The structure of the large regulatory α subunit of phosphorylase kinase examined by modeling and hydrogen‐deuterium exchange

Abstract 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.