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Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex.

ABSTRACT: The four integral delta subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca(2+) sensitivity to the enzyme, but bind independently of Ca(2+). In addition to binding Ca(2+), an obligatory activator of PhK's phosphoryltransferase activity, the delta subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the delta subunits can be exchanged for exogenous CaM. In this study, a CaM double-mutant derivatized with a fluorescent donor-acceptor pair (CaM-DA) was exchanged for delta to assess the conformational substates of PhKdelta by single molecule fluorescence resonance energy transfer (FRET) +/-Ca(2+). The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca(2+), as observed by alterations of the compact, mid-length, and extended populations of their FRET distance distributions. Specifically, the combined predominant mid-length and less common compact conformations of PhKdelta became less abundant in the presence of Ca(2+), with the delta subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaM's Ca(2+)-dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, +/-Ca(2+), as well as from exogenous CaM bound to the PhK complex as delta'. The distinction between delta and delta' is that the latter binds only in the presence of Ca(2+), but stoichiometrically and at a different location in the complex than delta.

SUBMITTER: Priddy TS

PROVIDER: S-EPMC2206654 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex.

Priddy Timothy S TS Price E Shane ES Johnson Carey K CK Carlson Gerald M GM

Protein science : a publication of the Protein Society 20070601 6

The four integral delta subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca(2+) sensitivity to the enzyme, but bind independently of Ca(2+). In addition to binding Ca(2+), an obligatory activator of PhK's phosphoryltransferase activity, the delta subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the delta subunits can be exchanged for exogenous CaM. In ...[more]

PMID: 17525461

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Project description:Previous studies have demonstrated a calcium-dependent interaction of calmodulin (CaM) and Fas that is regulated during Fas-induced apoptosis in several cell lines, including cholangiocarcinoma, Jurkat cells, and osteoclasts. The binding of CaM and Fas has been identified on residues 231-254 of Fas; the V254N point mutation decreases the CaM/Fas binding, and the C-terminal deletion mutation increases the CaM/Fas binding. Recent studies have shown that CaM is recruited into the Fas-mediated death-inducing signaling complex (DISC) in a calcium-dependent manner. However, the molecular mechanisms whereby Fas mutations and CaM/Fas binding might regulate Fas-mediated DISC formation are unknown. In this study we investigated the binding thermodynamics and conformation of the CaM/Fas complexes with combined explicit solvent molecular-dynamics simulations and implicit solvent binding free-energy calculations. The binding free-energy analysis demonstrated that the Fas V254N point mutation reduced its binding affinity with CaM. In contrast, the Fas mutant with the deletion of the 15 amino acid at the C-terminus increased its binding to CaM. These observations are consistent with previous findings from biochemical studies. Conformational analyses further showed that the Fas V254N mutation resulted in an unstable conformation, whereas the C-terminal deletion mutation stabilized the Fas conformation, and both mutations resulted in changes of the degree of correlation between the motions of the residues in Fas. Analysis of the CaM/Fas complex revealed that CaM/Fas binding stabilized the conformation of both CaM and Fas and changed the degree of correlated motion of the residues of CaM and Fas. The results presented here provide structural evidence for the roles of Fas mutations and CaM/Fas binding in Fas-induced DISC formation. Understanding the molecular mechanisms of CaM/Fas binding in Fas-mediated DISC formation should provide important insights into the function of Fas mutations and CaM in regulating Fas-mediated apoptosis.

Dataset Information

Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex.

Publications

Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex.

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