Insights into ubiquitylation and arginine methylation by HUWE1 and PRMT5

Abstract

Post-translational modifications allow diversification of proteins beyond the canonical 20 amino acids. These modifications, such as ubiquitylation and methylation, play key roles in mammalian development and disease physiology, and thus constitute a major class of therapeutic targets in human disease. However, developing therapeutics against these enzymes requires a clear understanding of the molecular mechanisms and structural underpinnings of their function. HUWE1 is a ~3,900 amino acid HECT ligase responsible for regulating a plethora of cellular processes, ranging from protein orphan quality control to apoptosis and DNA damage response. We solved the cryogenic electron microscopy (cryo-EM) structure of full-length HUWE1, revealing a solenoid ring structure with accessory domains located above or below the ring. Reported patient mutations dispersed along the entirety of the HUWE1 ring architecture show hypomorphic effects, providing a molecular understanding of patient mutations outside of the active site. The structure of HUWE1 in complex with its substrate, DDIT4, illuminates how the armadillo repeat regions of HUWE1 can capture both peptide and phosphorylated substrate degrons and provides a basis for HUWE1 substrate recognition. PRMT5 is a methyltransferase that regulates a large number of cellular pathways through methylation of substrates such as histone tails and spliceosome proteins. PRMT5 functions as a hetero-octamer with its obligate binding partner, WDR77, and uses substate adaptors pICln and RIOK1 to recruit and methylate the C-terminal tails of SmD1 and RPS10, respectively. We show that substrate adaptors compete for binding to PRMT5 and through cryo-EM, we find that substrate adaptor and substrate are bound to PRMT5 through two peptide motifs. Substrate adaptors, while not necessary in vitro, help enhance methylation. These studies illuminate the biochemical nature of PRMT5 interactors and the importance of peptide motifs in regulating PRMT5 activity. Finally, we utilize proteomics-based approaches and in silico structure prediction to discover novel PRMT5 interactors. We find that ZNRD2 is a novel interactor of PRMT5 that utilizes its N-terminal disordered region to bind to the same interface where the canonical substrate adaptors, pICln and RIOK1, bind. ZNRD2 interacts with incompletely assembled CCT subunits, and methylation of CCT7 by PRMT5 prevents binding with the E3 ligase APPBP2. Collectively, these findings uncover novel PRMT5 interactors and pave the way towards expanding our understanding of PRMT5 biology. Altogether, we provide unprecedented structural and mechanistic insight into the architecture of the full-length HECT ligase HUWE1, how patient mutations affect multiple aspects of HUWE1 ligase activity, and how HUWE1 utilizes its various auxiliary domains to recruit and ubiquitylate substrates. Next, we determine the biochemical mechanisms of substrate adaptor and substrate recruitment to the PRMT5 methylosome, showing that substrate adaptors, while bound flexibly to PRMT5, nonetheless function to enhance substrate methylation by acting as an additional anchor point. Leveraging the structural insights gained from PRMT5, we discover ZNRD2 as a novel interactor with PRMT5. ZNRD2 interacts with unassembled CCT subunits, implicating a role as a potential substrate adaptor for CCT7 methylation by PRMT5. Methylation of CCT7 by PRMT5 further abrogates binding to the APPBP2 E3 ligase, This body of work sheds light onto how various enzymes responsible for depositing different PTMs in the context of substrate recognition and PTM catalysis, which may pave the framework for developing targeted therapeutics against these enzymes, which are both frequently dysregulated in human disease.