Date of Award

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cell Biology and Biochemistry

Research Advisor

Brenda A. Schulman, Ph.D.

Committee

Linda M. Hendershot, Ph.D. Lawrence M. Pfeffer, Ph.D. Stephen White, Ph.D. Jie Zheng, Ph.D.

Abstract

Posttranslational modification of macromolecules by ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo and NEDD8 regulate a vast array of processes in the cell. Transfer of UBLs to their target generally occurs by a series of molecular handoffs down an E1‑E2‑E3 cascade. We are interested in understanding how E1‑E2 pairs interact and mediate UBL transfer. To this effect, we studied two E1‑E2 pairs: the Sumo pathway (Sumo utilizes a canonical E1 and E2) and the Atg8 pathway (Atg8 is a UBL involved in autophagy that utilizes a non-canonical E1‑E2 pair).

Sumo conjugation is initiated by the heterodimeric Aos1‑Uba2 E1 enzyme (in humans called Sae1‑Uba2), which activates Sumo's C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. In this study we determined crystal structures of the C‑terminal ubiquitin-fold domain (ufd) from yeast Uba2 (Uba2ufd), alone and in complex with Ubc9. The overall superimposition of Uba2ufd and Ubc9 between yeast and human suggests conservation of fundamental features of Sumo conjugation. Docking the Uba2ufd-Ubc9 and prior full‑length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the Nedd8 cascade demonstrate that UBL cascades generally utilize some parallel E1‑E2 interaction surfaces. In addition, the structure of the Uba2ufd‑Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9‑E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3‑catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades.

Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin‑like protein (UBL) Atg8 during autophagy. Here we report that the unique N‑terminal domain of Atg7 (Atg7NTD) recruits a unique "flexible region" from Atg3 (Atg3FR). The structure of an Atg7NTD‑Atg3FR complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, which mutational analyses show is important for Atg8 conjugation. The structure, along with homology of the Atg7 C-terminal domain (Atg7CTD) to canonical E1s and their bacterial antecedents, allows modeling of a full‑length, dimeric Atg7~Atg8‑Atg3 complex. The model and supporting biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic Cys of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a novel E1~UBL‑E2 architecture for enzymes mediating autophagy.

DOI

10.21007/etd.cghs.2011.0310

Comments

One year embargo expired May 2012

Share

COinS