Proc. Natl. Acad. Sci. USAVol. 91, pp. 2071-2075, March 1994Biophysics

Structure of the gene V protein of bacteriophage fl determined bymultiwavelength x-ray diffraction on the selenomethionyl protein

(protein structure/x-ray crystallography/syncrotron radiation/DNA binding protein/protein-DNA complex)

MATTHEW M. SKINNER*, HONG ZHANGt, DALE H. LESCHNITZER*, YUE GUANt, HENRY BELLAMYt,ROBERT M. SWEET§, CARLA W. GRAY$, RUUD N. H. KONINGSII, ANDREW H.-J. WANGt**,AND THOMAS C. TERWILLIGER*"***Life Sciences Division, MS M880, Los Alamos National Laboratory, Los Alamos, NM 87545; tBiophysics Division and Department of Cell and StructuralBiology, University of Illinois, Urbana, IL 61801; tStanford Synchrotron Radiation Laboratory, Stanford University, Stanford, CA 94305; §BiologyDepartment, Brookhaven National Laboratory, Upton, NY 11973; Program in Molecular and Cell Biology, University of Texas at Dallas,Richardson, TX 75083; and 1Departments of Biophysical Chemistry and Molecular Biology, University of Nijmegen, The Netherlands

Communicated by Jiri Jonas, October 18, 1993 (received for review April 10, 1993)

ABSTRACT The crystal structure of the dimeric gene Vprotein of bacteriophage fl was determined using multiwave-length anomalous diffraction on the selenomethionine-containing wild-type and isoleucine-47 methionine mutantproteins with x-ray diffraction data phased to 2.5 A resolution.The structure of the wild-type protein has been refined to an Rfactor of 19.2% using native data to 1.8 A resolution. Thestructure of the gene V protein was used to obtain a model forthe protein portion of the gene V protein-single-stranded DNAcomplex.

Gene V protein of bacteriophage fltt is a member of a classof proteins involved in DNA replication that bind to single-stranded nucleic acids with high affinity and cooperativity butlittle sequence specificity (1-4). Gene V protein coats thesingle-stranded DNA (ssDNA) intermediate in bacteriophagefl DNA replication, forming an ordered superhelical protein-DNA complex (1, 5-8). This protein-DNA complex facili-tates packaging ofthe ssDNA into new phage particles. GeneV protein also binds with some specificity to a translationaloperator sequence on phage fl gene II mRNA (9-12).The gene V protein provides a general model for protein-

ssDNA interactions that are strong, yet not sequence-specific. Gene V protein binding to single-stranded nucleicacids and to oligonucleotides has been studied using chemicalmodification, spectroscopic techniques, and mutagenesis(13-24). The structure of the protein-ssDNA complex hasbeen studied using electron microscopy and solution scatter-ing methods and is found to consist of a regular left-handedsuperhelix in which the gene V protein dimers are arrayed onthe outside of the superhelix and the ssDNA strands areinside (8, 25). The structure of the gene V protein is also ofinterest because its small size and the large number ofmutants available have made it a useful model for determin-ing effects ofamino acid substitutions on protein stability andfunction (23, 26, 27).A model for the crystal structure of the wild-type (WT)

gene V protein has been reported (28), but recent NMRstudies have demonstrated that the positions of amino acidsinvolved in the segments of antiparallel (-structure are notcompatible with those in the model (21), and a new determi-nation of the structure was necessary. The multiwavelengthanomalous diffraction (MAD) technique (29) was ideallysuited for this purpose, as the WT gene V protein containstwo methionine residues (Met-1 and Met-77) that might besubstituted in vivo in Escherichia coli by selenomethionine.Here we report the determination of the gene V protein

structure using the MAD technique, the refinement of thestructure using x-ray diffraction data on the WT gene VproteinAt and a model for protein-protein contacts in thegene V protein-ssDNA superhelical complex.

MATERIALS AND METHODSModeling of the Protein Portion of Gene V Protein-ssDNA

Complex. The gene V protein dimer was placed with itsinternal two-fold axis ofsymmetry perpendicular to the axis ofthe superhelix to be generated and Phe-73 pointing eithertoward or away from the helix axis. The outer surface of thedimer (not including the DNA-binding wing of the protein, seeFig. 2A) was adjusted to be 40 A from the helix axis. Adjacentdimers were generated by rotation about the superhelix axis by450 and translation along this axis by 10 A so as to make aleft-handed superhelical array. The number of contacts be-tween atoms in two adjacent dimers violating van der Waalscontacts by more than 1 A were counted for values of rotation(6) about the dimer axis from 00 to 1800 in increments of 10.

RESULTSStructure Determination and Refinement. Multiwavelength

x-ray diffraction data from two selenomethionine "deriva-tives" ofgene V protein provided phasing information for theWT structure (Table 1). Patterson functions for the seleno-methionine-containing wild-type gene V protein (Se-WT)data using anomalous or dispersive differences identified asingle site (Met-1 was found to be disordered; see below).Patterson functions for the selenomethionine-containinggene V protein with Ile-47 replaced with Met (Se-I47M) datausing anomalous differences yielded a two-site solution, oneof which corresponds to the site found in the Se-WT struc-ture. The electron density map based on MAD phases andWT structure factor amplitudes had an overall figure of meritof 0.63 to 2.5 A (Table 2 and Fig. 1). This map was readilyinterpretable and had clear density for backbone atoms of allof the 87 residues except for residue 1, the loop at positions21-24, and residues 85-87 and had clear side chain density

Abbreviations: Se-147M, selenomethionine-containing gene V pro-tein with isoleucine-47 replaced with methionine; MAD, multiwave-length anomalous diffraction; Se-WT, selenomethionine-containingwild-type gene V protein; ssDNA, single-stranded DNA; WT, wildtype.**To whom reprint requests should be addressed.ttThe Ffphages fl, fd, and M13 are very closely related, and the geneV proteins of all these phages are identical.

#4The coordinates of the refined gene V protein model (1BGH) havebeen deposited in the Protein Data Bank, Chemistry Department,Brookhaven National Laboratory, Upton, NY 11973.

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Table 1. Data collection statisticsProperty Se-WT Se-147M

Resolution, A 2.6 2.5Number of observations (at A.) 17,878 3099Unique reflections 2617 2018Completeness, % 94 65Rmerge, % 3.0 9.2R,-e (F at A. to native), % 10.5 22.7Mean AaIIo,Ab, % of mean F at Aa 5.0 7.0Mean Adisp, % of mean F at Aa 3.7 6.6A., A 0.9000 0.9000Ab, A 0.9794 0.9792AC, A 0.9797 0.9794A41 Aa -1.6 -1.6Ab -7.7 -8.6AC -9.6 -9.7Af" Aa 3.4 3.3Ab 5.8 4.9AC 2.2 2.9Optimum data collection energies were found by taking x-ray

fluorescence spectra of the crystals (30). Wavelengths differ slightlybetween the two experiments because absolute wavelengths werenot precisely determined. Unique reflections refer to the number ofreflections measured that are not symmetry-related. R factors usedhere are given by Rmerge = SIFobs - FavgI/Y Favg and Rsce = E: Fobs- Fal/l ' IFobs + Fnats, where nat, obs, and avg refer to native,observed, and average, respectively. Selenomethionine-containingSe-WT and Se-I47M were obtained by growing E. coli DL41 withplasmid pTT18 in the presence of selenomethionine (24, 31). Gene Vproteins were purified to homogeneity and space group C2 crystalswere grown as described (24, 32). Cell dimensions of the native geneV protein crystals were a = 76.08, b = 27.97, c = 42.36 A, and /3 =103.170, with one monomer in the asymmetric unit. Both the Se-WTand Se-147M crystals had unit cell dimensions that were isomorphicto the native crystals. X-ray diffraction intensities for four Se-WTcrystals were collected at 277 K at Stanford Synchrotron RadiationLaboratory beam line 1-5AD (33), and intensities for six Se-147Mcrystals were collected at 298 K at beam line X-12C at the NationalSynchrotron Light Source (30). Both facilities employed a Si (111)monochromator. The monoclinic crystals were mounted with theirb* axis parallel to the capillary, allowing simultaneous collection ofBbvoet pairs. The data collection procedure at beam line 1-SADallowed the collection of diffraction data in rotation increments of0.10 at each wavelength before proceeding to the next rotation, anoptimal procedure for measuring dispersive differences (those be-tween AC and A). Intensities for twoWT crystals were collected usingmonochromated Cu Ka radiation on a Rigaku diffractometer, withdata from X to 2.25 A for crystal 1 and data from oo 4.2 A and from2.26 A to 1.8 A for crystal 2. Data from the two crystals were initiallyscaled based on 583 reflections at low resolution. The scaling R (onF) was 10.8%. After initial refinement using data to a resolution of1.8 A, the data sets were rescaled using an overall thermal factor of-2.65 A2 based on a comparison of Fc calculated from the model tothe two sets of data. The native data set was 98% complete to 1.8 A,with F > 0 for 89%6 of the reflections and F > 2 a for 75%. A--, Aband Asp are defined in the legend to Table 2. 4f ' and 4f"' are thedispersion and absorption components, respectively, of the anoma-lous scattering.

beyond C'O for 50 side chains. The position of residue 47 waspositively identified using the location of the selenium site Bfrom the Se-I47M crystals. The chain was traced in bothdirections from position 47, usingNMR data on the alignmentof (-strands (21). Selenium site A was found to correspond toMet-77.The starting model was refined by simulated annealing

using X-PLOR (39). Simulated annealing-omit maps (38) werecalculated at the start of refinement and after refinement at1.8 A to allow rebuilding with minimal bias toward the model.The R factor at the conclusion of this procedure was 23.1%with all backbone atoms except residues 1 and 87 and allcorresponding side chains except residues 20-22, where the

thermal factors for the protein backbone averaged 63 A2. Thisloop was the most difficult region of the molecule to fit. Atotal of 34 ordered water molecules were identified usingdifference Fourier maps. The finalR factor for the model was19.2% for data with F > 2 or from 8 A to 1.8 A. The rmsdeviations in bond lengths and angles are 0.014 A and 2.90,respectively. The average temperature factor for main chainatoms is 20 A2, and for all protein atoms it is 21 A2. To obtainan unbiased assessment of the improvement of the model inthe early stages of refinement where data from 1.8 A to 2.2A were not used, a free R value (40) was calculated for 2018reflections with F > 2 o from 1.8 A to 2.2 A. This high-resolution free R value decreased from 46.3% at the start ofrefinement to 31.9%o after refinement to 2.2 A, indicating thatthe model was substantially improved by the refinementprocedure.Gene V Protein Structure. The fold of the gene V protein is

shown in Fig. 2 Upper. The two monomers are related by acrystallographic twofold axis of symmetry. The monomer,which contains 87 residues, is largely composed of /-struc-ture, with 63 of the 83 4, q, angles in the /-sheet region of aRamachandran plot (42). The bulk of the protein, 58 of the 87residues, is arranged as a five-stranded antiparallel /-sheet(,/8, /35, /31, /33, /34) and two antiparallel /ladder loops (/32and 33 and also (6 and (37) that protrude out from this sheet(43). The right-handed twist of the antiparallel /3-structuresresults in a distorted (3-barrel. The remainder ofthe moleculeis composed of 31o helical regions (residues 7-11 and 65-67),/3-bends (residues 21-24, 50-53, and 71-74), and one five-residue loop (residues 38-42; see Fig. 2 Lower).The antiparallel /-ladder composed of strands /32 and /3

has been referred to as the "DNA binding wing" (28, 44)because it contains residues thought to be near bound nucleicacids. This loop includes residues 15-30 (Fig. 2 Upper). Thesecond antiparallel /-ladder, composed of strands /36 and ,/7,forms much of the contact surface between the two mono-mers in the dimer. Strands /36 and ,/7 from the two monomersare related by the crystallographic twofold axis, and each hasbeen referred to as a "dyad loop" (ref. 28; Fig. 2 Upper). A,/3bulge at residue 79 positions the C terminus near the Nterminus at the back of the molecule.

DISCUSSIONGene V Protein Structure Determined Using MAD Tech-

niques. Met-77 in the Se-WT protein as well as Met47 andMet-77 in the Se-147M mutant protein were well ordered andprovided MAD phasing information and identified the loca-tions of these two side chains in the structure. The structureobtained here is similar to the previously published structureof the gene V protein (28), except that residues 1-40 and72-79 differ in position from the previous model by 1-3 aaresidues, resulting in an rms difference in backbone atomicpositions between the models of 5 A. Aside from this differ-ence in sequence register, the principal differences in back-bone positions between the models occur at the two 31ohelical regions, at the five-residue loop at residues 38-42, andat the loop between strands /36 and ,/7. The structure reportedhere is in full agreement with the solution structure of geneV protein recently determined by three-dimensional NMRtechniques (P. J. M. Folkers, R.N.H.K., and C. W. Hilbers,unpublished results).Modeling the Gene V Protein in the Gene V Protein-ssDNA

Superhelical Complex. Gene V protein forms an orderedsuperhelical complex when it binds to ssDNA (8). Thestructure ofthis complex is ofinterest because it may providea model for protein-ssDNA contacts, and it could provide abasis for understanding the cooperativity of binding of geneV protein to ssDNA. Cooperative contacts between adjacentgene V protein dimers in the gene V protein-ssDNA complex

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Table 2. Refined parameters for selenium atoms in gene V protein crystalsFractional coordinates FH/E Harker peak

Crystal Site x y z Occupancy B, A2 (acentric) height/a,Se-WT A 0.485 0.500 0.093 0.72 49.0 1.4 11.1Se-147M B 0.138 0.338 0.222 0.65 15.3 0.8 3.6Harker peak height is the ratio of the value of the Patterson function at the lowest expected self-vector to the rms of the

origin-removed map. FH/E is the average ratio of the rms calculated heavy atom structure factor amplitudes to the rmslack-of-closure error (34). Anomalous differences at Ab (A-o,Ad and dispersive differences between AX and A. (Adisp) wereused to construct a pseudo-multiple isomorphous replacement set of data (35). This pseudo-multiple isomorphousreplacement data set consisted of FPH and A.. for the Se-WT and Se-147M data sets, where FPH = Fp + a AdiSp and Aano= (3 A--, Ab. The parameter a is equal to the ratio of the real part of the scattering factor for selenium at Aa to the differencebetween the scattering factors at Ac and Aa, (3 is the ratio of the imaginary part of the scattering factor for selenium at Aato that at Ab, and Fp is a native structure factor amplitude, taken from the WT data. Heavy atom parameters were refinedwith HEAVY (34), using origin-removed Patterson refinement except that relative values of y for sites 1 and 2 were refinedusing phase refinement. The refined occupancies of both sites A and B are less than unity. The reason for this is probablythat values ofAf ' and Af"' were determined at the precise wavelengths where they were at their extrema, while during actualdata collection, the x-ray wavelength varied by as much as one-third the difference between Ab and Ac. In an effort tomaintain independence of the two derivatives, only site A was used in analysis of the Se-WT data, and only site B was usedfor the Se-I47M data. In a test, including site A and B yielded very little difference in the final phase angles and the qualityof the electron density map. B, selenium site at position 47 in the Se-I47M data set.

are thought to include at least Tyr-41 and possibly also Arg-16and Glu-40 (refs. 19 and 45; T.C.T., unpublished observa-tions). Side chains that are involved in ssDNA bindinginclude Tyr-26, Leu-28, and Phe-73 (13-24, 26, 45, 46). Thecomplex between gene V protein and ssDNA under moderatesalt conditions is a rod-like left-handed superhelical structurewith a variable pitch of 6-12 nm, averaging about 8 nm, andan outer diameter of about 8 nm, with about eight gene Vprotein dimers per turn of the superhelix and a ratio of aboutfour nucleotides per gene V protein monomer (refs. 8, 16-18,25, 47; C.W.G., unpublished observations). It is presumedthat a cross section of the rod-like structure would intersecttwo strands ofssDNA running in opposite directions (1). Twomolecular models of the complex have been proposed (48,49), but both disagree with experimental information on thestructures, most notably with the left-handed nature of thesuperhelical complexes.The crystal structure of the gene V protein and the helical

parameters described above were used to model the proteinpart of the protein-ssDNA complex. This modeling involvedtwo parameters, the orientation of the gene V protein dimerabout its twofold axis and the identification of which of twopossible surfaces of the gene V protein faces toward the helix

axis (see Materials and Methods). The number of pairs ofatoms in adjacent dimers in the model that violated van derWaals contact distances by more than 1 A was used toidentify plausible values of these parameters. When thesurface of the gene V protein containing Tyr-26, Leu-28, andPhe-73 was placed facing away from the helix axis (so thatbound ssDNA would be wrapped around the outside of theprotein in the superhelix), no values of the rotation anglewere found that yielded fewer than 95 such contacts. On theother hand, when this surface ofthe protein was placed facinginward, one value of the rotation angle yielded just 7 nonal-lowed contacts (Fig. 3 Left). As the structure of the proteinin the complex is not expected to be identical to that in thecrystal, this small number of disallowed contacts seems quitereasonable. This model suggests a physical basis for obser-vations on the ssDNA-binding properties of the gene Vprotein. The side chains of residues Tyr-26 and Leu-28 fromone dimer are, in this model, within about 6 A,of the sidechain of residue Phe-73 of an adjacent dimer, forming anintermolecular binding site (Fig. 3 Right). This arrangementis consistent with the observation that all three side chains areclose to bound oligonucleotides. The side chains of residuesArg-16, Glu-40, and Tyr-41 are all within 6 A of an adjacent

FIG. 1. (Left) MAD-phased electron density map with refined gene V protein model displayed using TOM-FRODO (36, 37). Theregion fromresidues 53 to 58, including Tyr-56, is shown. Contour is at lr. (Right) Simulated annealing-omit map (38) of the region shown in Left carriedout at 1.8 A. Contour is at 3a.

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DYAD LOOP

FIG. 2. (Upper) Ribbon diagram (41) of gene V protein dimer structure. The strands of the /-sheet for one monomer are labeled. (Lower)Topology diagram for one monomer ofgene V protein. Dotted lines indicate a single hydrogen bond, and heavy solid lines indicate two hydrogenbonds between the indicated residues.

dimer, consistent with effects of mutations at these sites onthe cooperativity of gene V protein binding to ssDNA (23,45). As the helical parameters used here are not preciselyknown, the contacts between dimers in the model are notprecisely defined. For example, the contacts would beslightly different ifthe helical pitch were 6 nm instead of8 nm.Additionally, the loop consisting of residues 15-30 would beexpected to be at least somewhat flexible in solution. Moreextensive modeling will be required to obtain a plausible setof interactions between bound ssDNA and the gene Vprotein, but the current model suggests that a strand ofssDNA would run approximately from the location of one

group ofthe three residues Tyr-26/Leu-28/Phe-73 to the nextin the superhelix. The distance from one Tyr-26 to the nextis about 15 A in this model, a distance readily spanned by fournucleotides in a partially extended conformation. The ssDNAwould then be near a number of other side chains that couldpotentially interact with it, including residues Arg-16, Arg-21,Gln-22, Lys-24, Glu-30, Lys-46, Glu-51, Gln-72, and Arg-80(43).

We thank Yi-gui Gao for crystallization of the WT gene V protein,Yi-gui Gao and Sriram Mahalinga for assistance in data collection,Paul Phizackerley for useful advice, and Elizabeth Manning and

tI/y.i.

FIG. 3. (Left ) Model of gene V protein portion of protein-sst)NA superhelix. Two anda half turns of the superhelix containing 20 gene V protein dimers are shown. Alternatedimers are colored green and magenta. (Right) Selected contacts between two adjacentgene V protein dimers in the superhelix. Residues E40' and Y41' are part of the secondmonomer in the dimer containing residues Y26 and L28. and residue F73 is on the adjacentdimer. The dimers are related by a twofold axis of symmetry For each side chain shownthere are three symmetry-related side chains that are not illustrated. It is envisioned thatthe path of the two strands of ssDNA that are part of this complex would run from lowerleft to upper right. one passing near thc residues L28. Y26 'F7" shown and one throughthe symmetry-related group of residues (not shown) at the lower part of the figure. In thismodel, each dimer has internal twofold svmmetrv. and each pair of adjacent dimers iSrelated by twfofold symmetry.

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David Cheng for technical assistance. We gratefully acknowledgesupport from the National Institutes of Health (to T.C.T., A.H.-J.W., and C.W.G.) and from the Laboratory Directed Research andDevelopment program of Los Alamos National Laboratory (toT.C.T.). Some of the work for this project was done at StanfordSynchrotron Radiation Laboratory, which is operated by the De-partment of Energy, Office of Basic Energy Sciences, Division ofChemical Sciences. The Stanford Synchrotron Radiation LaboratoryBiotechnology Program is supported by the National Institutes ofHealth, Biomedical Resource Technology Program, Division ofResearch Resources. Further support is provided by the DepartmentofEnergy, Office ofHealth and Environmental Research. Diffractiondata for this study collected at Brookhaven National Laboratorywere obtained at the Biology Department single-crystal diffractionfacility at beamline X12-C in the National Synchrotron Light Source.This facility is supported by the U.S. Department of Energy, Officeof Health and Environmental Research, and by an equipment grantfrom the National Science Foundation.

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