NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide

Abstract

The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

Emerging roles of the corepressors NCoR1 and SMRT in homeostasis

“…In addition to HDAC3, these partners include the proteins G protein pathway suppressor (GPS2) and transducing b-like 1 (TBL1) and its homolog, TBL-related 1 (TBLR1), which all together form the core repression complex (Oberoi et al 2011). In addition to binding with repressive transcription factors, NCoR1/SMRT can also recruit SMRT/ HDAC1-associated repressor protein (SHARP), a repressor protein that can bind RNA and also contains motifs that are conserved in corepressors, such as the receptor interaction domain (RID) motif (Shi et al 2001;Ariyoshi and Schwabe 2003;Mikami et al 2012; see below).…”

Structural Insights into the Recruitment of SMRT by the Corepressor SHARP under Phosphorylative Regulation

“…We next determined the solution structure of the SPOC/ p-SMRT-C8 complex using heteronuclear NMR to reveal how phosphorylation contributes to interaction. The use of chemically synthesized and partially labeled SMRT peptide facilitated the NMR assignment process (Mikami et al, 2013). Together with the two-dimensional (2D) 13 C, 15 N-filtered (F2) TOCSY experiment, we could assign 1 H chemical shifts in an unlabeled part of the SMRT peptide, such as Hb, Ha, and amide H of pS2522.…”

“…The recombinant SPOC domain (aa 3496-3664) of human SHARP was expressed and purified as described previously (Mikami et al, 2013). Briefly, proteins were expressed in Escherichia coli strain BL21 Rosetta (DE3) (Novagen) as a fusion protein with glutathione S-transferase (GST).…”

“…The 8-residue phosphorylated SMRT peptide p-SMRT-C8 (YETL(pS)D(pS)E), corresponding to aa 2518-2525 of human SMRT, derived by solid-phase synthesis, was purchased from TORAY Research Center. Commercially available 13 C-and 15 N-uniformly labeled F-MOC amino acid precursors (aspartic acid, glutamic acid, leucine, and tyrosine [CIL]) were used for peptide synthesis and were able to obtain DELY-selective 13 C/ 15 N-labeled phosphorylated peptide (Mikami et al, 2013). The solution structure of the SPOC/p-SMRT-C8 complex was determined using 0.9 mM complex solution in a buffer comprising 30 mM HEPES (pH 7.5), 1 mM DTT, and 0.1 mM EDTA with 93% H 2 O/7% 2 H 2 O or 99.9% 2 H 2 O.…”

“…The side-chain resonance assignments were obtained from five spectra: 3D C(CO)NH, 3D HC(CO)NH, 3D HCCH-TOCSY, 4D HC(CO)NH, and 1 H-13 C CT HSQC. The methyl resonances in 35/38 isopropyl groups of leucine and valine residues and 41/114 b-methylene groups were assigned in a stereospecific manner as previously reported (Mikami et al, 2013). The unlabeled part of the SMRT peptide was assigned by a 2D 13 C, 15 N-filtered (F2) TOCSY experiment using 13 C, 15 N-labeled SPOC domain/unlabeled p-SMRT-C8 complex sample.…”