The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms

Abstract

Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.

Figure 1.

Mutational analysis of sNASP histone binding specificity. (A) Schematic diagrams of the sNASP variants. Each of the constructs also contains an NH₂-terminal His-tag. (B) For the biochemical assays, the sNASP constructs were expressed from E. coli, purified by Ni²⁺-chelate chromatography and resolved by SDS–PAGE using Coomassie blue staining. Proteins were visualized by Coomassie blue staining. (C) Surface plasmon resonance (SPR) binding sensorgrams displayed for various concentrations of sNASP-N injected over a H3.3/H4 tetramer sensor chip (black lines). The data were globally fit to a 1:1 Langmuir binding model (red lines) and the residuals plotted above the kinetics plot. For SPR measurements, the sNASP variants were further purified over a size-exclusion chromatography (SEC) column described in the ‘Materials and Methods’ section.

Figure 2.

Binding of sNASP to histones in vivo. U2OS cell lines were isolated that allow for the Tet-inducible expression of His-tag full-length sNASP or various mutant forms of sNASP (as indicated at the top of each column). Whole cell extracts were made from each cell line and passed through a Ni²⁺-chelate affinity column. After extensive washing, proteins bound to the column were eluted with buffer containing a high concentration of imidazole. An aliquot of the whole cell extract (IN, 10%), the flow-through fraction (FT, 10%) and bound (elution) fraction (EL, 25%) were resolved by SDS–PAGE and analyzed by western blot probed with the antibodies indicated to the right of the blots.

Figure 3.

sNASP can bind to linker and core histones simultaneously. (A) The Ni²⁺-chelate column elution fraction isolated from the full-length sNASP whole cell extract was resolved by size exclusion chromatography (Superose 6). The indicated fractions were analyzed by western blots and probed using antibodies that recognized sNASP, histone H1.2, histone H4 and histone H2B (as marked). Arrows on the top of the blots indicate the void volume and elution position of molecular weight standards. (B) The Ni²⁺-chelate column elution fraction isolated from the full-length sNASP whole cell extract shown in Figure 2 was immunoprecipitated with an α-H3 antibody. The input flow-through and bound fractions were analyzed as described in Figure 2.

Figure 4.

Structural model of a sNASP monomer generated by Rosetta (25). (A) Ribbon representation of sNASP highlighting the TPR motifs (TPR1 in blue from residues 39–75, TPR3 in yellow from residues 196–236, and TRP4 in purple from residues 242–278), coiled-coil domain (CCD in green from residues 281–323), and the glutamates residues, drawn as CPK models, mutated to lysines in the sNASP-12E/K construct. The NH₂- and COOH-termini are labeled accordingly. The predicted binding sites of the linker and core histone sites are indicated. (B) The linear Poisson–Boltzman equation was solved for sNASP using APBS (30) with 150 mM monovalent salt at 25° C. The solvent accessible surfaces areas are displayed and colored blue (+5 kT/e) and red (−5 kT/e). The electrostatic potential gradient for sNASP is displayed as a blue (+2 kT/e) and red mesh (−2 kT/e). Predicted structures and electrostatic potentials were viewed and rendered using PyMOL (31). The ribbon and electrostatic diagrams are oriented in the same manner and the two structural views are related by an 180° rotation around the vertical axis.