Structure of the Spt16 middle domain reveals functional features of the histone chaperone FACT

Abstract

The histone chaperone FACT is an essential and abundant heterodimer found in all eukaryotes. Here we report a crystal structure of the middle domain of the large subunit of FACT (Spt16-M) to reveal a double pleckstrin homology architecture. This structure was found previously in the Pob3-M domain of the small subunit of FACT and in the related histone chaperone Rtt106, although Spt16-M is distinguished from these structures by the presence of an extended α-helix and a C-terminal addition. Consistent with our finding that the double pleckstrin homology structure is common to these three histone chaperones and reports that Pob3 and Rtt106 double pleckstrin homology domains bind histones H3-H4, we also find that Spt16-M binds H3-H4 with low micromolar affinity. Our structure provides a framework for interpreting a large body of genetic data regarding the physiological functions of FACT, including the identification of potential interaction surfaces for binding histones or other proteins.

FIGURE 1.

Spt16-M forms a double pleckstrin homology domain. A, domain organization and structures of Spt16-Pob3. Boundaries are indicated for N-terminal (N), dimerization (D), middle (M), and C-terminal (C) domains (8, 9). Known structures include Spt16-N (PDB code 3BIT (11)), Spt16-M (this study), Pob3-N/D (PDB code 3F5R), and Pob3-M (PDB code 2GCL (9)). B, ribbon representation of Spt16-M colored as a gradient from the N (blue) to the C terminus (red). Disordered residues are denoted by dashed lines. The region deleted in the crystallized Spt16-M construct is denoted by gray dashed lines. C, Spt16-M colored by domain: PH1 (slate blue), PH2 (pale green). Spheres depict mutants previously identified (16) including spt16-6 (P920L, cyan), spt16-7 (T848I, T849I, D850Y, gold), spt16-9A (G836S, P838S, blue), and spt16-11 (T828I, P859S, pink). Asp-850 is eclipsed by Thr-849 from this view. D, sequence and secondary structure of Spt16-M with coloring as in panel B. Residue numbering is indicated to the left with dots above every 10th residue. Residues highlighted in panel C are shown in colored font.

FIGURE 2.

Spt16-M is similar to other double PH domains and contributes to binding H3-H4. A, comparison of Spt16-M with the double PH domains of Pob3-M (gray, PDB code 2GCL, Ref. 9) and Rtt106 (purple, PDB code 3FSS, Ref. 13). B, electrostatic surface potential (−3 to 3 kT) of Spt16-M, Rtt106-PH, and Pob3-M. Molecular surface charge maps were prepared using PyMOL APBS tools (10) using a 2 Å solvent radius. C, EMSA showing the mobility of Oregon green-labeled Xenopus laevis H3-H4 in the absence (first lane) and presence of Spt16-Pob3 (50–1200 nm). Complexes of Spt16-Pob3 bound to H3-H4 are indicated. The asterisk indicates a minor complex form of unknown composition with identical dose-response characteristics as the main form. D, binding of Spt16-M and Spt16-M* (includes residues Gly-774–Ser-780) to S. cerevisiae H3-H4. Normalized fluorescence polarization values are plotted as a function of increasing concentrations of Spt16. A.U., absorbance units. E, quantitation of an EMSA (performed as in panel C) for H3-H4 binding to Spt16-Pob3 and (Spt16-11)-Pob3. Error bars indicate standard error.

FIGURE 3.

Location of previously identified SPT16 mutations and the structural basis of the spt16-11 phenotype. A–D, surface (A and C) and sphere (B and D) representations of Spt16-M mutations that suppress H3-L61W (orange (15)) or cause derepression of SER (green (18)). Glu-857 (blue) was identified in both studies, mutated to either a Gln (15), or a Lys (18). E and F, F_o − F_c omit map contoured at 3× r.m.s.d. around residues Thr-828 and Pro-859. Protein and solvent in the figure were deleted from the model, and random 0.1 Å shifts were applied to the rest of the structure followed by two cycles of refinement.