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The structure of the large regulatory ? 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, (????)4 , and 325 kDa of unique sequence (the mass of an ???? protomer). The ?, ? and ? subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic ? 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 ? and ? subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, ?. Here, we have modeled the full-length ? 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 ? to comprise two major domains: an N-terminal glycoside hydrolase domain and a large C-terminal importin ?/?-like domain. This structure is similar to our previously published model for the homologous ? 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 (????)4 PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter-subunit contact sites for ? in the quaternary structure of PhK; of particular interest is a low-exchanging region in the C-terminus of ? that is known to bind the regulatory domain of the catalytic ? subunit.

SUBMITTER: Rimmer MA

PROVIDER: S-EPMC5775172 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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

Rimmer Mary Ashley MA Nadeau Owen W OW Yang Jianyi J Artigues Antonio A Zhang Yang Y Carlson Gerald M GM

Protein science : a publication of the Protein Society 20171121 2

Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ)<sub>4</sub> , 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 o ...[more]

PMID: 29098725

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Project description:Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.

Dataset Information

The structure of the large regulatory ? subunit of phosphorylase kinase examined by modeling and hydrogen-deuterium exchange.

Publications

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

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