Structure and location of the regulatory β subunits in the (αβγδ)4 phosphorylase kinase complex

Abstract

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)(4) complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)(2) lobes joined with D(2) symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)(4) complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)(4) kinase core, because a β(4) subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.

FIGURE 1.

Cross-linking of PhK with DFDNB. Left panel, PhK (lane 1) was cross-linked with DFDNB (lane 2) and resolved by SDS-PAGE. Right panel, parallel samples were transferred to PVDF membranes and probed with mAbs against all of the subunits. All major conjugates cross-reacted only with anti-β and anti-α mAbs and not with anti-γ or anti-δ (δ = integral calmodulin subunit) mAbs. Cross-linking of PhK by DFDNB resulted in the formation of four major β-containing conjugates that corresponded by mass and cross-reactivity to a β₄ tetramer (501 kDa), αβ dimer (264 kDa), intermolecularly cross-linked ββ_x1 (250 kDa), and intramolecularly cross-linked ββ_x2 (250 kDa) dimers. As discussed under “Results,” the ββ_x2 dimer migrates faster than its theoretical mass of 250 kDa would predict. Progressing from top to bottom, hatchmarks to the left of column 1 correspond to molecular mass markers with masses (kDa) of 500, 279, 251, 164, 121, 117, 77, 64, and 52.

FIGURE 2.

MS/MS analysis of the signal at m/z 1149.102 identifying a conjugate comprising residues 48–56 and 1021–1028 of the regulatory β subunit of PhK. The composition of the ions identifying the cross-linked peptide and chemical structure of the cross-link are shown. Lowercase letters denote ions arising from amide cleavages of the peptide backbone and are color-coded for each peptide in the conjugate pair (black for residues 48–56 and green for 1021–1028). Intact covalent links formed between peptide fragments are indicated by a forward slash (/). For singly charged ions, it should be noted that one of the two peptide ions covalently attached to either position of the ring is a neutral product of the indicated backbone amide cleavage. Lines bisecting the bonds between the ring carbons and NO₂ nitrogens in the cross-link structure represent the loss of nitro groups, indicated in several fragment ions (40, 41). Heavy black bars between each peptide indicate residues cross-linked and are consistent with a pool of peptides, containing either Lys-1025/Tyr-51 or Lys-1025/Lys-53 cross-linked side chains.

FIGURE 3.

Theoretical three-dimensional model of the PhK β subunit. Hierarchical protein structural modeling of the β subunit was carried out using I-TASSER (46). X-ray crystal structures of Aspergillus awamori glucoamylase (blue and gray ribbon traces) and human PP2A PR65/A subunit (yellow trace) were used to thread, respectively, residues 41–670 and 671–1092 of the multidomain β subunit primary sequence (61, 65). The remaining N-terminal residues (1–40, blue trace) were modeled ab initio using QUARK (60). Models were constrained using the DFDNB cross-linking results, forcing the approach of the Lys-1025 and Tyr-52 α-carbons. Side chains of the DFDNB cross-linked residues (red) and the distances between the Lys-1025/Lys-51 and Lys-1025/Lys-53 cross-linked pairs (arrows) are indicated in a magnified view of the cross-link site linking the N and C termini of β.

FIGURE 4.

β₄ subcomplex revealed by nano-ESMS of autophosphorylated PhK. Nondenaturing MS of intact phosphorylated PhK yielded two charge state series centered at 12,500 and 16,000 m/z, the latter of which corresponds to the intact hexadecameric PhK complex (discussed in detail in Ref. 72). The former charge state series (blue circles), with charges ranging from +46 to +50, yielded a measured mass of 507,062 ± 55 Da that was in close agreement by mass with only one combination of subunits, namely a β₄ tetramer (mass_Theo = 500,338 Da).

FIGURE 5.

Docking the β model in the PhK cryo-EM envelope. A, magnified view of the β subunit docked in the three-dimensional cryo-EM envelope (green mesh) solved for the native PhK complex (11). The β model is shown positioned in the EM envelope, proximal to the interconnecting bridge region of PhK (11), using the known location of an anti-β subunit-specific mAb epitope (mapped to residues 703–815) as a marker (16). All known solvent-accessible structures (phosphorylatable serines 11, 26, and 700; cross-linked residues Lys-1025, Tyr-51, and Lys-53 and the anti-β epitope) are shown located at the periphery of the EM envelope. B, three predominant orientations of PhK are shown, revealing an optimal fit for the β model in the bridge region of PhK. C, β core in PhK, modeled by docking a single color-coded β structure in each of the four bridges of the EM envelope with overall D₂ symmetry.