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Biochem Biophys Res Commun
2019 Jan 15;5083:864-870. doi: 10.1016/j.bbrc.2018.11.150.
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The structural basis of human Spt16 N-terminal domain interaction with histone (H3-H4)2 tetramer.
Jiang H
,
Xu S
,
Chen Y
,
Li H
,
Tian L
,
Zhou H
,
Zhao Z
,
Yang C
,
Zhong Z
,
Cai G
,
Su D
.
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FACT (Facilitates Chromatin Transactions) is a heterodimeric protein complex involved in RNA polymerase II transcription elongation, playing essential roles in chromatin remodeling during transcription, replication, and DNA damage repair. The FACT subunit hSpt16 is essential for nucleosome reorganization. The N-terminal domain of hSpt16 (hSpt16-NTD) was recently described as a histone (H3-H4)2-binding domain; however, its mode of interaction remains unknown. In this study, we solved the structure of hSpt16-NTD437 at 2.19 Å and found that a long-disordered region (hSpt16-LDR), after the main body of hSpt16-NTD, is a novel histone-binding motif. Furthermore, hSpt16-LDR interaction with (H3-H4)2 is H3 N-terminal tail-independent. Therefore, Spt16-NTD is a histone H3-H4-specific binding domain with a distinct mechanism of interaction between histones and histone chaperones.
Fig. 1. Structural overview of hSpt16-NTD437 and its conformation in solution.
(A) Stereo view of hSpt16-NTD437 with secondary structure elements labelled. All α-helices are colored green, while β-sheets are yellow. (B) Analytical gel filtration of hSpt16-NTD437 (red line) with peak positions of molecular weight standards indicated by gray dotted lines. (C) Cross-linked hSpt16-NTD437 and GST were analyzed by SDS-PAGE. (D) Analytical ultracentrifugation confirmed that hSpt16-NTD437 in solution is in monomeric form. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. hSpt16-NTD510 interacts with histones (H3-H4)2.
(A) GST pulldown assays of recombinant proteins GST-hSpt16-NTD510 and GST-hSpt16NTD437 with histone (H3-H4)2 in increasing salt concentrations. GST protein was used as a negative control. (B) Trace amounts of hSpt16-NTD510 and hSpt16NTD437 binding with (H3-H4)2 under 1 M NaCl salt concentration conditions were detected using western blot. The hSpt16-NTD510 and hSpt16-NTD437 proteins used in the assays assessed by anti-GST antibody are shown in the upper panel, while histone (H3-H4)2 tetramers assessed with anti-H3 antibody are shown in the lower panel. (C–D) Analytical gel-filtration chromatography to determine the interaction between hSpt16-NTD437 (C, blue line) and hSpt16-NTD510 (D, blue line) with different versions of (H3-H4)2 (red line, green line, and black line for (H3-H4)2, (H3S1-H4)2, (H3S2-H4)2, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Binding affinity measurements of histones to hSpt16-NTD510 and hSpt16-NTD437 by ITC.
ITC was performed by titrating histones into a sample cell containing either hSpt16-NTD510 or hSpt16-NTD437. The top panel shows heat change upon ligand titration. The bottom panel shows the integrated data and ITC isotherm (solid line) fitted to a single-site binding model. ITC curves and fitted affinity of (A) (H3-H4)2 to hSpt16-NTD437, (B) (H3-H4)2 to hSpt16-NTD510, (C) H2A-H2B to hSpt16-NTD437, and (D) H2A-H2B to hSpt16-NTD510. The Kd and N values fitted from the curve are indicated in the panel.
Fig. 4. Spt16-LDR at the C-terminal of Spt16-NTD510 is a histone (H3-H4)2 binding domain.
(A) Diagram of Spt16 components, with C-terminal long-disordered region (Spt16-LDR, residues 438-510AA) highlighted in the green box. (B) Sequence alignment of Spt16-LDR in different species. The highly conserved sites are colored yellow, and particularly the residues in red is completely conserved. (C) Crystal structure of hSpt16-NTD437 (PDB: 5XM2), colored in green, (D) crystal structure of hSpt16-NTD510 (PDB: 5E5B), colored in blue, (E) Superposition of hSpt16-NTD437 and hSpt16-NTD510. (F) Far-UV CD spectrum of hSpt16-NTD510 (red line) and hSpt16-NTD437 (blue line) in solution state. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
