Dataset Information

Mass Spectrometric Analysis of Surface-Exposed Regions in the Hexadecameric Phosphorylase Kinase Complex.

ABSTRACT: Phosphorylase kinase (PhK) is a 1.3 MDa (????)4 enzyme complex, in which ???? protomers associate in D2 symmetry to form two large octameric lobes that are interconnected by four bridges. The approximate locations of the subunits have been mapped in low-resolution cryo-electron microscopy structures of the complex; however, the disposition of the subunits within the complex remains largely unknown. We have used partial proteolysis and chemical footprinting in combination with high-resolution mass spectrometry to identify surface-exposed regions of the intact nonactivated and phospho-activated conformers. In addition to the known interaction of the ? subunit's C-terminal regulatory domain with the ? subunit (calmodulin), our exposure results indicate that the catalytic core of ? may also anchor to the PhK complex at the bottom backside of its C-terminal lobe facing away from the active site cleft. Exposed loops on the ? and ? regulatory subunits within the complex occur at regions overlapping with tissue-specific alternative RNA splice sites and regulatory phosphorylatable domains. Their phosphorylation alters the surface exposure of ? and ?, corroborating previous biophysical and biochemical studies that detected phosphorylation-dependent conformational changes in these subunits; however, for the first time, specific affected regions have been identified.

SUBMITTER: Rimmer MA

PROVIDER: S-EPMC5015440 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Mass Spectrometric Analysis of Surface-Exposed Regions in the Hexadecameric Phosphorylase Kinase Complex.

Rimmer Mary Ashley MA Artigues Antonio A Nadeau Owen W OW Villar Maria T MT Vasquez-Montes Victor V Carlson Gerald M GM

Biochemistry 20151113 46

Phosphorylase kinase (PhK) is a 1.3 MDa (αβγδ)4 enzyme complex, in which αβγδ protomers associate in D2 symmetry to form two large octameric lobes that are interconnected by four bridges. The approximate locations of the subunits have been mapped in low-resolution cryo-electron microscopy structures of the complex; however, the disposition of the subunits within the complex remains largely unknown. We have used partial proteolysis and chemical footprinting in combination with high-resolution mas ...[more]

PMID: 26551836

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Project description:Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.

Project description:Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (?, ?, ?, and ?). The catalytic protein kinase subunit within this complex is ?, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the ? subunit, the predominant subunit responsible for PhK's activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple ? subunits, arguing for their self-association within the activated complex. Evidence for the theoretical ???? protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall stability of the complex, with the phospho-activated conformer being more labile, in concordance with previous hypotheses on the mechanism of allosteric activation of PhK through perturbation of its inhibitory quaternary structure.

Dataset Information

Mass Spectrometric Analysis of Surface-Exposed Regions in the Hexadecameric Phosphorylase Kinase Complex.

Publications

Mass Spectrometric Analysis of Surface-Exposed Regions in the Hexadecameric Phosphorylase Kinase Complex.

Similar Datasets

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