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X-ray crystallography shows SPOC is a versatile phosphoserine reader domain in gene expression regulation

SPOC domain-containing proteins play a crucial role in recognising and interacting with phosphorylated serines of RNA polymerase II (Pol II) and other proteins. A combination of X-ray crystallography and other techniques unveiled how the SPOC domains of PHF3, DIDO, RBM15 and SHARP coordinate the regulation of transcription and co-transcriptional processes.

SPOC (Spen paralogue and orthologue C-terminal domain) is a protein domain found in various proteins involved in different steps of gene expression regulation, including the human proteins PHF3, DIDO, RBM15 and SHARP [1].

PHF3 SPOC was recently established as a reader of phosphorylated Pol II C-terminal domain (CTD) [2]. Pol II CTD, a flexible unstructured tail consisting of multiple seven-amino-acid YSPTSPS repeats, undergoes dynamic phosphorylation and plays a crucial part in coordinating different stages of transcription. Two basic surface patches on PHF3 SPOC play a pivotal role in the recognition of phosphorylated serine residues on Pol II CTD. Despite an overall low sequence conservation, these patches are conserved in DIDO, RBM15 and SHARP, notably featuring a highly conserved arginine residue

(Figure 15a). Fluorescence anisotropy binding studies reveal that all four SPOC domains bind phosphorylated Pol II CTD peptides, albeit with varying affinities and specificities. While PHF3 and DIDO SPOC predominantly bind Pol II CTD peptides phosphorylated on two serine-2 residues in adjacent repeats, RBM15 and SHARP show a preference for serine-5 phosphorylated peptides and exhibit a comparatively lower affinity (Figure 15b).

SHARP SPOC was previously shown to bind a phosphorylated LSD-motif in the nuclear corepressor proteins SMRT and NCoR with substantially higher affinity than the Pol II CTD peptides. To define the structural determinants for the difference in affinity, X-ray crystallography data were collected at beamline ID30A-1 to obtain a high-quality structure of the SHARP SPOC domain in complex with the phospho-serine-5 CTD peptide. Comparative structural analysis reveals that, while SMRT/NCoR and Pol II CTD essentially occupy the same binding surface on SHARP SPOC, acidic residues within SMRT/NCoR that are absent from the Pol II CTD form additional hydrogen bonds with an arginine residue of SHARP, strengthening the binding and explaining the relatively higher affinity for SMRT compared to Pol II CTD (Figure 15c,d).

According to mass spectrometry-based interaction studies, PHF3 and DIDO SPOC domains primarily mediate interactions with Pol II and the transcription elongation machinery, while RBM15 and SHARP SPOC

Fig. 15: a) Electrostatic surface potential of SPOC domains.

Conserved basic patches are highlighted. b) Affinities of SPOC domains to different

phosphorylated CTD peptides. c) X-ray crystal structure of SHARP

SPOC and phospho-serine-5 Pol II CTD (PDB 7Z1K). d) Nuclear

magnetic resonance spectroscopy structure of SHARP SPOC and SMRT

phosho-serine-2522 (PDB 2RT5). Additional hydrogen bonds between

SMRT D2523 and E2525 and SHARP R3548 are highlighted.