Inactive Oxyanion hole in UFC1 T106I impairs UFM1 conjugation to substrate

Ufmylation is posttranslational modification essential for regulating vital cellular processes. Prevention of this modification leads to a wide range of cellular dysfunction and diseases. Akin to ubiquitin modification system, Ufmylation involve a cascade of three enzymes E1 UBA5, E2 UFC1, and E3 UFL1, that covalently tag UFM1 to a target protein. We recently deciphered the mechanism of UFM1 transfer from UBA5 to UFC1. An earlier report says that a biallelic UFC1 T106I mutation leads to infant encephalopathy disease because of impaired UFM1 transfer to UFC1. Invitro thioester assay showed no defect in basic pH. The determined Mutant structure showed no difference compared to UFC1 WT except for the abolition of the hydrogen bond between the Thr106 side chain and the back bone Nitrogen of Lys108. However, the structure is kept intact due to the hydrophobicity of non-polar Isoleucine. With the structure and activity not affecting UFC1 T106I charging with UFM1, Invitro conjugation activity showed that the mutant is inactive to transfer UFM1 to E3 UFL1 and subsequently to a substrate’s active lysine. Thr106 is a component of a TAK motif that replace HPN motif in other E2 systems. The Asn play a role of oxyanion hole that stabilizes the negatively charged intermediate due to the attack on the E2~UB by the substrate lysine. We suggest that the back bone nitrogen of Lys108 of TAK motif is the oxyanion hole which is stabilized by the hydrogen bond with Thr106. The mutation to isoleucine causes an inactive oxyanion hole that consequently arrests ufmylation.