Crystal structures reveal catalytic and regulatory mechanisms of the dual-specificity ubiquitin/FAT10 E1 enzyme Uba6

Abstract

The E1 enzyme Uba6 initiates signal transduction by activating ubiquitin and the ubiquitin-like protein FAT10 in a two-step process involving sequential catalysis of adenylation and thioester bond formation. To gain mechanistic insights into these processes, we determined the crystal structure of a human Uba6/ubiquitin complex. Two distinct architectures of the complex are observed: one in which Uba6 adopts an open conformation with the active site configured for catalysis of adenylation, and a second drastically different closed conformation in which the adenylation active site is disassembled and reconfigured for catalysis of thioester bond formation. Surprisingly, an inositol hexakisphosphate (InsP6) molecule binds to a previously unidentified allosteric site on Uba6. Our structural, biochemical, and biophysical data indicate that InsP6 allosterically inhibits Uba6 activity by altering interconversion of the open and closed conformations of Uba6 while also enhancing its stability. In addition to revealing the molecular mechanisms of catalysis by Uba6 and allosteric regulation of its activities, our structures provide a framework for developing Uba6-specific inhibitors and raise the possibility of allosteric regulation of other E1s by naturally occurring cellular metabolites.

Insights into the ISG15 transfer cascade by the UBE1L activating enzyme

“…1c, d). Secondary structures were not visible, however the model resembled previously described E1 structures [13][14][15][16][17][18] and therefore allowed for the placement of individual domains. The UBE1L catalytic cysteine domain (i.e.…”

“…Recently crystal structures of the E1 enzyme UBA6 in complex with ubiquitin and FAT10 were described 17,18 . Like ISG15, FAT10 contains two tandem ubiquitinlike domains fused through a short linker.…”

Bacterial antiviral defense pathways encode eukaryotic-like ubiquitination systems

“…The closest structural relatives of E1 BilD are the human ubiquitin/FAT10 E1 UBA6 (Cα r.m.s.d. 3.5 Å over 401 residue pairs spanning IAD and AAD) 24 and the heterodimeric human NEDD8 E1 NAE1-UBA3 (Cα r.m.s.d. 3.6 Å over 221 residue pairs between E1 BilD IAD and NAE1 IAD, and Cα r.m.s.d.…”

“… (a) Domain schematic of E. aridi E1 BilD and the H. sapiens ubiquitin/FAT10 E1 UBA6 (PDB ID 7SOL) 24 . IAD: inactive adenylation domain; AAD: active adenylation domain; CYS: catalytic cysteine-containing domain; FCCH: first catalytic cysteine half-domain; SCCH: second catalytic cysteine half-domain (equivalent to CYS); UFD: ubiquitin fold domain.…”

“…IAD: inactive adenylation domain; AAD: active adenylation domain; CYS: catalytic cysteine-containing domain; FCCH: first catalytic cysteine half-domain; SCCH: second catalytic cysteine half-domain (equivalent to CYS); UFD: ubiquitin fold domain. (b) Structures of E. aridi E1 BilD (left) and H. sapiens UBA6 (PDB ID 7SOL) 24 , with domains colored as in panel (a) and catalytic cysteines shown as spheres and colored yellow. The Cα r.m.s.d.…”