Differential Affinity Cross-linking of Phosphorylase Kinase Conformers by the Geometric Isomers of Phenylenedimaleimide

Abstract

Phosphorylase b kinase (PbK) from skeletal muscle is a highly regulated oligomer consisting of four copies of four distinct subunits (␣␤␥)␦ 4 . The ␥ subunit is catalytic, and the remaining subunits are regulatory. To characterize effector-induced changes in the quaternary structure of the enzyme, we utilized the ortho-, meta, and para-isomers of phenylenedimaleimide (PDM), which in addition to having different geometries, also vary 2.5-fold in their cross-linking spans. Even at concentrations equivalent to the ␣␤␥␦ protomers of PbK, all three isomers caused specific, rapid, and extensive cross-linking of the holoenzyme to form primarily ␣␤ dimers, plus smaller amounts of ␤␥␥ and ␣␥␥ trimers. The formation of these three conjugates was nearly totally inhibited by a 10-fold molar excess over PDM of N-(o-and p-tolyl)succinimide, which are chemically inert structural analogs of PDM. This inhibition suggests that PbK has binding sites for PDM and that PDM acts as an affinity crosslinker in binding to these sites prior to forming crosslinked conjugates. The largest effect on cross-linking in progressing from o-to p-PDM was on the ␣␥␥ trimer, which is preferentially formed by the p-isomer. Activation of the enzyme by either phosphorylation or the allosteric activators ADP and GDP resulted in large increases in the amount of ␣␥␥ formed, small increases in ␤␥␥, and little change in ␣␤. When cross-linked in the presence of the reversibly activating nucleoside diphosphates, PbK remained activated after their removal, indicating that cross-linking had locked it in the active conformation. Our results provide direct evidence for perturbations in the interactions of the catalytic ␥ subunit with the regulatory ␣ and ␤ subunits upon activation of PbK.Through allosteric and covalent modification sites on its three regulatory subunits, phosphorylase b kinase (PbK) 1 integrates neural, hormonal, and metabolic signals to modulate glycogenolytic flux in skeletal muscle (for review, see Refs. 1 and 2). Although the ␣, ␤, and ␦ (calmodulin) regulatory subunits clearly control the activity of the catalytic ␥ subunit, little is known concerning the mechanisms through which they exert this control, including the extent to which their regulatory influence on ␥ is direct versus indirect; this is especially the case for the larger regulatory subunits, ␣ and ␤. Phosphorylation (3) or proteolysis (4) of ␣ causes increased activity of ␥ within the (␣␤␥␦) 4 holoenzyme, but evidence for a direct ␣-␥ interaction that is altered by this activation has not been observed. Likewise, multiple means of activating the holoenzyme cause common conformational changes in the ␤ subunit (5-7); but again, no evidence for alteration of a direct ␤-␥ interaction has been observed. In two previous studies, cross-linking was used successfully to detect changes in the cross-linking patterns of both the ␣ (8) and ␤ (5) subunits upon activation of the holoenzyme; but with the cross-linkers used, the observed changes involved only a second ␣ or ␤ subunit to form homod...

The Structural Effects of Endogenous and Exogenous Ca2+/Calmodulin on Phosphorylase Kinase

“…Enzymes and Proteins-Nonactivated and autophosphorylated PbK used in this study were described in the accompanying report (31). All experiments described herein were repeated a minimum of three times using three different PbK preparations.…”

“…All experiments described herein were repeated a minimum of three times using three different PbK preparations. Phosphorylase b and bovine serum albumin were obtained as described (31), as were the four mAbs and their detection conjugates. Bovine brain CaM was isolated as described previously (32).…”

CrossSearch, a User-friendly Search Engine for Detecting Chemically Cross-linked Peptides in Conjugated Proteins

“…To minimize the number of candidate ions observed in the analysis of large proteins by chemical cross-linking, we have used an alternative approach to the labeling strategies by simply using a minimal stoichiometric excess of cross-linker, which for convenience we term MSEC. The MSEC approach works particularly well with the PhK complex because it binds small organic reagents (a descriptor for most cross-linkers) with high affinity (23)(24)(25) as do many other proteins (for a review, see Ref. 26).…”

“…26). We have shown previously that PhK specifically binds small hydrophobic cross-linkers, such as the geometric isomers of phenylenedimaleimide (25) and GMBS (23), and that non-active structural analogs of those cross-linkers inhibit its cross-linking by the parent compound (25). Using the MSEC approach with GMBS, we demonstrate herein that this reagent cross-links the hexadecameric PhK complex to form sufficient amounts of a ␤␥ dimer for analysis and that the formation of this conjugate occurs with a tolerable amount of monoderivatized products.…”

In Silico characterization of phosphorylase kinase: Evidence for an alternate intronic polyadenylation site in PHKG1

“…When compared to the sequences of the α and β regulatory subunits, the additional 21 residues added to γ v contain a 7 amino acid stretch (PTSPRVL) that is also partially conserved in the β subunit (residues 824-830). Given the fact that another region of β has previously been identified to inhibit the catalytic activity of γ (residues 420-436) [43], and that β makes numerous contacts with γ in the holoenzyme [44], it is reasonable to speculate thatthis portion of γ v may also be inhibitory. Furthermore, the fact that γ has been shown to self-associate between resides 66-110 [37] and form dimers [40,45], suggests a potential inhibitory mechanism through the formation of γ-γ v heterodimers.…”