Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity.
Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1 throught UBL thioester intermediate, and generating a thioester-linked E2 throught UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2 throught NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.
Huang
Danny T
DT
Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Hunt
Harold W
HW
Zhuang
Min
M
Ohi
Melanie D
MD
Holton
James M
JM
Schulman
Brenda A
BA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D000075687:NEDD8 Protein; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000378:metabolism*; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D004952:Esters; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D013438:Sulfhydryl Compounds; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D044763:Ubiquitin-Conjugating Enzymes; D044764:Ubiquitin-Activating Enzymes