Structural study of UFL1-UFC1 interaction uncovers the importance of UFL1 N-terminal helix for ufmylation

Abstract

Ufmylation, a protein modification by Ubiquitin-like (UBL) protein UFM1, plays a crucial role in several cellular processes including DNA damage response, protein translation and ER homeostasis. To date, little is known how the enzymes responsible for this modification coordinate their action. Here we have studied the details of UFL1 (E3) activity, its binding to UFC1 (E2), and its relation to UBA5 (E1), using a combination of structural modeling with Alphafold2, X-ray crystallography, NMR, and in vitro biochemical activity assays. Guided by an Alphafold2 model, we generated an active UFL1 fusion construct that includes its cofactor DDRGK1, and solved the first crystal structure of this critical interaction. This fusion construct also unveiled the importance of the N-terminal helix of UFL1 for its binding to UFC1, which was validated by ITC and NMR experiments. Importantly, the binding site suggested by our structural model of the UFL1-UFC1 interaction reveals a conserved interface, and suggests a competition for binding to UFC1 between UFL1 and UBA5, which we reconfirmed by NMR.