C.R. Clapier et al. Drosophila)] are unlikely to modify interactions between NCP particles, as the residues concerned face solvent regions internal to the octamer core. The other two (Ser113 ? Ala in H2A and Ala121 ? Ser in H2B) are converse substitutions which localize to the outer face of the NCP; their net effect is the displacement of a single hydroxyl group across the face of the NCP by 45 A˚ (or 75% of the octamer’s diameter), which probably has no more than a modest effect on inter-NCP interactions. The remaining seven substitutions differentiating Dro- sophila from Xenopus localize to the hydrophobic core of H2A and to the H2A-H2B dimer interface (Fig. 1, high- lighted in cyan). The residues cluster into two groups, located on opposite sides of the pseudodyad [Fig. 3(A)]. Remarkably, all the substitutions are of a mutually com- pensatory nature. Two substitutions are juxtaposed in histone H2A at positions mediating interactions between the a2 and a3 helices. The converse nature of these sub- stitutions, Ile62 ? Val and Val87 ? Ile, allows for the net translocation of a methyl group without perturbing the spatial coordinates of the protein backbone [Fig. 3(B)]. A similar phenomenon is observed for Xenopus residues H2A-Thr59 and H2B-Val38, which in Drosophila are Ala and Ile residues, respectively [Fig. 3(C)]. These two positions are juxtaposed in the dimer interface, such that the gain and loss of a methly group are mutually offset. The Thr59 hydroxyl group forms an intrachain hydrogen bond with the backbone helix a2, and so its loss in Drosophila is unlikely to affect dimer stability. Finally, in Xenopus, H2A residue Leu55 is sandwiched between Val30 of the same chain and H2B residue Val66. All three positions are substituted in Drosophila in such a way as to preserve the volume occupied by side chains in the hydrophobic core: replacement of Leu55 by the more slender Met is countered by replacement of the two valines by bulkier isoleucines [Fig. 3(D)]. Their compensatory nature suggests that these substi- tutions are unlikely to influence the kinetics or stability of H2A-H2B dimer formation, and hence the dynamics of nucleosome assembly/disassembly. More generally, the phenomenon of counterbalanced substitutions may partly account for the poor correlation observed between sequence conservation and the number of intrachain con- tacts mediated by histone residues,34 because two or more poorly conserved residues may coevolve to preserve inter-residue contacts. Patterns in histone evolution The core histones are among the best-conserved pro- teins known. The sequence conservation is notably greater in structured (histone-fold) regions than in the histone tails (Fig. 1). Our analysis of Drosophila and Xen- opus NCPs suggests that the sequence divergence in structured residues should have little impact on histone octamer assembly, histone–DNA interactions, or inter- 6 PROTEINS NCP interactions. Clearly, NCP evolution has been tightly constrained since the speciation event that sepa- rated the vertebrate and invertebrate clades. In contrast, yeast and higher eukaryotes exhibit consid- erably more differences in their histone-fold sequences (Fig. 1), suggesting that histone evolution underwent a burst prior to the appearance of metazoa, only to stag- nate thereafter. Although yeast and metazoan NCP struc- tures differ little at the mononucleosomal level, substan- tial differences in crystal packing interactions suggest that they may exhibit different internucleosomal interactions in vivo.23 This may be a reflection of the significantly lower requirements for DNA compaction of the much smaller yeast genome compared to that of metazoa. Mutational studies of histones (both in vivo and in vitro) have made it clear that maintaining nucleosome structure cannot entirely account for the extreme degree of histone sequence conservation. By corollary, sequence changes in histone mutants or variants are of little struc- tural, but of decisive functional consequence. Histones account for a large percentage of the nucleosome’s exposed surface–a highly sculpted, differentially charged landscape that interacts with many nuclear factors35 and that likely mediates nucleosome–nucleosome interactions to form chromatin higher order structure. Thus, unlike globular proteins, exposed surface residues are exception- ally constrained, and can only mutate if compensatory changes minimize the effects. ACKNOWLEDGMENTS We thank Peter B. Becker for plasmids leading to the expression of the recombinant D. melanogaster histones; and ESRF/EMBL colleagues for access and support to the ESRF beamlines. 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