Mass Spectrometric Analysis of Surface-Exposed Regions in the Hexadecameric Phosphorylase Kinase Complex

Abstract

Phosphorylase kinase (PhK) is a 1.3 MDa (αβγδ)4 enzyme complex, in which αβγδ protomers associate in D2 symmetry to form two large octameric lobes that are interconnected by four bridges. The approximate locations of the subunits have been mapped in low-resolution cryo-electron microscopy structures of the complex; however, the disposition of the subunits within the complex remains largely unknown. We have used partial proteolysis and chemical footprinting in combination with high-resolution mass spectrometry to identify surface-exposed regions of the intact nonactivated and phospho-activated conformers. In addition to the known interaction of the γ subunit’s C-terminal regulatory domain with the δ subunit (calmodulin), our exposure results indicate that the catalytic core of γ may also anchor to the PhK complex at the bottom backside of its C-terminal lobe facing away from the active site cleft. Exposed loops on the α and β regulatory subunits within the complex occur at regions overlapping with tissue-specific alternative RNA splice sites and regulatory phosphorylatable domains. Their phosphorylation alters the surface exposure of α and β, corroborating previous biophysical and biochemical studies that detected phosphorylation-dependent conformational changes in these subunits; however, for the first time, specific affected regions have been identified.

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

“…At 15 s, over 80% of the domain undergoes low exchange, with only the N‐terminal residues 3–17 and a region from 362–372 exhibiting medium exchange and residue 220 high exchange (Table ). The extent incorporation measured for residue 220 (resolved using overlapping peptides covering residues 218–242) is consistent with its location in a short loop in our model that was previously suggested by partial proteolysis . This predicted short loop connects two helices that correspond to helices 6 and 7 in GH‐15 structures.…”

“…All but 3 residues at the termini of Variable subdomain 2 constitute a region (967–1064) that is unique to α (i.e., not present in β) and which contains the regulatory phosphorylatable region (970–1030) . Adjacent to the N‐terminus of this domain, residue 967 has been identified as part of an exposed loop by partial proteolysis, which is consistent with our model. The C‐terminus of this domain, residues 1035–1066, occurs within a region (1037–1078) that rapidly (15 s) undergoes exchange to a medium level, which is maintained through 6 h (Table ); our model predicts this stretch to be a loop‐short helix‐loop structure.…”

“…This hypothesis of surface exposure is supported by our high exchange data [e.g., peptide 723–749, Fig. (D)], as well as partial proteolysis of the complex, which exposed a loop (699–748) overlapping this region. This loop was the most rapidly proteolyzed region of α in the PhK complex, and was targeted by a variety of proteases having different specificities.…”

“…The C‐terminus of this domain, residues 1035–1066, occurs within a region (1037–1078) that rapidly (15 s) undergoes exchange to a medium level, which is maintained through 6 h (Table ); our model predicts this stretch to be a loop‐short helix‐loop structure. The previous identification of an exposed loop end by partial proteolysis at residue 1041 occurs at a distorted helix‐loop (1037–1041) in the model (Fig. ).…”

“…HDX was used to examine the structural and functional regions of the α subunit, either predicted or determined experimentally . Analyses of the deuterium content for peptides that overlapped specific regions in the primary structure allowed us to further resolve the extent incorporation occurring at multiple or even single amino acids at discrete sites within these regions.…”