Refinement of Eco RI Endonuclease CrystalStructure: A Revised Protein Chain Tracing

The original model of Eco RI endonucle-ase (1) proved refractory to crystallographicrefinement, whereby model coordinateswere adjusted to optimize the fit to theobserved diffraction data (the best R factorfor the original model is 0.25). We havenow obtained five additional isomorphousderivatives by cocrystallizing the protein withthe halogenated oligonucleotides: U'CGC-GAATTCGCG, TCGCGAAU'TCGCG, TC-GCGAATU'CGCG, TCGCGAATICGC'GandBU kICGAAUB,TCGCBrG (2) (Eco RIsite underlined). The heavy atoms were lo-cated by difference Patterson and Fouriermethods (with the use of phases from theoriginal platinum derivative); halogen at-oms were found at the locations expectedfrom the earlier structure (a minor exceptionis the first derivative, which suggests thatthe unpaired thymidine may be in the synconformation). The phasing power for theplatinum derivative is 2.09; those for thehalogen derivatives are 1.76, 2.11, 1.75,1.67, and 1.96, respectively. The mean fig-ure of merit is 0.59 for all six derivatives to2.8 A resolution.The new data yielded a multiple isomor-

phous replacement (MIR) electron densitymap that suggests a different connectivitybetween elements of secondary structure inthe protein (3). A model based on thatconnectivity has refined robustly by the X-PLOR and Konnert-Hendrickson methods(4) to an R factor of 0.20 (no orderedsolvent has been included in this model, norhave coordinates for the first 16 amino acidresidues, which appear disordered) (5). Theroot-mean-square deviations in bond dis-tance, angle distance, and 1-4 dihedral dis-tance are 0.016 A, 0.031 A, and 0.031 A,respectively, in the current cycle of Konnert-Hendrickson. The real space R factor (6),and Ramachandran plots are well behaved.The building block decomposition ofUngeret al. is standard (7), an indication that thebackbone geometry is consistent with otherprotein structures that have been well deter-mined to high resolution. Coordinates forCX, atoms ofthe protein and all atoms (otherthan hydrogen) of the DNA have beendeposited with the Brookhaven Data Bank.We also cocrystallized the protein with a

longer oligonucleotide [15 nucleotides (nt)],

with the sequence TCGTGGAATTCCACG(space group R32, a = b = 128.1 A, c =148.1 A). We obtained a K2PtCl4 derivative(phasing power 1.76) and an iodine deriva-tive by means of a synthetic oligonucleotidein which the first C is replaced by C' (phas-ing power 1.55). Anomalous scattering datawere collected for both derivatives; theanomalous phasing powers are 1.63 and1.55, respectively. The average figure ofmerit is 0.62 to 3.5 A resolution. The MIRelectron density map of the 15-nt complexwas analyzed independently from that ofthe13-nt complex described above. A chaintracing was obtained for the protein withinthe 15-nt complex; this tracing is essentiallyidentical to that obtained for the 13-ntcomplex (8). Final analysis of the 15-ntcomplex awaits further refinement.The new chain tracing (Fig. 1) is based on

some new elements of electron density and anew interpretation that alters the assignmentof specific amino acid residues to some ofthe original features. Of particular note are(i) the extended chain motif, (ii) the 13bridge, (iii) the placement of Glu"', and(iv) a 1-loop-a topological motif.The extended chain motif (Met'37 to

Ala142) is a segment ofextended polypeptidechain that runs through the major groove ofthe DNA, roughly parallel to the DNAbackbone (Fig. 2). Although the 4-* tor-sion angles of this segment are in the 1region of the Ramachandran plot, the con-formation is not sufficiently regular to be anisolated strand of 1 sheet. The motif isanchored at one end by the inner recogni-tion helix, while its other end is fixed by theinner arm. There are several contacts be-tween residues in the extended chain andsome of the bases within the Eco RI recog-nition site. These are in addition to contactsfrom the recognition helices (5). The seg-ment of the gene that codes for this motif isa mutational hotspot (9), which is not sur-prising given its structure. Most of thecontacts from the extended chain motif aremade to pyrimidine bases, consistent withthe suggestion of Heitman and Model thatthe protein interacts with pyrimidines (10)as well as with purines. An extended chainmotifhas not been seen in previously report-ed DNA-protein cocrystal structures.

The 13 bridge is part of the loop connect-ing 131 to 12, which includes an a helix (a3)and a pair of antiparallel 13 strands (13' and13") that are not part of the main 13 sheet(11); a3 and especially the segment fromIle73 to Asn85 pack primarily against thetwofold-related subunit rather than theirown subunit. Specifically, the 13 bridge isformed by 1' and 1" because they span thegap between the globular units. 1" is in closeproximity to the DNA backbone (Fig. 3),forming one side of the cleft where theDNA backbone is bound.The current model places Glu"' near the

scissile bond of the DNA. Mutations at thislocus have been identified by Modrich andco-workers that reduce the cleavage activityof the enzyme but do not alter (or evenenhance) DNA binding (12). It is interest-ing that Glu"'l is part of 133, specifically thecarboxyl-terminal portion, which runs paral-lel to the DNA backbone in a mannersimilar to the 13 bridge.The 1-loop-a motif can be seen in the

topological similarity of the loops that con-nect 13 to a4 and 134 to a5 (Fig. 1). Theseloops form the arms, which are segments ofstructure that extend from the central do-mains and nearly surround the DNA (Fig.2). The inner arm is the loop connecting 13to the extended chain motif; it contacts theDNA backbone. It is buttressed by the outerarm, which is the loop connecting 14 to a5,as in the previous model. This topologicalsymmetry, which is at the heart of thenucleotide binding fold, suggests that 13-loop-a may be a general topological motiffor nucleic acid binding.Some of our previous discussion (1) was

based on direct observation of the electrondensity map, particularly the spatial relationsof the major structural elements (doublehelix, a helices, and 13 sheets). These conclu-sions have not changed because they areindependent of any interpretation that re-sulted in the assignment of amino acid resi-dues to specific features of electron density.The central kink, which unwinds the DNAby approximately 25 degrees, is a prominentfeature of all the electron density maps. Thehelical recognition motif, which consists ofaparallel bundle offour a helices penetratingthe major groove ofDNA, is similarly unaf-fected. This motif is different from those ofhelix-turn-helix, zinc finger, and leucine zip-per.The current model retains the topology in

which a five-stranded, largely parallel 13sheet is surrounded by a helices (Fig. 1).The order and direction of the strands with-in the 13 sheet remain unchanged. The anti-parallel motif (131-133) is still the foundationfor the strand scission site, and the parallelmotif (133-135) is likewise the foundation for

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New model

A Outer arm

Fig. 1. (A) The topological structure of the new chaintracing for Eco RI endonuclease. Strands of ,B sheet arerepresented by arrows and a helices by cylinders. Thestrands of the major 1 sheet are shown with Arabicnumerals sequentially from the amino terminus; strandsin loop regions are indicated with sequential Romannumerals. Similarly, major a helices are shown withsequential Arabic numerals, while two short helices areindicated with Roman numerals. The amino acid residuenumbers at the beginning and end of each element ofsecondary structure are indicated. (B) The old chaintracing is shown with similar labeling. (C) The three-dimensional flow ofthe polypeptide chain ofone subunitof the enzyme is shown in stereo figures generated byPriestle's program (13). The second subunit, which isidentical, is not shown for clarity. a helices are represent-ed by coiled ribbons, strands of ,B sheets by arrows, andloops by narrow ribbons. The arm is toward the lowerright portion of the figure, approximately in the plane ofthe figure. The 1 bridge (see text) and a3 projects out ofplane ofthe figure toward the viewer and is located to thecenter left. (D) A similar view including a rough approxi-mation to the location ofthe DNA backbones, which arerepresented by ,B arrows.

the direct protein-DNA contacts. Helices a4and a5 play a central role in the sequence

specific interaction and are also referred toas the inner and outer recognition helices,respectively. The similarity between the par-

allel motif and nucleotide binding fold isthus enhanced.

In summary, new isomorphous derivativedata suggest a revised chain tracing for thecrystal structure Eco RI endonuclease thathas refined satisfactorily. The central kink,which unwinds the DNA, is unaffected bythe new data; likewise the major groove ispenetrated by a parallel bundle of four a

helices. Principal changes in the protein

include altered connectivity through loopsand helices at the surface ofthe complex thatnonetheless retains the original largely paral-lel five-stranded a-1 topology. The new datareveal a bridge that interacts with theDNA backbone, a repeated P-loop-a topo-logical motif for nucleic acid recognition,and an extended chain motif that runs

through the major groove and contacts

some of the bases in the Eco RI recognitionsite.

*Present address: Agouron Pharmaceuticals, Inc., 11025North Torrey Pines Road, La Jolla, CA 92037.tTo whom correspondence should be addressed.

YOUNGCHANG KiMJOHN C. GRABLEROBERT LOVE*

Departments of Biological Sciences andCrystallography,

University ofPittsburgh,Pittsburgh, PA 15260PATRICIA J. GREENE

Department ofBiochemistry and Biophysics,University of California,

San Francisco, CA 94143JOHN M. ROSENBERGt

Departments of Biological Sciences andCrystallography,

University ofPittsburgh

SCIENCE, VOL. 249

B Outer arm

C

D

Old model

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Fig. 2. (A) The Cat atoms from the segments ofpolypeptide chain that form the principle recogni-tion elements are shown against the DNA. Thefour recognition helices, two from each subunit,are yellow, and the extended chain motifs fromeach subunit are pink. The molecular axis oftwofold symmetry is horizontal, in the plane ofthe figure. (B) CG, atoms for one entire subunit ofEco RI endonuclease are shown against the DNA.The recognition helices and extended chain motifare yellow and pink, respectively, as in the previ-ous panel. The arm is orange, and the rest of thepolypeptide chain is green. (C) Both subunits ofthe protein dimer are shown with the DNA,colored as in the previous panel.

REFERENCES AND NOTES

1. J. A. McClarin, C. A. Frederick, B.-C. Wang, P.Greene, H. W. Boyer, J. Grable, J. M. Rosenberg,Science 234, 1526 (1986).

2. U' is 5-iododeoxyuracil, C' is 5-iodocytosine, andUBr and CBF are the corresponding 5-bromopyrimi-dines. Some of the halogenated oligonucleotidescontain base analogs in the recognition site, andfurther analyses ofthe structures of these derivativesare required to detect subtle changes that couldaffect DNA recognition.

3. Some but not all of the connectivity changes sug-gested by the new diffraction data were anticipatedby electron density maps obtained with phases calcu-lated from fragments of the original structure. Un-fortunately, those maps were ambiguous and analy-sis of them proved incondusive. Complete nativedata were re-collected to 2.7 A resolution on aNicolet arc detector as were data for the platinum

derivative (1), but the resultant maps were equallyincondusive; hence the need to obtain additionalisomorphous derivatives.

4. A. Brunger, J. Kuriyan, M. Karplus, Sience 235,458 (1987); W. Hendrickson, Methods Enzymol.115, 252 (1985).

5. Y. Kim, P. J. Greene, J. M. Rosenberg, in prepara-tion.

6. C-I. Branden and T. A. Jones, Nature 343, 687(1990); Y. Kim, unpublished program.

7. R. Unger, D. Harel, S. Wherland, J. L. Sussman,Proteins 5, 355 (1989).

8. J. Grable, Y. Kim, P. J. Green, J. M. Rosenberg, inpreparation.

9. S. D. Yanofsky, R. Love, J. A. McClarin, J. M.Rosenberg, H. B. Boyer, P. J. Greene, Proteins 2,273 (1987).

10. J Heitman and P. Model, ibid. 7, 185 (1990).11. We use the term loop in the sense of Richardson [J.

S. Richardson, Adv. Protein Chem. 34, 167 (1981)],

Fig. 3. A cross section of the complex showingthe ,B bridge. The view is down the average DNAhelix axis; only those atoms at the approximatedepth of the 13 bridge are shown. The DNA isblue, the 1 bridge is red, the visible portions ofthe recognition helices are yellow, and the rest ofthe protein is green. ," is the red strand that isclose to the DNA backbone. 13 can also be seen;it is approximately vertical and follows the DNAbackbone in the lower right portion ofthe figure.

which is the stretch of polypeptide chain that inter-connects the strands of the principal ,B sheet orcrossover helices, the latter being those a helices thatpack against the 13 sheet and are also necessary forthe 1 strands to connect in a parallel rather thanantiparallel direction. By this definition loops oftencontain a helices or 13 hairpins (or both) that do notcontribute to forming the core of the a-1 domain.

12. K. King, D. Wright, P. Modrich, Fed. Proc. 45,1913 (1986); K. King, S. J. Benkovic, P. Modrich,J. Biol. Chem. 264, 11807 (1989).

13. J. P. Priestle, J. Appl. Crystallogr. 21, 572 (1988).14. We thank R. Unger and J. Sussman for calculating

the subunit decomposition of our structure. Sup-ported by NIH grant GM25671 and grantDMB890026P for computer time at the PittsburghSupercomputing Center.

3 July 1990; accepted 16 August 1990

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Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracingYC Kim, JC Grable, R Love, PJ Greene and JM Rosenberg

DOI: 10.1126/science.2399465 (4974), 1307-1309.249Science 

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