The regulatory α and ß subunits of phosphorylase kinase directly interact with its substrate, glycogen phosphorylase.

The selective phosphorylation of glycogen phosphorylase (GP) by its only known kinase, phosphorylase kinase (PhK), keeps glycogen catabolism tightly regulated. In addition to the obligatory interaction between the catalytic γ subunit of PhK and the phosphorylatable region of GP, previous studies have suggested additional sites of interaction between this kinase and its protein substrate. Using short chemical crosslinkers, we have identified direct interactions of GP with the large regulatory α and ß subunits of PhK. These newfound interactions were found to be sensitive to ligands that bind PhK.

Cryoelectron microscopy reveals new features in the three-dimensional structure of phosphorylase kinase.

Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (alphabetagammadelta)(4). PhK comprises two arched octameric (alphabetagammadelta)(2) lobes that are oriented back-to-back with overall D(2) symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 A. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90 degrees instead of 68 degrees, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.

A Ca(2+)-dependent global conformational change in the 3D structure of phosphorylase kinase obtained from electron microscopy.

Phosphorylase kinase (PhK), a Ca(2+)-dependent regulatory enzyme of the glycogenolytic cascade in skeletal muscle, is a 1.3 MDa hexadecameric oligomer comprising four copies of four distinct subunits, termed alpha, beta, gamma, and delta, the last being endogenous calmodulin. The structures of both nonactivated and Ca(2+)-activated PhK were determined to elucidate Ca(2+)-induced structural changes associated with PhK's activation. Reconstructions of both conformers of the kinase, each including over 11,000 particles, yielded bridged, bilobal structures with resolutions estimated by Fourier shell correlation at 24 A using a 0.5 correlation cutoff, or at 18 A by the 3sigma (corrected for D(2) symmetry) threshold curve. Extensive Ca(2+)-induced structural changes were observed in regions encompassing both the lobes and bridges, consistent with changes in subunit interactions upon activation. The relative placement of the alpha, beta, gamma, and delta subunits in the nonactivated three-dimensional structure, relying upon previous two-dimensional localizations, is in agreement with the known effects of Ca(2+) on subunit conformations and interactions in the PhK complex.

Three-dimensional structure of phosphorylase kinase at 22 A resolution and its complex with glycogen phosphorylase b.

Phosphorylase kinase (PhK) integrates hormonal and neuronal signals and is a key enzyme in the control of glycogen metabolism. PhK is one of the largest of the protein kinases and is composed of four types of subunit, with stoichiometry (alphabetagammadelta)(4) and a total MW of 1.3 x 10(6). PhK catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a (GPa) and the stimulation of glycogenolysis. We have determined the three-dimensional structure of PhK at 22 A resolution by electron microscopy with the random conical tilt method. We have also determined the structure of PhK decorated with GPb at 28 A resolution. GPb is bound toward the ends of each of the lobes with an apparent stoichiometry of four GPb dimers per (alphabetagammadelta)(4) PhK. The PhK/GPb model provides an explanation for the formation of hybrid GPab intermediates in the PhK-catalyzed phosphorylation of GPb.

Direct visualization of the calmodulin subunit of phosphorylase kinase via electron microscopy following subunit exchange.

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.

Structure of phosphorylase kinase. A three-dimensional model derived from stained and unstained electron micrographs.

Phosphorylase kinase, the first protein kinase discovered, is a key regulatory enzyme in glycogen metabolism. Although its biochemical properties are well characterized, details of its three-dimensional structure and subunit topology are yet to be elucidated. This study describes four characteristic views of the hexadecameric holoenzyme (alpha 4 beta 4 gamma 4 delta 4) as observed in both negatively stained and unstained electron micrographs. The predominant views are the widely reported "butterfly" with two wing-like lobes connected by thin bridges, and the previously described "chalice", composed of "cup" and "stem" segments. Two additional views, a "cube", similar to the previously reported "tetrad", and a "cross" or "X" are less common, but illustrate the overall geometry of the particle. Based on these images, the first three-dimensional model of the enzyme has been constructed. It is composed of four identical protomers that associate with D2 symmetry to form the two major structural elements (the two lobes). Two protomers in a head to head arrangement make up each symmetrical lobe; to complete the holoenzyme, one lobe is inverted and placed perpendicular to the other. Thus, the overall structure has three 2-fold axes of symmetry, and the arrangement of the four protomers approximates a tetrahedron. Each lobe of the model corresponds to a wing of the butterfly projection. Two projections form the chalice: in the intra-lobe orientation, one lobe forms the cup and the other forms the stem, and in the inter-lobe view, one-half of each lobe contributes to each segment of the image. The cube and cross projections result from 90 degrees rotations from the butterfly orientation. In the cube, the distal portions of each lobe are projected separately. In the cross, one lobe is crossed over and is above the other. This model both accounts for and predicts all of the observed microscopic images.

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy.

In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy. Scanning tunneling microscopy and atomic force microscopy have been used to view complexes between the activating enzyme phosphorylase kinase and its substrate phosphorylase b. Changes in the size and shape of phosphorylase kinase were observed when it bound phosphorylase b.

Viewing molecules with scanning tunneling microscopy and atomic force microscopy.

Two new microscopic techniques make it possible to obtain images of biologically interesting molecules directly in air, vacuum, or under water. Scanning tunneling microscopy and atomic force microscopy both have the capacity to visualize atoms on the surface of rigid structures and provide details of molecular structure for lipids, proteins, carbohydrates, and nucleic acids. In addition to providing visualizations of individual molecules, these scanning probe techniques allow direct imaging of complexes between molecules or between molecules and higher-order subcellular structures such as membranes and cytoskeletal components. Both microscopes can be operated under a variety of ambient conditions ranging from high vacuum to above atmospheric pressure. Specimens need not be dry; both techniques have been used to image molecules in aqueous media under nearly physiological conditions. It is proposed that as these techniques mature they will allow direct observation of many molecular interactions under physiological conditions or even in vivo while they are occurring within the cell.

Scanning tunneling microscopy of the enzymes of muscle glycogenolysis.

Scanning tunneling microscopy (STM) has been used to examine the structures of the skeletal muscle enzymes phosphorylase and phosphorylase kinase. The interaction of these two proteins represents the last step in the process of signal transduction which results in muscle glycogen being converted into metabolic energy for use in muscle contraction. Phosphorylase b has a molecular weight of 97,000 and the dimer is seen by STM to have dimensions of 11 X 5.7 nm. Phosphorylase b has a tendency to form linear arrays of dimers on the graphite surface used as the support for STM imaging. Phosphorylase kinase is imaged as a butterfly-like object with lateral dimensions of 36 X 27 nm. The molecular thicknesses given by scanning tunneling microscopy for these two non-conducting molecules is significantly less than expected. The height measurement in STM is dependent not only on the surface topology of the object being imaged, but also on the electronic work function of the object compared to that of the graphite surface on which it lies. In addition to the individual proteins, a complex between phosphorylase and phosphorylase kinase has been observed by scanning tunneling microscopy.