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1172 nature structural biology • volume 7 number 12 • december 2000

In bacteria, periplasmic and outer-membrane proteins are syn-thesized in the cytoplasm as precursor proteins that contain N-terminal signal sequences (preproteins)1. They are first recog-nized and bound by SecB and then targeted to the membrane forexport via the interaction of SecB with SecA, a peripheral mem-brane protein at the site of translocation2–7. The binding of pre-proteins to SecB enables them to exist in a translocationcompetent state that will neither aggregate nor fold8. These rolesin protein export are demonstrated in vivo by the cytoplasmicaccumulation of pulse-labeled precursor species in a strain thatis devoid of SecB9 and in vitro by showing that SecB is requiredfor the translocation of precursors into inverted vesicles of cyto-plasmic membrane10.

Despite extensive research on these early events in proteintranslocation, a central question remains unanswered: what is thestructural feature of SecB that allows it to bind to preproteins aswell as to membrane bound SecA for translocation? There is arapidly growing body of information pertinent to the recognitionof partially unfolded proteins by molecular chaperones. Crystalstructures of GroEL11 and DnaK12 reveal that both proteins usehydrophobic structural elements to bind and stabilize non-nativepolypeptides. Like GroEL and DnaK, SecB binds and stabilizespreproteins that are in a non-native conformation with theirhydrophobic structural core exposed to the solvent13. However,SecB is unique in that it is a small protein of 17–20 kDa that hasno enzyme activity. Although SecB can bind to a spectrum ofnon-native polypeptides in vitro, in vivo it seems to selectivelybind only to polypeptides that are destined for translocation14.Models for the mode of action of SecB have been proposed basedon biochemical and kinetic data. However, the lack of a high reso-lution structure of SecB has impeded further understanding ofSecB function. We have used X-ray crystallography to determinethe three-dimensional structure of SecB from the bacteriumHaemophilus influenzae to 2.5 Å resolution. This structure hasenabled us to build a molecular model of how SecB binds non-native polypeptides and interacts with its downstream targetSecA.

Tertiary structure of SecBSecB exists as a homotetramer in solution (the subunits are heretermed A to D). The monomer of SecB has a simple α + β fold(Fig. 1). Apart from the disordered N-terminal 14 amino acids,

the first half of the sequence (residues 15–96) folds into a four-stranded antiparallel β-sheet. The sheet has a typical Greek-keytopology, with the first two strands (β1 and β2) located at theopposite sides of the sheet. The loop (crossover loop) connectingthese two strands crosses over at one end of the sheet and includesa short 310-helix. The second half of the molecule contains twoantiparallel α-helices. Helix α1 is a long eight-turn helix and runsdiagonally underneath the β-sheet. Because the β-sheet has a typ-ical left-handed twist, it intimately wraps around the helix.Packing between these two structural elements forms thehydrophobic core of the molecule. The second helix (α2) con-nects to α1 through a long 11-residue loop. This loop (helix-con-necting loop) has an extended conformation and ispredominantly hydrophobic; it is one of the highly conservedregions in SecB. Together with strand β2 and the crossover loop,the helix-connecting loop forms a large hydrophobic groove onthe side surface of the molecule (Fig. 2b, left). We propose thatthis is the peptide binding surface of SecB (see below). The lengthof helix α2 varies greatly among the different subunits (fromthree turns in subunit D to six turns in subunit B). After the thirdturn, the helix no longer packs against the rest of the moleculeand becomes freestanding in solution. The structural differencesin α2 from the four subunits reflect the differences in their indi-vidual crystal packing environments. Proton NMR spectroscopyconfirms that the C-termini of the SecB subunits are solventexposed and highly mobile15.

Quaternary structure of SecBThe tetrameric SecB molecule is organized as a dimer of dimers.Two monomers pair together mainly via strand β1 and helix α1to form a dimer (Fig. 2a). The prominent feature of the dimer isan eight-stranded antiparallel β-sheet. The dimer is primarilystabilized by hydrogen bonds between the two antiparallel β1strands as well as by the burial of hydrophobic side chains. Twodimers then associate to form a tetramer with the four long α1helices sandwiched between two antiparallel β-sheets (Fig. 2b).The tetramer has an overall rectangular shape with a slenderwaist where four helices meet to form the dimer–dimer interface.Examination of the dimer–dimer interface shows that it consistsof mainly polar interactions involving side chains from the fourα1 helices. When the four subunits of the tetramer are superim-posed, the root mean square (r.m.s.) deviation of the main chain

Crystal structure of the bacterial protein export chaperone SecBZhaohui Xu, John D. Knafels and Kae Yoshino

SecB is a bacterial molecular chaperone involved in mediating translocation of newly synthesized polypeptidesacross the cytoplasmic membrane of bacteria. The crystal structure of SecB from Haemophilus influenzae showsthat the molecule is a tetramer organized as a dimer of dimers. Two long channels run along the side of themolecule. These are bounded by flexible loops and lined with conserved hydrophobic amino acids, which define asuitable environment for binding non-native polypeptides. The structure also reveals an acidic region on the topsurface of the molecule, several residues of which have been implicated in binding to SecA, its downstream target.

Department of Biological Chemistry, The University of Michigan Medical School, 1301 E. Catherine Road, Ann Arbor, Michigan 48109, USA.

Correspondence should be addressed to Z.X. email: [email protected]

© 2000 Nature America Inc. • http://structbio.nature.com©

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nature structural biology • volume 7 number 12 • december 2000 1173

Cα atoms is between 0.45 Å and 0.72 Å. The major conforma-tional variations between subunits are in the loops and the C-terminal helices.

Independent biochemical, biophysical and genetic data onSecB are consistent with our crystal structure. Most of thesestudies have been performed on Escherichia coli SecB. To helpfacilitate the structure interpretation, we use the one-letter codefor all E. coli residues and the three-letter code for all H. influen-zae residues (refer to Fig. 1a). The L75Q (Ile 84) and E77K(Glu 86) mutations in E. coli have no effect on tetrameriza-tion16,17. These residues are located on strand β4 but their sidechains point away from the interface (Fig. 2a, green residues). Incontrast, the C76Y (Cys 85), V78F (Val 87) and Q80R (Gln 89)mutations shift the tetramer–dimer equilibrium in solutiontowards the dimer. These residues are also located on strand β4but their side chains point towards the core of the molecule(Fig. 2a, red residues). Although they do not participate directly

in the interface, it is conceivable that these drastic mutationscould affect the overall folding of the subunit, leading to the disruption of the dimer–dimer interface.

The peptide binding siteThe peptide binding site of SecB must satisfy three biochemicalcriteria. First, it must be located on the surface of SecB18. TheSecB tetramer has a very stable quaternary structure over a rangeof solution pHs, with an estimated tetramer–dimer equilibriumconstant well below 20 nM (ref. 17). It is unlikely that the bindingof peptide would require a change in the oligomerization state ofSecB. Second, the binding site must have an overall hydrophobiccharacter. It is generally believed that the presence of a signal pep-tide in the preprotein slows the folding of the polypeptide, thusexposing the non-native features for SecB to bind19,20. Isothermaltitration calorimetry has demonstrated that hydrophobic interac-tion is the major driving force for SecB binding to non-native

Fig. 1 The structure of the SecB monomer. a, Multiple sequencealignment of bacterial SecB proteins. Sequences were alignedusing ClustalW33. Invariant residues are shaded and outlined.Homologous residues are also outlined. Secondary structureelements are indicated underneath the sequence: α-helices aredrawn as blue cylinders; β-strands as yellow arrows; and otherelements as gray lines. Residues that line subsite 1 of the pep-tide binding groove are outlined by purple boxes. Residues thatline subsite 2 of the peptide binding groove are outlined bycyan boxes. b, Ribbon drawing of the SecB monomer. The sec-ondary structure elements are labeled and colored as in (a): α-helices are drawn as blue coils; β-strands as yellow arrows;and other elements as thick gray lines. c, Stereo view of a Cαtrace of the SecB monomer, with every 10th residue labeled. d, Experimental electron density map. Stereo view of an m |Fo| exp(iφmod) electron density map contoured at 1.5 σ andsuperimposed upon the refined model. m, |Fo|, and φmod are thefigure of merit, the observed amplitudes and modified MADphases after solvent flattening and phase extension to 2.5 Å,respectively. Molscript34 and POV-ray35 were used to produce(b,c); O32 was used to produce (d).

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polypeptides18. Third, the binding site should be flexible and plas-tic to accommodate a spectrum of peptides13,14.

Only one surface area on SecB can satisfy all three criteria(Fig. 3a). It is a groove formed between helix α2, strand β2, andthe crossover loop, with the helix-connecting loop as its floor.We propose that this groove is the peptide binding site of SecB.The tetramer contains four grooves, two on each side of the mol-ecule between the two β-sheets. The two grooves on the sameside of the tetramer fuse to form a 70 Å long continuous bindingchannel (Fig. 2b, right) so that the tetramer contains two chan-nels. Both ends of the channels are rather shallow, but the mid-section is deep, bounded by the loops of the two sheets.

Each groove can be roughly divided into two subsites (Fig. 3b).Subsite 1 constitutes the deep section of the channel. It has thecrossover loops of the two sheets as its wall and the first half of thehelix-connecting loops as its floor. Most of the amino acids liningthe surface of the channels are aromatic and conserved (Fig. 3b,purple residues). Because the loops are in a very extended confor-mation, both main chain and side chain atoms contribute to thesurface. They are structurally flexible, as shown by the conforma-tional fluctuation among the different subunits, and can providethe necessary plasticity for binding peptides. Subsite 2 is borderedby helix α2, strand β2 and the second half of the helix-connectingloop (Fig. 3b). It can be viewed as a floor extension of subsite 1.Many of the amino acids lining this site are hydrophobic but notaromatic (Fig. 3b, cyan residues). It is much shallower and con-siderably more open than subsite 1. In one of the subunits (sub-unit B), there are some extra electron densities in the spacebetween helix α2 and strand β2, which we have assigned to the N-terminus of the C subunit of a neighboring tetramer in thecrystal. The main chain of the polypeptide makes hydrogenbonds to strand β2 and becomes an additional antiparallel strandof the sheet. This is reminiscent of the peptide binding siteobserved in the structure of the phosphotyrosine binding (PTB)domain21. The PTB domain was initially identified as a peptidebinding domain that binds phosphotyrosyl containing peptidesin a manner different from that of the Src homology 2 (SH2)domain22. The structure of the PTB domain revealed that the pep-tide is bound between a four-stranded antiparallel β-sheet and an α-helix. In particular, the N-terminal part of the peptide formsantiparallel hydrogen bonds with the β-sheet while the Tyr con-taining NPxY recognition motif and the C-terminus binds to ahydrophobic pocket formed by the loop regions of PTB21. A com-parison between SecB and the PTB domain shows that the twoproteins have an interesting structural similarity; SecB has atopology similar to that of the C-terminal half of the PTB domain(Fig. 3c). This observation suggests that polypeptide binding toSecB and to the PTB domain could occur in a similar fashion.

We propose that the two peptide binding subsites of SecB rec-ognize distinct features in substrate polypeptides. Subsite 1 rec-ognizes hydrophobic/aromatic regions of the polypeptides while

subsite 2 binds to more extended regions of the polypeptides byforming regular main chain hydrogen bonds with them. In arecent study, Knoblauch et al.23 suggested that the ‘SecB bindingmotif ’ within polypeptide substrates is a nine-residue longsequence that is enriched in aromatic and basic residues. Thearomatic nature of subsite 1 makes it an ideal place to bind anaromatic residue rich polypeptide because it can be particularlystabilized by ring–ring stacking interactions. Also, the length ofsubsite 1 is sufficient to accommodate ∼ 10 amino acids. Subsite 1could thus structurally provide the recognition site for the ‘SecBbinding motif ’.

Binding of a substrate peptide to SecB has been shown tochange the proteolysis pattern of SecB by protease K13. The pro-tected site is between L141 (Met 150) and Q142 (Asn 151) in E. coliSecB24. In the crystal structure, the site is shown to be located at aposition where helix α2 starts to extend out of the core moleculeand this is suitably exposed for proteolytic cleavage. The positionis very close to the peptide-binding subsite 2. The presence of abound polypeptide could easily change the accessibility of the site.

The SecA binding siteBesides acting as a molecular chaperone, SecB is also a targetingfactor in protein translocation. It specifically recognizes mem-brane bound SecA. Upon binding to SecA, it transfers the boundpolypeptide to SecA for membrane translocation25. It is knownthat the C-terminal 22 residues of SecA are involved in bindingSecB26,27. This region is rich in Arg and Lys residues and is, there-fore, positively charged. Mutagenesis studies and biochemicalcharacterization has implicated residues Asp 27, Glu 31, Ile 84and Glu 86 of SecB as important for the SecB–SecA interac-tion7,16. These four positions are well conserved throughout theSecB family, with Asp 27 and Glu 31 invariant, and Ile 84 and Glu86 undergoing only the most conservative changes. They are spa-tially clustered on the solvent exposed side of the β-sheet (Fig.

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Fig. 2 The quaternary structure of the SecB molecule. a, Ribbon draw-ings of the SecB dimer. Both drawings follow the same color scheme as inFig. 1b. The drawing on the left is in the same orientation as in Fig. 1b.Residues that have been studied in mutagenesis experiments16,17 arehighlighted and colored according to their effects on SecB’s functions(see text). b, Ribbon drawings of the SecB tetramer in two orthogonalviews. Each subunit in the tetramer is colored with a different color: A,green; B, yellow; C, red; D, blue. The drawing on the left is in the sameorientation as in Fig. 1b. The dimensions of the molecule are indicatedand one of the proposed peptide binding channels iis schematically rep-resented by a hatched rectangle. This figure was produced using pro-gram Molscript34 and POV-ray35.


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2a, green residues), forming a large acidic patch on the surface ofthe molecule (Fig. 4). The charge and position of this regionmakes it a good candidate for the SecA binding site. In addition,deletion of the C-terminal α2 helix causes a modest defect in theSecB–SecA interaction15. Helix α2 is highly solvent exposed andcontains several acidic residues, and could thus constitute part ofthe SecA binding site.

SecA exists as a dimer in solution while SecB is a tetramer, andit has been puzzling how these two molecules might interact.From a symmetry point of view, the fact that SecB is organized asa dimer of dimers provides an easy solution. Each subunit of SecAcould interact with the acidic region formed by one dimer ofSecB. Furthermore, the binding of SecA to the β-sheet and helixα2 could change the conformation of these structural elements.This would then directly affect the peptide binding site, leading tothe release of polypeptide from SecB. The precise details of theinteraction between SecB and SecA can only be answered when astructure of the SecB–SecA complex is available.

In summary, we have shown that the SecB tetramer is orga-nized as a dimer of dimers. The peptide binding surface is locat-ed on the side of the molecule and between two β-sheets. LikeGroEL and DnaK, this peptide binding surface is hydrophobic innature. In fact, GroEL and DnaK could partially replace SecBin vivo by stabilizing translocation competent polypeptides28.They could not, however, stimulate translocation as SecB does.As general molecular chaperones in the cell, the peptide binding

affinities of GroEL and DnaK are modulated by their intrinsicATPase activities. SecB, on the other hand, has evolved to usealmost its entire molecular surface for the two functions it per-forms: binding polypeptide and binding SecA. It does not haveany molecular mass devoted to additional enzymatic activitybecause its peptide binding affinity is modulated by binding toSecA, the downstream target in the translocation pathway. Thus,SecB has been engineered by nature to become a translocationspecific molecular chaperone.

MethodsProtein expression and purification. Full length SecB was clonedfrom the H. influenzae genome using the polymerase chain reaction(PCR). N-terminal His8-tagged protein was expressed in E. coliBL21(DE3) cells. Tagged SecB was purified from sonicates using aNi2+-NTA column (Qiagen). Tobacco etch virus (TEV) protease diges-tion removed the His tag, and the protein was passed over anotherNi2+-NTA column. Ion exchange chromatography on a Source-Q col-umn (Pharmacia) was the final purification step. Selenomethionine(SeMet) SecB was prepared essentially as above, except thatB834(DE3) cells (Novagen) were used for expression, and weregrown in SeMet containing defined media.

Crystallization and data collection. Crystals of H. influenzaeSecB were obtained at room temperature using hanging drop vapordiffusion with 20 mg ml-1 SecB solution in 20 mM Tris-HCl (pH 7.5).The reservoir consisted of 2.0 M (NH4)2SO4, 10% (v/v) ethanol,100 mM PIPES (pH 6.5); drops of 4 µl contained a 1:1 mixture of pro-

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Fig. 3 The proposed peptide binding channel. a, The solvent accessible surface of SecB. On the left, the exposed surface is colored based on theunderlying atoms: all backbone atoms, white; all noncharged polar and charged chain atoms (Asn, Gln, Ser, Thr, Cys, Asp, Glu, Arg, Lys and His), blue;all hydrophobic side chain atoms (Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp and Met), yellow. On the right, the exposed surface encompassing the two pro-posed peptide binding subsites is highlighted. b, Ribbon drawing of the SecB tetramer viewed from the side of the molecule. The orientation is thesame as in (a). The two subsites are shown in two zoom-in views. Residues lining subsite 1 are colored purple and those lining subsite 2 are coloredcyan. For the purpose of clarity, only one subunit was drawn in each of the zoom-in views. The residues lining the two sites are all hydrophobic withthe exception of Thr 53. c, Schematic drawing of a PTB domain and a SecB monomer. The shared structural motif is highlighted in gray. The peptidebinding sites are represented by hatched rectangles. Grasp36 was used to produce (a); Molscript34 and POV-ray35 were used to produce (b).


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tein and reservoir solution. The crystals were cryoprotected in asolution of 2.0 M Li2SO4, 20 mM PIPES (pH 6.5) and 18% (v/v) glyc-erol. Frozen crystals were used for data collection under a nitrogenstream at 110 K. Crystals belonged to the space group P43212 withcell constants a = b = 126.3 Å, c = 148.4 Å (Table 1). The asymmetricunit contained one SecB tetramer molecule with a solvent contentof 65%. All data were collected on BioCARs beamlines 14-BM-C and14-BM-D at the Advanced Photon Source using an ADSC Quantum 4CCD detector (Table 1). Multiwavelength anomalous diffraction(MAD) data were collected at four wavelengths (Table 1). All datawere processed using DENZO and intensities were scaled usingSCALEPAK29 (Table 1).

Structure determination and refinement. Experimental phaseswere obtained using the MAD method30, with SeMet derivatives(Table 1). Eleven of the 16 selenium sites expected for the SecBtetramer molecule were found using the Patterson heavy atomsearch method as implemented in CNS31. All four N-terminal SeMetswere disordered. Subsequently, heavy atom parameters wererefined and MAD phases were calculated in CNS (Table 1). The initial

Fig. 4 The proposed SecA binding site. The orientation is orthogonal tothat in Fig. 3. The drawing on the left is the solvent accessible surface ofthe SecB tetramer. The surface encompassing Asp 27, Glu 31, and Glu 86 iscolored green; the surface encompassing Ile 84 is colored yellow. Thesefour residues have been shown to be important for SecB’s interaction withSecA7,16. The drawing on the right is the same surface except that it is col-ored based on the electrostatic potential of the molecule (ranging from -10 to +10kT). Figure produced using Grasp36.

Table 1 Crystallographic data statistics

Data collection and MAD phasing statisticsCrystal Space group Cell dimensionsNative P43212 a = b = 126.34 Å, c = 148.33 ÅSeMet P43212 a = b = 126.33 Å, c = 148.37 Å

Crystal dmin (Å) No. of measurements No. of unique reflections Completeness (%)1 I / σI1 Rsym (%)1,2

Native 2.5 515,992 42,096 99.7 (99.0) 20.3 (3.5) 4.8 (29.2)SeMet λ1 (1.0031 Å) 2.9 280,177 50,309 98.5 (98.8) 21.7 (4.7) 4.9 (26.7)SeMet λ2 (0.9793 Å) 2.9 277,281 50,198 98.4 (97.5) 18.4 (3.4) 5.6 (30.9)SeMet λ3 (0.9791 Å) 2.9 274,508 50,081 98.2 (96.0) 17.6 (3.0) 5.8 (33.1)SeMet λ4 (0.9574 Å) 2.9 270,029 49,852 98.0 (93.9) 16.9 (2.6) 6.1 (36.2)

Observed diffraction ratios3

λ1 λ2 λ3 λ4

λ1 0.036 0.056 0.060 0.054λ2 0.045 0.046 0.063λ3 0.068 0.064λ4 0.071

MAD phasing power4 and figure of meritλ1 → λ1- λ1 → λ2+ λ1 → λ2- λ1 → λ3+ λ1 → λ3- λ1 → λ4+ λ1 → λ4- FOM5

100–2.9 Å 0.31 1.65 1.80 1.43 1.75 0.42 0.71 0.623.0–2.9 Å 0.12 0.70 0.72 0.45 0.52 0.10 0.23 0.32

Refinement statisticsNumber of reflections (working / test) 36,144 / 4048Number of nonhydrogen atoms 4489Resolution (Å) 45.0–2.5 Rcryst / Rfree (%)6 24.2 / 30.2Bond length deviation (Å) 0.012Bond angle deviation (º) 1.8Average B-factor (Å2) 59.2

1Values in parentheses are for the specified high resolution bin except where indicated.2Rsym = ΣhΣi|Ii(h) - <I(h)>| / Σh<I(h)>, where Ii(h) is the ith mesurement and <I(h)> is the weighted mean of all measurements of I(h).3Values for the observed anomalous diffraction ratios are <∆|F|2>1/2 / <|F|2>1/2, where ∆|F| is the Bijvoet difference at one wavelength (diagonal ele-ments) or the dispersive difference at the two wavelengths (intersecting at an off-diagonal element). Values were computed at 45.0–2.9 Å resolu-tion.4MAD phasing power is defined as [<|Fh1 – Fhi|2> / ∫φ Pλ1→λi(φ)(||Fλ1|eiφ + Fhi - Fh1| - |Fλi|)2dφ]1/2 for individual lack-of-closure expressions between the reflec-tions of the reference wavelength λ1, its Friedel mate (indicated by λ1-) and the other wavelengths (indicated by λ i+ and λ i-). Fhi are the heavy atomstructure factors and Pλ1→λi(φ) is the corresponding phase probability distribution.5Figure of merit.6R = Σ(|Fobs| - K|Fcalc|) / Σ|Fobs|. Rfree is the R-value obtained for a test set of reflections that consisted of a randomly selected 10% subset of the diffrac-tion data used during refinement or σA value calculations.


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model containing residues 15–152 was constructed from electrondensity maps obtained by MAD phasing and subsequent solventflattening and histogram matching along with phase extension to2.5 Å resolution, as implemented in CNS. The high quality of theexperimental map and known selenium sites allowed unambiguoustracing of the protein backbone and most of the side chains usingO32. All refinement procedures were carried out using CNS, withprogress measured using cross-validation with a 10% randomlyselected test set. Initial refinement consisted of several iterations oftorsion angle dynamics simulated annealing using the maximum lik-liehood target function with the experimental phases as a priorphase distribution (MLHL), followed by model rebuilding in O. Laterrefinement consisted of rounds of selecting chemically reasonablewater molecules in phase-combined σA-weighted 2Fo - Fc maps, con-jugate gradient minimization, individual restrained atomic B-factorrefinement, and model rebuilding using experimental, σA-weight-ed, and phase combined σA-weighted 2Fo - Fc maps, utilizing unmod-ified MAD phases. The refinement of the four protomers weretightly restrained by noncrystallographic symmetry except for theloops and helix α2, where structural deviations were apparent inthe electron density map. Data from 20–2.5 Å were used with bulksolvent correction. The final structure includes residues 15–151 of all

four subunits and 63 solvent molecules. The N-terminal 14 residueswere not visible and were assumed to be disordered. Additionalresidues can be visualized at the C-terminus for three subunits,which are stabilized to varying degrees by crystal contacts. Statisticsare shown in Table 1.

Coordinates. Atomic coordinates and structure factors have beendeposited in the Brookhaven Protein Data Bank (accession code1FX3).

AcknowledgmentsWe thank J. Stuckey for maintaining the X-ray facility at the University ofMichigan Medical School and for assistance on synchrotron data collection; M.Marletta and D. Peisach for access to their SGI graphic workstation duringstructure fitting and refinement; K. Brister for access and help at APS BioCARsbeamlines; J. Dixon, M. Marletta, K. Orth and R. Taussig for critically reading themanuscript. This work was supported by an NIH grant to Z.X. and the Universityof Michigan Biological Scholar Program.

Received 9 August, 2000; accepted 20 October, 2000.

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