Fig. 2. Structure of the SAP-1–SRF–DNA ternary complex. (A) View of the SAP-1 monomer and SRF dimer unobscured by the SRE DNA. The B-box of SAP-1 (yellow) connected by a flexible linker (dashed line, unobserved) to the ETS domain is bound to the SRF_B monomer (red) and adds an antiparallel β-strand flanked by short 3₁₀-helix segments to the four-stranded, antiparallel β-sheet of the SRF MADS-box. The SRF_A monomer (green) directly contacts the SAP-1 ETS domain. The entire DNA oligonucleotide (gray) is shown and the protein secondary structure elements are labeled. (B) View showing SAP-1 and SRF bound to opposite DNA faces. The SAP-1 ETS domain is bound in the EBS major groove and SRF is centered on the CArG-box minor groove.

Fig. 4. Structure of the SAP-1 B-box sequence element bound to SRF. (A) Model of the SAP-1 B-box superimposed in the experimental electron density. The 2F_o – F_c electron density map calculated at 3.15 Å resolution is contoured at 1.5σ. The amino acid F150 is labeled to indicate the B-box polypeptide chain. The density at the lower left corresponds to DNA and at the upper right to SRF. (B) SAP-1 polypeptide chain bound to SRF shown as an electrostatic potential surface (red negative and blue positive charge). With the exception of the interactions with the DNA phosphate groups, the interaction surface is predominantly hydrophobic. The three aromatic amino acids Y141, Y147 and F150 contribute substantially to the SAP-1–SRF interaction interface, and the analogous amino acids in Elk-1 were identified as the most important for its addition to the ternary complex (Ling et al., 1997). (C) Side chains of the SAP-1 B-box stabilizing its bound conformation. R138 and N139 make hydrogen bonds to DNA phosphate groups. Y141, I142 and Y147 make a hydrophobic cluster by which the 3₁₀3 helix and β-strand interact. L146 extends along the surface of SRF, and F150 protrudes into the hydrophobic cleft formed by the SRF βII strand, coil region and αII helix. L152 and L155 bind the 3₁₀4 helix to SRF.

Fig. 6. SAP-1 B-box and MATα2 MBM interactions with SRF and MCM1. (A) Structural alignment of human SAP-1–SRF–DNA and yeast MATα2–MCM1–DNA ternary complexes. C_α atoms of human SRF (amino acids 143–222, orange) and yeast MCM1 (amino acids 18–97, magenta) superimpose with an r.m.s.d. of 0.5 Å. The positions of amino acids in common for human SAP-1 (yellow) and yeast MATα2 (red) and interacting with the MADS-box are labeled from 1 to 8. The identity of the amino acids is given in the B-box position table in which the direction of chains is indicated. Positions 3, 1 and 7–8 in that order appear to be the most important for specificity of binding. Reversal of the MATα2 MBM sequence to match the SAP-1 B-box allows identification of a plausible MADS-box inter action sequence in the Fli-1 SBM2 sequence as shown in the table. (B) MADS-box amino acids important for B-box binding and specificity. The main chain path and side chains F150 and L146 are shown for the SAP-1 B-box (yellow). The van der Waals surface of the SRF_B monomer (red) is shown with amino acids important for specificity highlighted. The SRF cluster of V194, I206 and I215 (yellow) contacts SAP-1 F150. SRF M169, K170 and Y173 (cyan) bind helix 3₁₀3, and SRF L219 and N220 (cyan) bind helix 3₁₀4. The SRF amino acids A198, R200, L202 and Q203 (violet) were found to be important for the discrimination of binding by accessory proteins (Mueller and Nordheim, 1991).

Fig. 1. Sequences of the protein and DNA components used for the human SAP-1–SRF–DNA crystals. (A) SAP-1 ETS domain through the B-box (amino acids 1–156) and SRF core domain (MADS-box plus SAM domains, amino acids 132–223). The terminal amino acids and the SAP-1 linker region disordered in the structure are printed in gray. The N-terminal methionine of the wild-type SAP-1 sequence was replaced by glycine followed by serine (amino acids 0–1). Secondary structure elements are indicated below the sequences where the larger tubes represent α-helices, smaller tubes 3₁₀-helices and arrows β-strands. (B) SRE consensus sequence with demarcation of protein–DNA interactions. The EBS (GGA core) and the CArG sequence are on a gray background. The base pair numbering starts at the 2-fold symmetric sequence center of the CArG-box with the two different strands labeled W and C. Amino acids contacting DNA are grouped by secondary structure elements with monomer SRF_A in green, monomer SRF_B in red and SAP-1 in orange. Hydrogen bond interactions are shown as solid lines and hydrophobic contacts as dotted lines.

Fig. 3. Structural alignments of the SAP-1–SRF–DNA ternary complex with the SRF–DNA and SAP-1–DNA binary complexes. The ternary complex colors are as in Figure 2. The binary complex components have colors corresponding to their labels shown in parentheses. (A) SRF–DNA (Pellegrini et al., 1995) and SAP-1–SRF–DNA (r.m.s.d. 0.66 Å for 164 C_α atoms). The amino acids Y158 and H193 involved in binding the extremities of the DNA site have different side chain conformations in the two structures. DNA helical axes for the ternary (pink) and binary (orange) complexes were calculated with CURVES (Lavery and Sklenar, 1988). (B) SAP-1–DNA (Mo et al., 1998) and SAP-1–SRF–DNA (r.m.s.d. 0.90 Å for 88 C_α atoms). The overlap of the two protein chains is nearly exact except at the N- and C-termini. In contrast, the conformations of the DNA differ significantly outside of the central GGA sequence.

Fig. 5. SAP-1 ETS domain interaction with the SRF MADS-box. (A) The amino acids in the hydrophobic interface between the SAP-1 α3 helix and the SRF αI helix are shown. (B) Electron density of the interaction interface. The simulated-annealing, 2F_o – F_c composite omit map is contoured at 1.0σ.