Proximal regions of the catalytic γ and regulatory β subunits on the interior lobe face of phosphorylase kinase are structurally coupled to each other and with enzyme activation

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

“…We report nearly full coverage of the subunit by threading with templates corresponding to GH-15 family members and the protein phosphatase 2A (PP2A) subunit PR65/A (PP2AA). We directly demonstrate for the intact complex by top-down MS and chemical cross-linking that the ␤ subunits compose the central bridge region of PhK and that rigid-body docking of their theoretical counterparts in the individual bridges of the native PhK cryo-EM envelope correlates well with the known structural details for this subunit in the complex (7,16,30).…”

“…Autophosphorylated PhK was prepared as described previously (33). The mAbs against the ␣, ␤, and ␥ subunits of PhK were previously described (15,16), and the anti-calmodulin mAb was from Zymed Laboratories Inc.. All other secondary conjugates were from Southern Biotechnology. Truncation mutants of the PhK ␤ subunit were constructed as described previously (7).…”

“…Reconstructions of images of PhK from EM reveal it to be a D 2 symmetrical structure of two lobes (10 -12), each composed of two ␣␤␥␦ protomers packed head-to-head with the lobes associated through interconnecting bridges (13)(14)(15)(16). The number of bridges reported for PhK from EM fields of the negatively stained complex on solid grids varies from a minimum of two to a maximum of four (13,17), with reconstructions of the stained kinase revealing only two large bridges between the lobes (10,12,18).…”

“…Although the primary structures are known for both ␣ and ␤ (20,21), essentially all higher order secondary (2°) and tertiary (3°) structures reported for these subunits are based on predicted rather than experimental data (22)(23)(24). In contrast, high resolution structural information is available for the catalytic core of the ␥ subunit and for the ␦ subunit (3,25); on the other hand, the small size and location of these subunits in the complex make them unlikely candidates for bridging the two (␣␤␥␦) 2 lobes (14,16). The ␣ subunit is theoretically large enough to span the distances calculated for the bridges (ϳ3 nm); however, its C terminus has been localized to the distal ends of the lobes, which would require the subunit to extend ϳ90 Å in a straight line through one lobe before traversing the space to the adjacent lobe (10).…”

“…Moreover, extensive digestion of this subunit in the complex has no effect on the bridges, whereas partial hydrolysis of both ␣ and ␤ disrupts the bridges, forming isolated lobes and smaller fragments in EM fields of the kinase (17). Proteolytic mapping of PhK also suggests ␣ to be more peripherally located in the complex than ␤ (26), and localization by immuno-EM of an epitope of the ␤ subunit close to the bridges makes it a more likely candidate for composing the interlobal bridges joining the two (␣␤␥␦) 2 lobes of PhK (16). Nevertheless, no evidence has been previously obtained to indicate the direct involvement of any of the four subunits of PhK in bridge formation.…”