The Calmodulin-binding Domain of the Catalytic γ Subunit of Phosphorylase Kinase Interacts with Its Inhibitory α Subunit

Abstract

Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca 2؉ ions, of the (␣␤␥␦) 4 phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory ␣ and catalytic ␥ subunits. The ␥ subunit is also known to interact with the ␦ subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca 2؉ ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the ␥ (residues 343-386) and ␣ (residues 1060 -1237) subunits interact. The association of this region of ␣ with ␥ was corroborated by the isolation of a crosslinked fragment of ␣ containing residues 1015-1237 from an ␣؊␥ dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of ␥ that we found to interact with ␣ has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156 -17163), we tested the influence of Ca 2؉ on the conformation of the ␣ subunit and found that the region of ␣ that interacts with ␥ was, in fact, perturbed by Ca 2؉ . The results herein support the existence of a Ca 2؉ -sensitive communication network among the ␦, ␥, and ␣ subunits, with the regulatory domain of ␥ being the primary mediator. The similarity of such a Ca 2؉ -dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the ␥ subunit of PhK.Phosphorylase kinase (PhK) 1 , a Ca 2ϩ -dependent enzyme involved in the regulation of glycogenolysis, is among the largest and most complex enzymes known. Structurally, PhK is composed of four copies each of four different subunits, (␣␤␥␦) 4 and has a mass of 1.3 ϫ 10 6 Da (for reviews see Refs. 1-3). Of the four subunits, ␥ is catalytic, whereas the remaining three are regulatory: ␣ and ␤ exert quaternary constraint on the activity of ␥, and ␦ is an intrinsic molecule of calmodulin (CaM). To fully understand how PhK integrates diverse physiological signals to regulate glycogenolytic flux in skeletal muscle, it is first essential to understand how intrasubunit and intersubunit interactions within the hexadecameric holoenzyme change in response to effector ligands, and in so doing, control its catalytic activity. Despite the increased availability of structural information regarding PhK, interactions associated with activation and involving specific regions of individual subunits, in particular the ␣ and ␤ subunits, have largely remained uncharacterized. In this study, we have focused on delineating interacting regions between the large ␣ and catalytic ␥ subunits to advance our understanding of how structural perturbations correlate with activation of this complex holoenzyme.By using chemical cross-linkers as s...

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

“…The first of these, the GH-15 family of glycosyl hydrolases, provided several good template matches that extended from residues 41 to 670. These results differed from two previous reports that predicted only the GH-15 (␣/␣) 6 catalytic core to be present in ␤ (22, 24), extending approximately through its first 480 residues (22). Just C-terminal to the GH-15 catalytic core, Carriere et al (23) predicted a small loop followed by an additional domain (termed B), which extended from residues 492 to 676; however, they were unable to achieve fold recognition for this domain.…”

“…An independent analysis of the primary structure of ␤ using the Pfam database also predicted a GHL fold for this region of the subunit (69). Our predicted domain structures for residues 40 -670 of the ␤ subunit mirrored that of the shorter domain predicted by Carriere et al (23) and are topologically similar to several domains observed in the template glucodextranase crystal structure (61), comprising an (␣/␣) 6 catalytic core connected to the B and C domains by a short loop (61).…”

“…The GH-15 thread coverage of ␤ is indicated in a two-color scheme, with blue and gray representing, respectively, the (␣/␣) 6 -barrel fold for the catalytic domain (A) and a mixed 2°structure corresponding to the B and C domains found in bacterial and archaeal glucoamy- lases and glucodextranases (61). The protein structurally closest to the best model of this region is Anthrobacter globiformus glucodextranase (PDB code 1ULV) (61), with a template modeling score of 0.8462 and root mean square deviation of 1.92 Å, indicating a good topographical match (62).…”

“…The catalytic ␥ subunit comprises an N-terminal catalytic core (residues 1-298) that has a typical protein kinase fold (2, 3) and a C-terminal regulatory domain (␥CRD) that binds both the intrinsic calmodulin (␦) subunit (4,5), as well as the regulatory ␣ subunit (6). The activity of ␥ in the (␣␤␥␦) 4 complex is tightly regulated by the ␣ (138.4 kDa), ␤ (125.2 kDa), and ␦ (16.7 kDa) subunits, which together promote constraining quaternary (4°) interactions on ␥ (44.7 kDa) that are attenuated by signaling molecules from metabolic, hormonal, and neural pathways that target each of the three regulatory subunits (7,8).…”

4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

“…S5, Table S1, and SI Methods). Formaldehyde, which has one of the shortest crosslinking spans (<3 Å) was used to yield specific covalent adducts between neighboring chaperonin subunits both within a single ring and across the rings (30,31). Given the similarity in molecular weight and isoelectric point of the TRiC subunits, and hence of their covalently linked dimers, we separated the cross-linked subunits by 2D-PAGE (Fig.…”