Octamer element and OCA-B 1-44 peptide used in crystallization of the ternary complex. (A) Sequence of the OCA-B 1-44 peptide used for crystallization. The boxed region indicates the portion of the peptide that is ordered in the structure. Lowercase letters below the amino acid sequence refer to elements of secondary structure in the crystal structure. (t) Turn; (H) helix. Boldface type highlights residues Val-22 and Val-24, which make base-specific contacts at the fifth and sixth positions of the octamer element. (B) Sequence of the octamer element and the oligonucleotide used for crystallization. The octamer site is underlined, and the A at position 5 of this site is circled.

Electron density for the interaction between the adenine at the fifth position of the octamer element and Val-22. An SA-omit electron density map (which gives an unbiased way of checking a structure) was calculated with σ^A-weighted 2Fo-Fc coefficients (Read 1986) after omitting both Val-22 and the entire Ade-5 nucleotide from the final model and subjecting this partial model to simulated annealing in XPLOR (Brunger 1996). Gly-21 (gray), Val-22 (gray and purple), Arg-23 (gray), and Ade-5 (green and blue) from the final model are shown in the context of the SA-omit electron density contoured at 1.5σ. (A) The view perpendicular to the plane of the Ade-5 base (green, same view direction as in Fig. 3C) illustrates the electron density for the pair of hydrogen bonds (dotted lines) between the polypeptide backbone of Val-22 and the adenine ring. (B) The view looking into the major groove of the octamer element (same view direction as in Fig. 3A) illustrates the electron density for the Val-22 side chain (purple).

Structure of the ternary complex of the OCA-B 1–44 peptide, the Oct-1 POU domain, and an octamer element. (A) Overview of the ternary complex showing the OCA-B peptide (purple), the Oct-1 POU domain (the POU homeodomain is in red; the POU-specific domain is in yellow), and DNA containing the octamer sequence (position 5 is in green). About 20 amino acids of the OCA-B 1–44 peptide are well-ordered in the crystal. The OCA-B peptide traverses the major groove at the A:T base pair at position 5 of the octamer site. At the amino terminus of the ordered region, the OCA-B peptide makes contacts with the POU homeodomain; at the carboxyl terminus of the order region the OCA-B peptide forms a short α-helix and makes extensive contacts with the POU-specific domain. (B) Schematic summary of protein–DNA and protein–protein contacts in the ternary complex. The OCA-B 1–44 peptide is represented by the shaded rectangle across the center of the DNA; the positions of the POU-specific domain and the POU homeodomain are suggested by dashed outlines. Close contacts (<4.0 Å) between the OCA-B peptide and the POU-specific domain are summarized in the box in the upper left; close contacts with the POU homeodomain are summarized in the box in the lower right. All major DNA contacts are shown for the OCA-B peptide, but only key base contacts between the Oct-1 POU domain and the DNA are shown. The complete set of DNA contacts for the Oct-1 POU domain is very similar to those observed previously in its structure with an octamer element only (Klemm et al. 1994). (C) Contacts between the OCA-B 1–44 peptide (purple) and bases 5 (green) and 6 (gray) of the octamer element. The POU-specific domain, the POU homeodomain, and other bases in the octamer are not shown. Hydrogen bond contacts between the polypeptide backbone of the OCA-B peptide residue Val-22 and the exocyclic amine and N7 of adenine at position 5 are shown with red dots [the relevant atoms at these positions are blue (N), red (O), and gray (C)]. Hydrophobic interactions between OCA-B Val-22 and Thy-6′, and between OCA-B Val-24 and both Thy-5′ and Thy-6′ are indicated with gray dots. Residues at the amino terminus (Ala-16–Pro-18, extended chain) and at the carboxyl terminus (Lys-29-His-38, helix) of the ordered part of the OCA-B peptide are represented according to their secondary structure. (D) Interaction between the hydrophobic surface of the OCA-B peptide helix and the hydrophobic pocket of the Oct-1 POU-specific domain. POU-specific domain (yellow) residues at the amino terminus of helices one (Leu-6, Leu-9) and four (Phe-57, Met-60), and at the carboxyl terminus of helix three (Leu-53) form a shallow hydrophobic pocket. The surface of the helix from the OCA-B peptide (purple; Val-28, Leu-31, Leu-32) docks against the hydrophobic pocket of the Oct-1 POU-specific domain. The helices of the POU-specific domain are labeled with Roman numerals. Bases for one strand of the octamer element are also indicated.

Mutations that attenuate the interaction of OCA-B with the complex of the Oct-1 POU domain and the octamer element are located at the peptide–protein interface. The positions of the POU-specific domain (yellow) and the POU homeodomain (red) are indicated by illustration of their Connolly solvent accessible surfaces (Connolly 1983). The OCA-B peptide (purple) is indicated by a ribbon. Residues deemed essential—via previous mutagenesis studies (Gstaiger et al. 1996; Babb et al. 1997; Sauter and Matthias 1998)—for ternary complex formation are shown in space-filling representation, and all of these are clustered at the peptide–protein interface seen in our crystal structure.