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. 2021 Jun 2;22(11):1908-1914.
doi: 10.1002/cbic.202100079. Epub 2021 Mar 31.

Biochemical Investigation of the Interaction of pICln, RioK1 and COPR5 with the PRMT5-MEP50 Complex

Adrian Krzyzanowski  1   2 , Raphael Gasper  3 , Hélène Adihou  4   5 , Peter 't Hart  6 , Herbert Waldmann  1   2
Affiliations

Biochemical Investigation of the Interaction of pICln, RioK1 and COPR5 with the PRMT5-MEP50 Complex

Adrian Krzyzanowski et al. Chembiochem. .

Abstract

The PRMT5-MEP50 methyltransferase complex plays a key role in various cancers and is regulated by different protein-protein interactions. Several proteins have been reported to act as adaptor proteins that recruit substrate proteins to the active site of PRMT5 for the methylation of arginine residues. To define the interaction between these adaptor proteins and PRMT5, we employed peptide truncation and mutation studies and prepared truncated protein constructs. We report the characterisation of the interface between the TIM barrel of PRMT5 and the adaptor proteins pICln, RioK1 and COPR5, and identify the consensus amino acid sequence GQF[D/E]DA[E/D] involved in binding. Protein crystallography revealed that the RioK1 derived peptide interacts with a novel PPI site.

Keywords: interfaces; peptides; protein-protein interactions; proteins.

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Conflict of interest statement

H.A. was employed by AstraZeneca.

Figures

Figure 1
Figure 1
Interaction between PRMT5 and its adaptor proteins. A) Schematic representation of the PRMT5–adaptor protein complexes with their methylation substrate. The PRMT5–MEP50 complex was visualised based on the PDB structure 4GQB. B) BLAST alignments for pICln, RioK1, COPR5 and DAB1. C) Results of the fluorescence polarization measurements for interaction of the identified peptide sequences with the full PRMT5–MEP50 complex, truncated TIM–MEP50 and isolated TIM barrel domain (n=3). D) Results of fluorescence polarisation assays for histone tail peptides and the truncated protein complex TIM–MEP50 (n=3).
Figure 2
Figure 2
Crystallographic elucidation of the PRMT5–RioK1 interaction. A) Structure of the isolated TIM barrel of PRMT5, co‐crystallised with the RioK1‐derived peptide 23 (PDB ID: 7BOC). B) Structure of the TIM barrel–peptide complex (PDB ID: 7BOC). The colours of the protein surface show the electrostatic potential (blue=positive, red=negative, white=neutral). C) Structure of 23 fitted onto the full PRMT5–MEP50 complex. Image generated through a superposition of the obtained TIM barrel–peptide crystal structure with the PDB structure 4GQB. D) Close‐up of 23 bound to the TIM barrel. E) Superposition of the RioK1‐derived peptides from PDB structures 7BOC (grey, this work) and 6V0N (yellow, Sellers et al.). [7]
Figure 3
Figure 3
Interaction between pICln/RioK1 and PRMT5 protein complexes. A) FP results for the interaction of Alexa488‐labelled pICln with PRMT5–MEP50 and TIM–MEP50 (n=3). B) FP results for Alexa488‐tagged RioK1 interacting with PRMT5–MEP50 and TIM–MEP50 (n=3). C) Comparison of the K D values obtained for the interactions between pICln/RioK1 and the PRMT5 protein complexes with FP and FIDA.
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