3 years ago

The Structure of the Large Regulatory α Subunit of Phosphorylase Kinase Examined by Modeling and Hydrogen-Deuterium Exchange

Owen W. Nadeau, Mary Ashley Rimmer, Jianyi Yang, Antonio Artigues, Gerald M. Carlson, Yang Zhang
Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ)4, and 325 kDa of unique sequence (the mass of an αβγδ protomer). The α, β and δ subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic γ subunit in response to diverse signaling molecules. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and β subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full-length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen-deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N-terminal glycoside hydrolase domain and a large C-terminal importin α/β-like domain. This structure is similar to our previously published model for the homologous β subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen-deuterium exchange results obtained for this subunit as part of the (αβγδ)4 PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter-subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low-exchanging region in the C-terminus of α that is known to bind the regulatory domain of the catalytic γ subunit. This article is protected by copyright. All rights reserved.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/pro.3339

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