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) , 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) 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.

Structural characterization of the catalytic γ and regulatory β subunits of phosphorylase kinase in the context of the hexadecameric enzyme complex

“…These results are consistent with a large body of evidence demonstrating that the β subunit undergoes significant conformational changes in the PhK complex . Differences in the HDX for the predicted (α/α) 6 barrels for the homologous α and β subunits provide additional evidence supporting differences in the modeled structures generated by us and others, with the barrel for α conserving more residues critical for GH catalytic function and that for β containing a small C‐terminal insert …”

“…). Similarly, we recently demonstrated that predicted helices in the GHL catalytic domain of the homologous PhK α subunit exchanged moderately . This pattern was consistent with the simulated unfolding of a related GH‐15 family member that unexpectedly underwent thermal denaturation at moderate temperatures, suggesting the α subunit's predicted (α/α) 6 barrel to be moderately dynamic.…”

“…Helices 1, 8, 11 and 7 are thought to be the first to unfold upon thermal denaturation of the GH‐15 (α/α) 6 barrel; however, exchange for the first three of these predicted helices was very low for the β subunit barrel. Additionally, exchange for helix 7 in the β subunit was predominately low, reaching medium levels only and at later time points, whereas in α its exchange varied from low to high, consistent with its partial melting in the complex . Similar to α, we observed that predicted helices 4 and 9 transitioned from low to medium levels of exchange in the β subunit barrel, despite being packed for the most part internally within each lobe in our model, suggesting β also to be moderately dynamic.…”

“…High resolution structures of CaM (δ) and the catalytic domain of the γ subunit (minus the γCRD) have been solved, but in the absence of PhK's other subunits. The α and β subunits are homologous, with two unique regions in α and one in β, and structural models for the individual subunits have been generated . These models for both isolated subunits show highly helical, two‐domain structures; however, the structure of neither subunit has been examined in the context of the (αβγδ) 4 hexadecameric complex.…”

“…The multiple subunits of PhK result in 325 kDa of unique sequence, making HDX challenging. Using a sufficiently long gradient to reduce the number of co‐eluting peptides, while still keeping the gradient sufficiently short to reduce back‐ exchange of the deuterons, allowed for significant coverage of the α, β and γ subunits, the first subunit being the subject of the preceding article and the latter two of this article.…”