographic structure, the overall fold of thisprotein is similar to that of the B. subtilis protein,but there are notable differences on both secondaryand tertiary structural level between the two pro-teins. Phosphorylation of the active site and proton-ation states of the active site His residues have beeninvestigated for the E. coli protein by high-resolutionNMR methods.24 This work has provided new insightinto the structure of phosphorylated IIAglc.

The global fold of B. subtilis IIAglc determined byX-ray crystallography15 is similar to that determinedby NMR.18 However, there are apparent local differ-ences between the NMR and the crystal structures.For example, the N-terminal region of the B. subtilisIIAglc forms a specific ‘‘hairpin’’ conformation in the

Abbreviations: IIAglc, glucose permease IIA domain; PTS,bacterial phosphotransferase system; NMR, nuclear magneticresonance; 3D, three-dimensional; 4D, four-dimensional; TPPI,time-proportional phase incrementation; FID, free inductiondecay; NOESY, nuclear Overhauser effect; HSQC, hetero-nuclear single-quantum correlation spectroscopy; HMQC, het-eronuclear multiple-quantum correlation spectroscopy; COSY,correlated spectroscopy.

Yuan Chen’s present address is Division of Immunology,Beckman Research Institute of the City of Hope, 1450 E.Duarte Road, Duarte, CA 91010.

*Correspondence to: Peter E. Wright, Department of Molecu-lar Biology, The Scripps Research Institute, La Jolla, CA 92037.

Received 28 April 1997; Accepted 17 November 1997

PROTEINS: Structure, Function, and Genetics 31:258–270 (1998)

r 1998 WILEY-LISS, INC.

crystal structure, but the NMR studies suggest thatthe N-terminal 12 residues are highly mobile. Todate, high-resolution NMR structures have not beenavailable for detailed comparison with the X-raycrystallographic structure.

NMR spectroscopy plays an important role inthree-dimensional structure determination of biologi-cal macromolecules. To generate protein structuresof high accuracy, it is necessary to obtain a largenumber of experimental restraints. Using multidi-mensional heteronuclear NMR methods, we haveidentified 2,137 NOE constraints and 95 torsionalconstraints. These new experimental restraints havebeen used to calculate the high-resolution solutionstructures of the B. subtilis IIAglc domain.

MATERIALS AND METHODSSample Preparation

The overexpression, uniform isotope labeling with15N or both 15N and 13C, and purification of the B.subtilis IIAglc have all been described previously.25,26

The sample was prepared at a concentration of 1–2mM in 90% H2O/10% D2O or 99.996% D2O (fromMSD Isotopes, Inc.) containing 10-mM potassiumphosphate buffer at pH 6.87.

NMR Experiments

All experiments were recorded at 308K on BrukerAMX-500 or AMX-600 spectrometers, equipped formultichannel operation. Simultaneous quadraturedetection was used in the acquisition dimension.Sign discrimination in the other dimensions wasachieved by either time-proportional phase incremen-tation (TPPI),27 States,28,29 or States–TPPI.30 Hetero-nuclear decoupling during acquisition was achievedby GARP-1 phase modulation.31 Chemical shiftswere referenced as described previously.16,18

J-modulated HSQC spectra32 was recorded withthe 15N-labeled sample in order to obtain 3JHNa

coupling constants. Twelve spectra were acquired,corresponding to the following delays (in seconds):0.000, 0.010, 0.020, 0.023, 0.025, 0.028, 0.031, 0.036,0.040, 0.050, 0.060, and 0.080. Spectral widths of 7ppm sampled over 1,024 data points and 68 ppmsampled over 256 data points were used in the v2

and v1 dimensions, respectively. Sign discriminationwas achieved by TPPI. In addition, two 2D 1H–15NHSQC–NOESY experiments33,34 were acquired attwo different mixing times: 20 ms and 40 ms. Thespectral widths were 25 ppm and 38 ppm in the v2

and v1 dimensions, respectively. Digital oversam-pling by a factor of two in the v2 dimension was usedto improve the dynamic range and the baseline.34 Atotal of 4,096 and 512 data points were collected inthe v2 and v1 dimensions, respectively. Water sup-pression was achieved using low-power irradiationof the H2O resonance during the recovery delay. Signdiscrimination in v1 was achieved by the States–TPPI method.29 A 3D 1H–15N NOESY–HMQC35–38

with a mixing time of 100 ms was recorded asdescribed previously.16

1H–13C 3D NOESY–HSQC spectra33,34 were re-corded with carrier frequency centered at 41.1 ppm(the center of the aliphatic 13C) and 126.3 ppm (thecenter of the aromatic 13C) using 15N and 13C doublylabeled IIAglc sample. A 4D NOESY–HMQC spec-trum39 with 13C carrier frequency centered at 41.1ppm was also recorded with this sample. All NOESYexperiments were run at a mixing time of 100 ms.The 4D NOESY–HMQC spectrum was acquired withspectral widths of 36.2 ppm, 8.0 ppm, 36.2 ppm, and25.0 ppm in the v1, v2, v3, and v4 dimensions,respectively. A total of 8, 128, 8, and 256 complexdata points were collected in the v1, v2, v3, and v4

dimensions, respectively. States–TPPI in the v1 andStates in the v2 and v3 dimensions were used for signdiscrimination. For the 3D NOESY–HSQC spectrumwith the 13C carrier placed at the center of aliphatic13C resonances, spectral widths of 8.0 ppm, 36.2ppm, and 25.0 ppm were used in the v1, v2, and v3

dimensions, respectively. For the 3D NOESY–HSQCexperiment with the 13C carrier centered on thearomatic 13C resonances, spectral widths were 8.2ppm, 20.7 ppm, and 25.0 ppm in the v1, v2, and v3

dimensions, respectively. For both experiments,States–TPPI in the v1 and States in the v2 dimen-sions were used for sign discrimination, and a totalof 256, 64, and 1,024 data points were collected in thev1, v2, and v3 dimensions, respectively. A 2D DQF–COSY spectrum and a homonuclear 2D TOCSYspectrum with a 30-ms isotropic mixing period wereacquired as described previously.16

All data were processed either on a Convex C-240computer or an SGI Indigo workstation using amodified version of the FTNMR software or FELIX2.05 (Hare Research, Inc.). Skewed and phase-shifted sine-bell weighting functions or a cosinefunction combined with a Lorentzian-to-Gaussiantransformation was applied before Fourier trans-form. Either a simple spline baseline correction or afirst-order polynomial baseline correction was ap-plied to the acquisition dimension after Fouriertransformation.

NMR Constraints

A total of 2,232 conformational constraints werederived from the NMR data. These include 2,137interproton distance constraints, 57 F constraints,and 38 x1 constraints. The NOE constraints werederived from the 3D 15N NOESY–HMQC spectrum,the two 13C NOESY–HSQC, and the 4D 13C NOESY–HMQC spectra.39 The 13C NOESY–HSQC33,34 spec-trum with the 13C carrier placed in the middle of thearomatic resonances was used to obtain NOE con-straints involving aromatic residues, which aremostly observed in the interior of the protein. Aro-matic spin systems could be assigned from the 3DNOESY–HSQC spectrum, since adjacent protons on

259HIGH-RESOLUTION NMR STRUCTURE OF IIAGLC

the same aromatic ring give particularly strongNOEs. Intraresidue NOEs from ring proton reso-nances to Cb proton resonances were used to makeassignments to specific aromatic residues in thesequence. Once the aromatic resonances had beenassigned, NOEs between aliphatic protons and aro-matic protons were also assigned. The assignmentswere confirmed by the existence of symmetry-relatedpeaks (NOE from aromatic protons to aliphaticprotons) in the 3D 13C NOESY–HSQC spectrumrecorded with 13C carrier in the middle of the ali-phatic resonances.

The upper limits of the distance constraints wereevaluated on the basis of cross-peak volumes. Theupper limits were divided into three categories: 3.0Å, 4.0 Å, and 5.0 Å. An extra 0.5 Å was added to theupper bound for constraints involving methyl groups.The lower limits of the NOE constraints were uni-formly set to 1.8 Å in the DISGEO calculation andset to 1.0 Å in the AMBER calculation. SequentialdaN(i,i 1 1) NOE cross-peaks were used to calibrateconstraints obtained in the 15N-edited NOESY spec-trum, and interstrand daa(i,j) NOE cross-peaks withinthe b-sheets were used to calibrate NOE constraintsobserved in aliphatic 13C-edited NOESY spectra.40

Intraresidue dde NOEs for Phe and Tyr were used tocalibrate NOE constraints derived from the aromatic13C-edited NOESY spectrum.

Torsional constraints were derived from vicinalcoupling constants. 3JHNa coupling constants weredetermined from a series of J-modulated HSQCspectra.32 For 3JHNa less than 5 Hz, the F torsionalconstraints were set as follows: for values of 4 Hz #3JHNa , 5 Hz, the constraints were set to 290° , F ,40°. For 3JHNa , 4 Hz, the constraints were set to260° , F , 0°. For 3JHNa greater than 8 Hz, theconstraints were set to 2160° , F , 280°. A total of57 F constraints were obtained in this manner.Measured J-coupling constants are listed in Table I.

For residues other than proline, x1 constraintswere determined and stereo-specific assignments ofthe Cb methylene protons were made for caseswhere the Ca–Cb bond was predominantly in one ofthe three low-energy staggered rotamer conforma-tions. 3Jab coupling constants of residues that havemethylene groups other than the AMX spin systemswere estimated qualitatively from the 3D 1H–15NTOCSY–HSQC spectrum.41,42 At least two fold differ-ences in the intensities of the two NH–CbH cross-peaks indicated rotamers corresponding to x1 5 180°or x1 5 260°. For AMX spin systems, 3Jab couplingconstants were evaluated from the DQF–COSY spec-trum. In cases where one of the CaH–CbH cross-peaks was strong and the other was missing, therotamers corresponding to x1 5 180° or x1 5 260° aresuggested. When both CaH–CbH cross-peaks weremissing, but both of which appeared strong in theNOESY spectrum, x1 near 60° was indicated. Forrotamers assigned to x1 5 60°, ratios of NH to CaH

and NH to CbH cross-peak intensities were used toconfirm the assignments.42 Stereo-specific assign-ments of the b-methylene protons were obtained

TABLE I. 3JHNa CouplingConstants (1/20.5 Hz)

Residue 3JHNa Residue 3JHNa

2 Ile 7.1 84Phe 5.03 Ala 6.2 86Ile 9.54 Glu 6.8 87Asp 5.46 Leu 5.9 88Thr 5.57 Gln 7.2 89Val 2.6

10 Ile 7.8 90Ser 3.712 Glu 7.0 91Leu 6.913 Glu 5.5 92Lys 6.014 Val 5.2 94Glu 4.915 Phe 8.1 96Phe 9.116 Val 9.8 97Thr 9.319 Ile 8.1 98Ser 8.720 Thr 8.3 99Phe 8.622 Glu 7.3 101Ser 9.223 Ile 6.4 104Asp 3.124 His 9.4 105Arg 6.527 Thr 5.3 107Glu 9.128 Asp 7.6 110Gln 3.829 Val 4.5 112Leu 8.831 Asp 6.9 113Leu 8.233 Val 7.1 114Glu 8.635 Ser 3.0 115Val 9.237 Lys 6.6 116Asp 7.538 Met 5.2 117Leu 3.439 Met 9.0 118Asp 5.141 Phe 7.6 119Ala 6.843 Phe 5.5 120Val 7.044 Ala 8.1 123Asn 9.645 Ile 9.0 124Val 8.346 Leu 8.0 126Ser 5.948 Ser 9.7 127Leu 8.149 Glu 7.9 129Thr 7.851 Ile 8.1 131Ile 9.252 Val 9.0 132Val 10.153 Val 9.0 133Phe 9.556 Val 5.3 138Glu 1.957 Arg 8.3 140Glu 6.759 Lys 8.2 141Thr 9.260 Ile 5.4 142Val 7.361 Leu 8.7 143Ser 9.162 Asn 7.6 144Ile 6.963 Val 9.3 145Lys 8.564 Phe 4.3 146Ala 7.266 Thr 8.4 147Ser 8.668 His 5.4 149Ser 7.069 Ala 7.9 150Val 9.770 Ile 7.3 151Asn 8.972 Leu 8.7 153Glu 5.773 Gln 8.8 155Glu 6.174 Ser 5.1 156Asp 6.975 Asp 6.7 157Ile 7.580 Ile 9.3 158Val 7.981 Leu 9.0 159Lys 8.882 Ile 8.9 160Ile 8.783 His 8.8 161Glu 8.8

260 Y. CHEN ET AL.

from the relative intensities of the intraresidue dNb

NOE cross-peaks in 20-ms and 40-ms 2D 1H–15NNOESY–HSQC spectra. The x1 angles were con-strained to the three rotamers: 160° 6 60°, 180° 660°, 260° 6 60°. A total of 38 x1 constraints wereobtained, as shown in Table II.

Stereo-specific assignments of Pro Cb-methyleneprotons were made on the basis that Hb3 is alwayscloser to the Ha proton. Stereo-specific assignmentsof sidechain NH2 protons in Asn and Gln were basedon the fact that Hd2/He2 is always closer to theHb/Hg methylene protons. Hydrogen bonding con-straints were introduced for slowly exchanging am-ide protons in well-defined b-sheet structures.16

Thirty-five hydrogen-bonding constraints were usedfor initial structure calculations using DISGEO, butwere omitted from the AMBER calculations.

Structure Calculations

Structures were generated using a modified ver-sion of the metric matrix distance geometry programDISGEO43 on CRAY-YMP. In the DISGEO calcula-tions, approximately 1,900 NOE-derived distanceconstraints and 35 hydrogen-bonding constraintswere used. Hydrogen-bonding constraints were in-cluded as distance constraints, defined as follows:HN to O distances were set to 1.8 to 2.0 Å, and N to Odistances were set to 2.7 to 3.0 Å. Methylene protonsor protons groups that were not stereo-specificallyassigned were replaced by pseudoatoms and theupper limits were corrected as described by Wuthrichet al.44 All NOE constraints that are redundant withthe covalent structure were filtered out prior tostructure calculation. ‘‘Sub’’ structures were embed-ded first followed by 2,000 steps of conjugate gradi-ent minimization. Then the complete structureswere embedded and followed by an additional 2,000steps of conjugate gradient minimization in four-dimensional space. Seventy-eight structures wereembedded and refined using DISGEO.

The 28 DISGEO structures with the smallest finalerror were selected for further refinement by re-strained molecular dynamics (MD) simulated anneal-ing using the SANDER module of the programAMBER 4.1. In the MD calculations, a total of 2,137NOE constraints was used, including the NOE con-straints used in the DISGEO calculation plus addi-tional NOE constraints identified later. The hydro-gen-bonding constraints used in the DISGEOcalculations were explicitly excluded from the simu-lated annealing calculations to allow sampling of thealternate hydrogen bonding. In addition, F and x1

dihedral angle constraints were included in the MDcalculation. For methylene protons that were notstereo-specifically assigned, methyl protons and aro-matic ring protons, r26 averaging of the NOE con-straints was used. Parabolic potential functions wereused for distance and dihedral angle constraints.The potential functions are flat between the givenlower limits and the upper limits and rise paraboli-cally. At 0.5 Å or 30° above distance or dihedral angleconstraint upper limits, the penalty function con-verts to a linear, rather than quadratic, form. Thelower limits of the distance constraints were set to 1Å; however, the all-atom force field used will keeptwo protons more than 1-Å apart.

The calculations were performed in vacuum with-out explicit solvent molecules. The net charges ofGlu, Asp, Arg, and Lys sidechains were reduced from61.0 to 60.25 and a distance-dependent dielectricconstant was used to compensate for not includingwater molecules explicitly. The force constant for thetranspeptide bond was set to 50 kcal/mol to enforceplanarity. Three cycles of simulated annealing wereperformed on each of the 28 DISGEO structures. Thestructures were heated from 0.01 to 1,200 K over 1

TABLE II. Stereo-SpecificAssignments*

Residue Hb2 (ppm) Hb3 (ppm) x1

12Glu 2.07 1.96 18015Phe 2.87 3.26 18024His 2.66 3.44 26038Met 2.12 2.06 18039Met 2.06 1.94 18041Asp 2.88 2.72 18046Leu 1.29 2.22 18048Ser 3.88 4.00 16049Glu 1.95 2.25 26059Lys 1.66 1.58 26064Phe 2.63 3.22 26067Lys 1.81 2.04 18068His 2.87 1.20 16073Gln 1.98 1.86 26074Ser 3.86 4.78 16079Glu 1.87 1.71 18081Leu 1.47 1.72 18084Phe 3.60 2.44 18090Ser 4.01 3.98 26091Leu 2.23 2.08 18096Phe 2.87 2.76 26099Phe 2.43 3.47 260

101Ser 3.81 3.59 260105Arg 1.84 2.02 260107Glu 1.59 2.12 260110Gln 2.26 1.80 180112Leu 1.58 1.35 260117Leu 1.89 1.42 260118Asp 2.58 2.70 180121Lys 2.01 1.97 160123Asn 2.58 3.08 260127Leu 1.52 1.58 180133Phe 2.98 3.22 260135Asn 2.65 3.11 160140Glu 2.35 1.65 260145Lys 1.50 1.75 260149Ser 3.88 3.76 180151Asn 2.57 2.68 260155Glu 2.10 1.99 180

*The chemical shifts are referenced as describedpreviously.16,18

261HIGH-RESOLUTION NMR STRUCTURE OF IIAGLC

Fig. 1. Stereo view showing the superposition of the best 16 solution structures of the B. subtilisIIAglc domain. The structures were superimposed to minimize the RMSD of backbone N, Ca, C8atoms of residues 16–161. Residues 1–12 are shown in red; residues 13–162 in pink.

Fig. 2. Plots of several parameters versus residue number. A:Total number of NOE constraints versus residue number. In B,RMSD (NMR) indicates average RMSD of the backbone heavyatoms of the best 16 NMR structures from the mean structure, andS2 indicates order parameters obtained by 15N relaxation measure-ments.19 In C, RMSD (NMR–X-ray) indicates average RMSD ofthe backbone heavy atoms of the 16 NMR structures from theX-ray crystallographic structure, and B-factor represents the aver-age temperature factor of the backbone atoms from the X-raycrystal structure15 of the B. subtilis IIAglc. In B and C, the structureswere superimposed to minimize the RMSD of backbone atoms N,Ca, C8 of residues 16–161.

262 Y. CHEN ET AL.

Fig. 3. Ribbon diagram of one of the solution structures of B. subtilis IIAglc. Residues 1–15 areshown in red; residues 16–162 in pink.

Fig. 4. Stereo view showing the hydrophobic core of an NMR structure. Backbone of the proteinis shown in pink. Sidechains of the hydrophobic aliphatic residues are shown in green, those of thearomatic residues in blue, and hydroxyl oxygen atoms in red.

263HIGH-RESOLUTION NMR STRUCTURE OF IIAGLC

ps, then equilibrated at 1,200 K for 2 ps, and finallycooling down slowly to 0.01 K over 12 ps. The weighton the NMR constraints was 3.2 kcal/mol for the first3 ps of MD simulation, and was then set to 32kcal/mol during the remaining 12 ps of simulatedannealing. The 20 best structures were selected onthe basis of minimal violations of NMR constraintsand covalent geometry. Then a fourth round of 15-pssimulated annealing for over a period of 15 ps wasperformed for these structures with the force con-stant for NMR constraint violations maintained at32 kcal/mol throughout. Two additional rounds ofsimulated annealing (over 15 ps each) were per-formed with the correct protonation states of His 68and His 83 (He protonation of His 68 and Hdprotonation of His 83), as suggested.24

RESULTS AND DISCUSSIONQuality of the NMR Structures

The 16-MD-refined structures of B. subtilis IIAglc

with the lowest NMR constraint violations and low-est AMBER energies were used for detailed analysisof the structure. Figure 1 shows superposition of thebackbone N, Ca, and C8 atoms of the 16 NMRstructures. The largest NOE constraint violationamong these structures is 0.33 Å, and no torsionalangle constraints are violated by more than 10°. Thebest 16 structures display only small deviations fromidealized covalent geometry (Table III). The struc-tures also have very good nonbonded contacts withnonbonded energies ranging from 2820 to 2778kcal/mol. AMBER energies range from 21,746.2 to21,666.1 kcal/mol for the best 16 structures. Allpeptide bonds are trans. The structures have goodbackbone conformation with 63% residues in themost favorable regions, 29% residues in additionallyallowed regions, and 4.3% in generally allowed re-gions of the F–C space according to the programProcheck-NMR.45

The backbone conformation of the IIAglc is welldefined, except at the N-terminus where residues1–15 are disordered. The average RMSD for allbackbone heavy atoms (C8, Ca, N) of residues inb-sheets, a-helices, and 310-helices (as specified inTable III) is 0.52 Å. The average RMSD for allbackbone heavy atoms of residues 16–162 is 0.70 Å.The overall high precision of the NMR structures isclearly correlated to the high density of experimentalconstraints. In regions of helices and b-sheets, asmany as 62 NOE constraints per residue have beenused in structural calculation. On average, 14 con-straints per residue have been identified. The num-ber of NOE constraints per residue and the backboneRMSD from the mean structure for each residue areshown in Figure 2.

Certain regions of the protein are less well defined,as shown by larger RMSD values. These regionsinclude residues 1–15, 56–58, 75–78, 90, 103–106,136–138, 147, 155–157, and 162. These regions occur

in loops and turns on the protein surface and havefewer NOE constraints per residue than average(Fig. 2A). In particular, no nonsequential NOEsinvolving the N-terminal 11 residues was observed.15N relaxation measurements19 reveal that some ofthese regions exhibit true dynamic disorders. Thus,residues 1–15, 102, 136–138, 139, 146–149, and 162all have order parameters (S2) that are significantlybelow average (Fig. 2B), suggesting that the in-creased RMSD in these regions arise from backboneflexibility. However, backbone RMSD and order pa-rameters do not correlate well in other regions; in

TABLE III. Structural Statistics

Number of NOE constraints

NOEs between CaH/NH or CaH/NH to CbH 497All other interresidue NOEs 689Intraresidue NOEs 951

Average number of distance constraint violationsper NMR structure

0.1–0.2 Å 32.50.2–0.3 Å 5.00.3–0.4 Å 0.69.0.5 Å 0

Number of distance constraint violationsin the X-ray structure

0.1–0.2 Å 160.2–0.3 Å 110.3–0.4 Å 140.4–1.0 Å 45.1.0 Å 44

Average RMSD from the mean structurefor the NMR structures

BackboneN, Ca, C8

All heavyatoms

Residue 16–162 0.70 Å 1.19 ÅResidues in secondary structures* 0.53 Å 1.01 Å

Average RMSD of the NMR strucutresfrom the X-ray structure

BackboneN, Ca, C8

All heavyatoms

Residue 16–162 1.44 Å 1.48 ÅResidues in secondary structures 1.14 Å 1.29 Å

Average molecular mechanics energiesof the NMR structures

Amber energy 5 21703.0 kcal/mol (ranges from 21746.2to 21666.1 kcal/mol)

Nonbonded 5 2802.4 kcal/mol (ranges from 2820.6 to2778.2 kcal/mol)

NOE energy 5 41.6 kcal/mol (ranges from 33.9 to 50.8kcal/mol)

Torsional constraints energy 5 3.4 kcal/mol (ranges from1.7 to 5.9 kcal/mol)

RMSD of bond length from ideal value 5 0.005 ÅRMSD of bond angle from ideal value 5 2.2°

*Residues in secondary structures: 19–24, 26–28, 32–35, 41–46, 52–54, 58–64, 71–74, 79–84, 96–98, 104–106, 110–115,117–123, 129–134, 140–144, 149–152, 158–161.

264 Y. CHEN ET AL.

TABLE IV. Hydrogen BondingAnalysis IIAglc

Donor Acceptor

A/B*(NMR

structure)

X-raystruc-ture

Slowex-

change

Backbone to backbone

16 Val N 55 Pro O 13/0 A Y17 Ser N 154 Gln O 13/1 A Y21 Gly N 150 Val O 15/0 A Y22 Glu N 46 Leu O 16/0 A Y23 Ile N 148 Gly O 11/0 A Y24 Hip N 44 Ala O 16/0 A28 Asp N 25 Pro O 12/0 B Y29 Val N 26 Ile O 13/2 B Y31 Asp N 29 Val O 16/0 A35 Ser N 31 Asp O 14/0 A36 Gly N 32 Gln O 15/0 A37 Lys N 34 Phe O 16/0 A38 Met N 36 Gly O 16/0 —38 Met N 33 Val O — B40 Gly N 37 Lys O 16/0 B41 Asp N 133 Phe O 11/5 — Y43 Phe N 131 Ile O 16/0 A Y44 Ala N 24 Hip O 16/0 A Y45 Ile N 129 Thr O 13/2 A Y46 Leu N 22 Glu O 16/0 A Y48 Ser N 20 Thr O 11/0 A49 Glu N 20 Thr O 1/9 —51 Ile N 49 Glu O 11/0 —52 Val N 113 Leu O 16/0 A Y53 Val N 18 Pro O 16/0 A Y54 Ser N 110 Gln O 10/0 A Y58 Gly N 107 Glu O 4/4 — Y58 Gly N 106 Val O — A Y59 Lys N 73 Gln O 9/1 A Y60 Ile N 104 Asp O 11/1 A Y61 Leu N 71 Gly O 10/1 A Y62 Asn N 71 Gly O 13/2 B Y64 Phe N 69 Ala O 14/1 A Y67 Lys N 64 Phe O 13/0 A70 Ile N 82 Ile O 5/8 A Y71 Gly N 62 Asn O 15/0 A Y72 Leu N 80 Ile O 16/0 A Y73 Gln N 59 Lys O 11/1 A Y74 Ser N 78 Arg O 8/1 A Y75 Asp N 57 Arg O — A77 Gly N 74 Ser O — A77 Gly N 75 Asp O 8/0 —80 Ile N 72 Leu O 16/0 A Y81 Leu N 132 Val O 16/0 A Y82 Ile N 70 Ile O 15/0 A Y83 Hid N 130 Pro O 6/6 A Y84 Phe N 68 Hie O 14/0 A Y86 Ile N 126 Ser O 12/0 A91 Leu N 87 Asp O 6/3 —91 Leu N 88 Thr O — A92 Lys N 89 Val O 12/0 A94 Glu N 91 Leu O 10/0 A96 Phe N 93 Gly O 15/0 A Y97 Thr N 114 Glu O 13/0 A Y99 Phe N 112 Leu O 15/0 A

100 Val N 112 Leu O 9/3 —102 Glu N 100 Val O 9/2 —104 Asp N 101 Ser O — A Y104 Asp N 102 Glu O 7/2 — Y106 Val N 58 Gly O — A Y107 Glu N 58 Gly — 7/1 — Y109 Gly N 54 Ser O — A Y110 Gln N 107 Glu O — A Y112 Leu N 52 Val O 16/0 A Y

TABLE IV. (Continued)

Donor Acceptor

A/B*(NMR

structure)

X-raystruc-ture

Slowex-

change

113 Leu N 52 Val O 4/12 A Y114 Glu N 97 Thr O 16/0 A Y115 Val N 50 Gly O 16/0 A Y116 Asp N 95 Gly O 16/0 A Y117 Leu N 115 Val O 15/0 —120 Val N 116 Asp O 16/0 B Y121 Lys N 117 Leu O 16/0 A Y123 Asn N 120 Val O 15/0 A124 Val N 121 Lys O 11/0 — Y129 Thr N 45 Ile O 14/0 A Y131 Ile N 43 Phe O 16/0 A Y132 Val N 81 Leu O 16/0 A Y133 Phe N 41 Asp O 16/0 A Y134 Thr N 79 Glu O 14/0 A Y136 Leu N 134 Thr O 14/0 —137 Ala N 135 Asn O 11/0 — Y139 Gly N 136 Leu O 9/1 —140 Glu N 136 Leu O 14/0 — Y140 Glu N 137 Ala O — A Y141 Thr N 161 Glu O 13/0 A Y143 Ser N 159 Lys O 16/0 A Y145 Lys N 157 Ile O 15/0 A145 Ala N 157 Ile O 15/1 A148 Gly N 146 Ala O 15/0 —150 Val N 21 Gly O 16/0 A Y153 Gly N 17 Ser O 14/0 A Y154 Gln N 151 Asn O — B Y159 Lys N 143 Ser O 16/0 A Y161 Gly N 141 Thr O 16/0 A Y

Between backbone and sidechain

17 Ser OG 151 Asn O 14/0 —19 Ile N 17 Ser OG — B Y31 Asp N 35 Ser OB 0/8 —33 Val N 31 Asp OD1 — A35 Ser OG 29 Val O 11/0 —48 Ser OG 20 Thr O 15/0 —54 Ser OG 110 Gln O 9/0 —56 Val N 54 Ser OG — B63 Val N 102 Glu OE2 — A76 Gly N 13 Glu OE2 — A78 Arg N 74 Ser OG — A79 Glu N 135 Asn OD1 — A85 Gly N 129 Thr OG1 13/0 B Y88 Thr OG1 84 Phe O 14/0 A

101 Ser N 104 Asp OD2 — A Y107 Glu N 110 Gln OE1 — A Y119 Ala N 116 Asp OD1 — B Y123 Asn ND2 119 Ala O 15/0 —126 Ser OG 125 Pro O 10/0 —129 Thr OG1 127 Leu O 14/1 —128 Met N 126 Ser OG — A Y135 Asn ND2 77 Gly O — A137 Ala N 140 Glu OE1 — A Y141 Thr OG1 161 Glu O 12/0 —142 Val N 41 Asp OD2 — A Y149 Ser OG 21 Gly O 14/0 —151 Asn N 154 Gln OE1 — A Y152 Arg N 20 Thr OG1 13/0 A Y

*Hydrogen bonds were classified into two categories based onthe H...X acceptor X 5 O, N) distance. In category A, H...X ,2.2 Å; in category B, 2.2 Å , H...X , 2.6 Å. In addition, theNH...X hydrogen bond angle had to be greater than 135° for ahydrogen bond to be counted.

265HIGH-RESOLUTION NMR STRUCTURE OF IIAGLC

particular, the small S2 values for residues Asp31–Phe 34 indicate large-amplitude conformationalfluctuations that are not reflected as backbone disor-der in the family of NMR structures.

Description of the Structure

The locations of the secondary-structural elementsin the refined NMR structure are in good agreementwith the previous low-resolution NMR structure17

and with the X-ray structure of B. subtilis IIAglc.15

The overall topology (Fig. 3) is an antiparallelb-barrel, made up from three b-sheets.17 On thebasis of backbone dihedral angles and hydrogen-bonding patterns, b-sheet 1 is formed from eightb-strands comprising residues 19–24, 41–46, 58–64,71–74, 79–84, 104–106, 129–134, and 149–152; b-sheet 2 is formed by residues 52–54, 96–98, and110–115; and b-sheet 3 consists of residues 140–144and 158–161. In addition, the structures contain fourshort helices. A 310-helix is formed by residues 26–28,whereas residues 32–35 form an a-helix, residues117–123 form an a-helix followed by a 310-helix, andresidues 88–91 adopt an irregular helical conforma-tion. The last helix is not well defined in the NMR

structures, with above average RMSD between back-bone heavy atoms. Slow conformational fluctuationsin this region are suggested by the absence of slowlyexchanging amide protons, indicating a lack of stablehydrogen bonding, and the occurrence of 15N ex-change broadening contributions, indicative of mo-tions on the millisecond-to-microsecond time scale.19

Most residues in regular secondary structures havewell-defined backbone conformations, as indicatedby small standard deviations of backbone F and Cangles.

The IIAglc structures have an extensive hydropho-bic core (Fig. 4). The central core formed by packingof the b-sheets contains residues Met 12, Phe 15, Pro18, Ile 19, Ile 23, Phe 43, Ala 44, Ile 45, Pro 47, Pro48, Val 52, Pro 55, Val 56, Ile 60, Val 63, Leu 72, Leu81, Ile 82, Phe 84, Phe 96, Val 100, Leu 112, Leu 113,Val 115, Leu 117, Val 120, Val 124, Met 128, Ile 131,Phe 133, Leu 136, Val 142, Ile 144, Ala 146, Val 150,Ile 157, Val 158, and Ile 160. A second hydrophobiccore is formed between the central b-sheet and thelarge loop formed by residues 24–42 (Fig. 4). Thiscontains the sidechains of Ile 26, Val 29, Pro 30, Phe34, Ile 80, Ile 86, Leu 127, Pro 130, and Val 123.

Fig. 5. Ramachandran plot for all residues in the 16 solution structures. The glycine residues areindicated with (1) symbols, and residues Lys 37, Met 38, Lys 67, Lys 92, and Glu 143 are indicatedby solid circles.

266 Y. CHEN ET AL.

Fig. 6. Stereo view showing the hydrophobic surface surround-ing the active site His residues of an NMR structure. Sidechains ofthe hydrophobic residues are shown in green, those of the polar

residues in orange, those of the positively charged residues inblue, and those of the negatively charged residues in red. Theactive site His residues are shown in cyan.

Fig. 7. A space-filling model showingthe interaction of the two active site Hisresidues in an NMR structure. Proteinbackbone is shown in yellow, carbonatoms in white, nitrogen atoms in blue,and hydrogen atoms in cyan.

Fig. 8. Superposi-tion of the X-ray crys-tal structure and oneof the NMR structures.The NMR structure isshown in pink and theX-ray structure in red.

Three hydrophilic residues, Ser 17, Thr 88, and Thr129, are also found in the hydrophobic core, and thesidechains of these residues form hydrogen bondswith backbone oxygen atoms of residues 151, 84, and127, respectively.

Sixty-seven out of 78 slowly exchanging amideprotons form hydrogen bonds in more than half ofthe best 16 NMR structures (Table IV). On the otherhand, the amide protons of some residues that are inhydrogen bonding in more than half of the NMRstructures were not identified as slowly exchangingamide protons. These residues mainly occur in turnsand loops on the surface.

The majority of the backbone dihedral angles ofthe 16 NMR structures have energetically favorablevalues. The Ramachandran plot of the best 16 solu-tion structures is shown in Figure 5. Most of theresidues have negative F dihedral angles. However,a few nonglycine residues appear to have positive Fangles in the solution structures. These residues areLys 37 (F 5 72.1 6 1.6°), Met 38 (30.3 6 5.6°), Lys67 (58.4 6 24.4°), Lys 92 (57.4 6 13.3°), and Glu 153(59.1 6 6.86°). All of these residues have dNa NOEintensities 5–10 times greater than the dNa NOEintensities of residues with apparent negative Fdihedral angles (with 3JHNa . 8 Hz or 3JHNa , 4 Hz)in the 40-ms 2D 1H–15N NOESY–HSQC spectrum.Also, the 3JHNa coupling constants of these residuesare 6.61, 5.2, 6.01, and 5.65 Hz for residues Lys 37,Met 38, Lys 92, and Glu 153, respectively. The 3JHNa

coupling constant of Lys 67 was not determinedbecause of resonance overlap. The experimentalNMR data are thus consistent with these residueshaving positive F values.

Structure of the Active Site

The active site for phosphoryl transfer is locatedon a concave surface formed by b-sheet 1, residues87–92, and residues 30–40 (Fig. 6). His 83 partici-pates directly in phosphoryl transfer, accepting aphosphoryl group from HPr and donating it to theIIAglc domain. This histidine is positioned near thecenter of the concave surface, in close proximity toHis 68, and surrounded by hydrophobic residues(Pro 30, Val 33, Phe 34, Met 38, Met 39, Phe 64, Pro65, Ile 70, Leu 81, Ile 86, Val 89, and Val 132). Thissurface of IIAglc has been identified as the bindingsite for HPr by observation of chemical shift changesupon complex formation.46 Polar residues at theperiphery of the binding surface (Fig. 6) may play arole in recognition of HPr. These polar sidechains(Asp 31, Gln 32, Asn 62, Thr 66, Glu 79, Asp 87, Ser90, Lys 92, and Thr 134) are shown in Figure 6. Onthe basis of molecular modeling, it has been proposedthat Asp 31, Gln 32, and Glu 79, and Asp 87 and Thr134 in particular, play a direct role in formation ofthe HPr–IIAglc complex.47 The analogous surface ofE. coli IIAglc has been shown to be involved incomplex formation with glycerol kinase.14

The two histidine residues, His 83 and His 68,which are involved in the phosphoryl transfer activ-ity, are close to each other in the 3D structure of B.subtilis IIAglc (Fig. 7). The conformation at the activesite in the NMR structure is very similar to that ofthe crystal structure.15 In 50% or more NMR struc-tures, the Nd–H atom of His 83 is hydrogen-bondedto the mainchain oxygen of Gly 85, and the Nd atomof His 68 accepts a hydrogen bond from the sidechainO–H atoms of Thr 66. The Ne atoms of His 83 ispartially exposed to solvent water molecules andavailable for nucleophilic attack by phospho-HPr.The Ne–H atom of His 68 interacts with Ne atoms ofHis 83, and is ready for hydrogen bonding to thephosphoryl group on His 83 in phosphorylated IIAglc.Since His 68 is crucial for phosphoryl transfer fromIIAglc to IIBglc, the hydrogen bond may play impor-tant roles in stabilizing the transition state.

Comparison With the X-RayCrystallographic Structure

The overall fold of the NMR structures and theX-ray crystallographic structure of the B. subtilisIIAglc, determined at 2.2-Å resolution,15 are verysimilar (Fig. 8), except for residues 1–15, which aredisordered in solution (Fig. 1). The backbone rootmean square differences between the X-ray andNMR structures and the X-ray temperature factorsare shown in Figure 2C. The largest RMS differencesbetween the X-ray and solution structures tend tooccur in areas that have above-average crystallo-graphic B-factors. The largest deviation between thecrystal and solution structures occur at the N-terminus. Residues 1–15 are disordered in the NMRsolution structures, and residues 2–13 are highlymobile as determined by 15N relaxation measure-ments.19 In contrast, only residues 1–3 are disor-dered in the crystal structure, and residues 4–13adopt a unique conformation, albeit with above-average backbone temperature factors. The N-terminal region forms part of the Q-linker, whichjoins the IIAglc domain to the membrane-boundIICBglc domains,5 and its flexibility is not unex-pected. Other regions where significant differencesoccur between the solution and crystal structuresinclude residues 75–77, 101–109, and 136–141; all ofthese regions correspond to exposed loops and allcontain residues with above-average temperaturefactors. The average RMSD between the 16 solutionstructures and the crystal structure is 1.14 Å for thebackbone heavy atoms and 1.88 Å for all heavyatoms among residues in regions that have regularsecondary structures (helices and b-sheets). Includ-ing all turns and loops, the average RMSD for allresidues 16–161 between the 16 solution structuresand the crystal structure increases to 1.44 Å forbackbone heavy atoms and 1.48 Å for all heavyatoms. There are 44 NOE constraints that areviolated by more than 1 Å in the X-ray crystallo-

268 Y. CHEN ET AL.

graphic structure. Only three of the violated con-straints are located in the middle of b-sheets; allother violations involve residues in turns and loopson the protein surface, and suggest true differencesbetween the crystal and solution structures in theseregions.

There is generally good agreement between back-bone dihedral angles in the NMR structures and theX-ray crystallographic structure. For most of theresidues in regions of well-defined conformation insolution structures, the backbone F and C angles inthe NMR structures and X-ray crystallographic struc-ture are very similar, and some small differences arewithin the uncertainty of the NMR structures, asshown in Figure 3. The residues that show persistentpositive F angles in the solution structures alsoshow similar F dihedral angles in the X-ray crystal-lographic structure, except for Met 38. The F angleof Met 38 is 267° in the X-ray structure, as opposedto 30.3 6 5.6° in the solution structures. Otherdeviations of the backbone dihedral angles in X-raycrystallographic structure that are larger than onestandard deviation from the average backbone dihe-dral angles in the solution structure are primarilylocalized to residues 76–78, 100–106, 135–139, and147–148. All these regions are located in turns orloops on the surface, and are relatively disordered inthe solution structures.

We conclude that the major features of the solutionstructure are very similar to the X-ray crystallo-graphic structure. The similarity includes the second-ary structure, overall tertiary fold, and details of theactive site structure. Observed structural differencesare mainly localized at the N-terminus, which ishighly flexible in solution, and loops and turns on theprotein surface. The solution structures of B. subtilisIIAglc provide additional insights into the conforma-tion of this important phosphoryl group transferprotein.

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