All ETDs from UAB

Advisory Committee Chair

Bingdong Sha

Advisory Committee Members

Chenbei Chang

Champion Deivanayagam

Fang-Tsyr Lin

Narayana Sthanam

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Sil1 is an Endoplasmic Reticulum (ER) localized protein. SIL1 was initially identified as a UPR-regulated gene. Later studies show Sil1 functions as the nucleotide exchange factor of ER lumenal Hsp70--Bip by directly interacting with the ATPase domain of Bip. Currently, the molecular mechanism how Sil1 catalyzes nucleotide exchange of Bip is still elusive. In this study we determine the complex structure of yeast S.cerevisia Sil1 and Bip (also called Kar2) ATPase domain at 2.3Å resolution by Single-anomalous dispersion (SAD) methods. The Sil1-Bip complex structure reveals that one sil1 molecule interacts with one Bip ATPase domain molecule to form the complex. Sil1 forms an elongated shape which is entirely comprised of α-helices including four Armadillo-like repeats. In the complex structure, Sil1 wraps around the subdomain IIb of the Bip ATPase domain and acts in a "clamp" model. The binding of Sil1 forces a ~13.5° outward rotation of subdomain IIb away from the nucleotide-binding cleft. The complex formation also induces a ~3.7° rotation of subdomain Ib to the opposite direction. These conformational changes open up the nucleotide-binding cleft of Bip ATPase domain, disrupt the hydrogen bonds between Bip ATPase domain and bound ADP, and eventually promote the ADP release and the subsequent ATP binding. A group of interface residues from Sil1 are mutated. Two single mutants H163A and E390A result in complete loss of Sil1-Bip interaction. All the other mutants compromise binding between two proteins to various extents. The ATPase activity assay results show all the Sil1 mutants lose the ability to stimulate the ATPase activity of Bip. The results are consistent with that of the reduced binding affinities between Sil1 mutants and Bip ATPase domain. Utilizing the structural and functional data, a reasonable mechanism for Sil1 functioning as the nucleotide exchange factor of Bip has been proposed.

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