Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10148-10152, November 1991 Biochemistry The nucleosomal core histone octamer at 3.1 A resolution: A tripartite protein assembly and a left-handed superhelix (nucleosome/chromatin/handshake motif/histone fold) GINA ARENTS*, RUFUS W. BURLINGAME*t, BI-CHENG WANGt, WARNER E. LOVE§, AND EVANGELOS N. MOUDRIANAKIS*¶l *Department of Biology and §Thomas C. Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, MD 21218; $Departments of Crystallography and Biological Sciences, The University of Pittsburgh, Pittsburgh, PA 15260; and IDepartment of Biology, University of Athens, Athens, Greece Communicated by Christian B. Anfinsen, August 30, 1991 ABSTRACT The structure of the octameric histone core of differed drastically from those derived from the crystallo- the nucleosome has been determined by x-ray crystaflography to graphic studies of Finch et al. (8) with the nucleosome core a resolution of3.1 A. The histone octamer is a tripartite assembly particle, and from those of Klug et al. (13) on the histone core in which a centrally located (H3-H4)2 tetramer is flanked by two of the nucleosome (hereafter called the MRC model). In this H2A-H2B dimers. It has a complex outer surface; depending on report, we present the results of our crystallographic rede- the perspective, the structure appears as a wedge or as a flat disk. termination of the structure of the histone octamer. It will The disk represents the planar projection of a left-handed pro- become apparent that the "shape and size" issue is now teinaceous superhelix with -28 A pitch. The diameter of the resolved; the outer dimensions of the histone octamer are particle is 65 A and the length is 60 A at its maximum and -10 consistent with the MRC model (length, 55 A; diameter, 70 A) A at its Ium extension; these dimensions are in agreement (13) and not with those reported earlier by us (length, 110 A; with those reported earlier by Klug et al. [Klug, A., Rhodes, D., diameter, 70 A) (11). The shape of the octamer is complex Smith, J., Finch, J. T. & Thomas, J. 0. (1980) Natwre (London) and, depending on the perspective, it can be described as a 287, 509-516]. The folded histone chains are elongated rather wedge or a flat disk (13) as well as a "tripartite assembly with than globular and are assembled in a characteristic "handshake" a central V-shaped (H3-H4)2 tetramer, flanked by two flat- motif. The individual polypeptides share a common central tened balls, the H2A-H2B dimers" (11). The increased length structural element of the helix-oop-helix type, which we name we reported earlier was the result of an apparent rotation of the histone fold. the electron density in the Fourier map about a "special position." This rotation, at the end, resulted in a much In all eukaryotic cells, the nuclear DNA is highly compacted "expanded" tetramer and distally displaced dimers, thus through its association with special proteins, the histones, generating an octamer with the erroneous length of 110 rather which both neutralize its electrostatic character and provide than 60 A. An extended and detailed account of the crystal- a scaffold for its path. This path must be compatible with the lographic analyses germane to this transformation will be several states oforganization the double helix experiences as published elsewhere (B.-C.W., J. Rose, G.A., and E.N.M., it progresses from interphase chromatin to the metaphase unpublished data). The results of our redetermination of the chromosome and back. At the first level of compaction, the structure of the histone octamer to a resolution of 3.1 A and histones and DNA are organized in repeating units (1) called an R value of 25.5% are presented here at two levels.** First, nucleosomes (2). Within each core nucleosome are two we present solid renderings of the shapes of the histone copies of each of the core histones H2A, H2B, H3, and H4 octamer to facilitate direct comparison with the earlier lower- in the form ofan octameric complex (3). The histone octamer resolution studies (8, 11, 13). Second, we outline salient has been shown by a variety of solution physical chemical recently found features ofthe folding ofthe four polypeptides experiments to be internally organized as a tripartite protein as well as their organization within the H2A-H2B dimer and assembly (4, 5). (H3-H4)2 tetramer subunits of the histone octamer. A de- Over the past decade, the structure ofboth the nucleosome tailed analysis will be presented in a forthcoming article. and the histone octamer has been the subject of intensive investigations. They have included small-angle neutron and MATERIALS AND METHODS small-angle x-ray diffraction (6, 7) of solutions of these assemblies, as well as x-ray crystallography of their ordered Structure Determination. The chicken erythrocyte histone states crystallized with the aid of either organic solvents (8, octamer forms large crystals in space group P3221 that 9) or high salts (10, 11). Our earlier crystallographic analysis diffract to better than 3.0-A resolution (10). Only one-half of of the chicken erythrocyte histone octamer described the the octamer-i.e., H2A-H2B-H3-H4-is present in the asym- octamer as having a "tripartite organization, that is, a central metric unit. This requires the molecular and crystallographic (H3-H4)2 tetramer flanked by two H2A-H2B dimers." Its twofold axes to coincide. A heavy atom derivative of the shape was described as an ellipsoid 110 A long and 65-70 A histone octamer crystals with tetrakis-(acetoxymercu- in diameter (11). At that level of analysis, the internal ri)methane was found that gave only one peak in the Patter- organization of the particle was in agreement with our earlier son map (11) at x = 0.322, y = 0.329, z = 0.992 (equivalent biochemical work that demonstrated the tripartite nature of the histone octamer in solution (4) and could accommodate Abbreviation: ISIR, iterated single isomorphous replacement. the histone-DNA contacts suggested by the studies of Mirz- tPresent address: Scripps Clinic and Research Foundation, La Jolla, abekov et al. (12). However, the shape and size of our model CA 92037. "To whom reprint requests should be addressed at: Department of Biology, The Johns Hopkins University, Baltimore, MD 21218. The publication costs of this article were defrayed in part by page charge **The a-carbon coordinates have been deposited in the Protein Data payment. This article must therefore be hereby marked "advertisement" Bank, Chemistry Department, Brookhaven National Laboratory, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Upton, NY 11973 (reference 1HIO). 10148 Downloaded by guest on September 30, 2021 Biochemistry: Arents et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10149 to z = -0.008), and the occupancy of this site was <50o Finally, the asymmetric unit of the new map contains no since the two -SH groups of the octamer are very close to redundant electron density-i.e., there is no partial repeat of each other on either side of the dyad. Thus, only one metal the dimer density near the tetramer. atom can be bonded to one but not to both of the cysteines. Initial Model. The interpretation ofthe map was facilitated Phases calculated by the iterated single isomorphous replace- by the fact that the asymmetric unit contains half of the ment (ISIR) procedure of Wang (15) resulted in the electron (H3-H4)2 tetramer and one H2A-H2B dimer and a single density map shown earlier (11). cysteine as residue 110 ofH3. A simple inspection ofthe map For reasons to be detailed elsewhere (B.-C.W., J. Rose, revealed one contiguous protein domain containing the mer- G.A., and E.N.M., unpublished data), the data were reex- cury of tetrakis-(acetoxymercuri)methane (11) and this was amined and the heavy atom parameters were reevaluated. assigned to the H3-H4 half-tetramer. The other separate but This process resulted in a slight displacement of the heavy closely apposed domain was assigned to the H2A-H2B dimer. metal position to a new site with x = 0.342, y = 0.344, z = It is interesting to note that the density of the dimer domain 0.007, and an improvement of the R(culliS) value for this site in the new map is nearly identical to the density for the from 51.3% to 48.0%o. Further examination of our earlier analogous dimer domain in the previous map. The overall results revealed that the minimum in R value calculated for quality of the present map and clear connectivities between the old site was actually a local minimum. Comparison ofthe secondary structure elements in it allowed for a straightfor- heavy metal difference Patterson (FPH - Fp)2 with theoretical ward tracing of the four chains. Patterson syntheses based on the old and new sites confirmed We have built a model for the histone octamer by using the the correctness ofthe new site; therefore, the old heavy metal program FRODO (16) on an Evans and Sutherland PS-390 and coordinates were abandoned. The ISIR procedure was used MicroVAX II minicomputer. Our initial model contained once again to calculate new phases for the original data by side-chain atoms for approximately two-thirds of the H2A- using the coordinates of the new heavy atom site, and a new H2B residues, while all others were built in as alanines. A electron density map was calculated. single round of refinement by simulated annealing using the By comparing the two Fourier maps, it is clear that the program XPLOR (17) reduced the crystallographic R value molecular boundary found in the old map is larger than that from 48% to 29% for the 10- to 3.5-A data set.