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

Nadeau, Owen W.; Lane, Laura A.; Xu, Dong; Sage, Jessica; Priddy, Timothy S.; Artigues, Antonio; Villar, María T.; Yang, Qing; Robinson, Carol V.; Zhang, Yang; Carlson, Gerald M.

doi:10.1074/jbc.m112.412874

Cited by 17 publications

(51 citation statements)

References 73 publications

(130 reference statements)

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“…Although the identification of the four β subunits as the four bridges of the PhK complex 16 places it more precisely than the other three subunits, which areas of it are surface-exposed is less understood. Partial proteolysis of the β subunit revealed three exposed loops, one of which was cleaved by all seven proteases and represents the N-terminus from position 1 to 32.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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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 mass spectrometry to identify surface-exposed regions of the intact nonactivated and phospho-activated conformers. In addition to the known interaction of the γ subunit’s C-terminal regulatory domain with the δ subunit (calmodulin), our exposure results indicate that the catalytic core of γ 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 α and β 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 α and β, 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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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“…32 These four bridges are composed entirely of the four β subunits. 33 When the enzyme becomes activated by phosphorylation, the ( αβγδ ) 4 PhK complex as a whole becomes less stable, but the four β subunits in the phospho-activated conformer avidly interact with one another. 34 With the nonactivated enzyme, the cationic activator Ca 2+ causes noteworthy structural changes in the β bridges, 35,36 and other activators of PhK, such as Mg 2+ and ADP, have been shown to cause conformational changes in the β subunits.…”

Section: Resultsmentioning

confidence: 99%

Activation of Phosphorylase Kinase by Physiological Temperature

et al. 2015

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In the six decades since its discovery, phosphorylase kinase (PhK) from rabbit skeletal muscle has usually been studied at 30 °C; in fact, not a single study has examined functions of PhK at a rabbit’s body temperature, which is nearly 10 °C greater. Thus, we have examined aspects of the activity, regulation, and structure of PhK at temperatures between 0 and 40 °C. Between 0 and 30 °C, the activity at pH 6.8 of nonphosphorylated PhK predictably increased; however, between 30 and 40 °C, there was a dramatic jump in its activity, resulting in the nonactivated enzyme having a far greater activity at body temperature than was previously realized. This anomalous change in properties between 30 and 40 °C was observed for multiple functions, and both stimulation (by ADP and phosphorylation) and inhibition (by orthophosphate) were considerably less pronounced at 40 °C than at 30 °C. In general, the allosteric control of PhK’s activity is definitely more subtle at body temperature. Changes in behavior related to activity at 40 °C and its control can be explained by the near disappearance of hysteresis at physiological temperature. In important ways, the picture of PhK that has emerged from six decades of study at temperatures of ≤30 °C does not coincide with that of the enzyme studied at physiological temperature. The probable underlying mechanism for the dramatic increase in PhK’s activity between 30 and 40 °C is an abrupt change in the conformations of the regulatory β and catalytic γ subunits between these two temperatures.

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“…CXMS provides a range of structural information, and the resolution of this information is dependent on how specifically a crosslinking (CX) site can be localized in the context of a protein target, with the identification of crosslinked amino acid side chains providing the highest structural resolution. CX sites may be used to determine the proximity of domains and amino acid side chains in protein monomers or complexes, to identify potential intramolecular or intermolecular protein binding sites, and to provide structural constraints for theoretical protein models (89–91). Many search algorithms and specialized reagents have been developed to enrich and enhance the detection of conjugates and more numerous side products in digests of crosslinked proteins (90,92–94), making this approach readily accessible to any researcher with access to MS and proteomics facilities.…”

Section: Part 3: Crosslinkingmentioning

confidence: 99%

“…In that study, phosphorylation of the complex also perturbed interactions of the subunits in the complex, resulting in preferential interactions among the regulatory β subunits, also detected by crosslinking in a previous study (152). In a parallel study, these investigators combined CXMS, immuno EM, cryoEM, modeling and biochemical data to determine the location of the regulatory β subunits in the PhK complex (89). The topology and location of the subunits in the connecting bridge region of the bilobal complex was determined using top-down MS, and CXMS was used to constrain an atomic model of the β subunit (generated by I-TASSER (153)) to facilitate its docking in the bridges of the cryoEM 3D structure.…”

Section: Part 3: Crosslinkingmentioning

confidence: 99%

Protein Structural Analysis via Mass Spectrometry-Based Proteomics

Artigues

Nadeau

Rimmer

et al. 2016

Modern Proteomics – Sample Preparation, Analysis and Practical Applications

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Modern mass spectrometry (MS) technologies have provided a versatile platform that can be combined with a large number of techniques to analyze protein structure and dynamics. These techniques include the three detailed in this chapter: 1) hydrogen/deuterium exchange (HDX), 2) limited proteolysis, and 3) chemical crosslinking (CX). HDX relies on the change in mass of a protein upon its dilution into deuterated buffer, which results in varied deuterium content within its backbone amides. Structural information on surface exposed, flexible or disordered linker regions of proteins can be achieved through limited proteolysis, using a variety of proteases and only small extents of digestion. CX refers to the covalent coupling of distinct chemical species and has been used to analyze the structure, function and interactions of proteins by identifying crosslinking sites that are formed by small multi-functional reagents, termed crosslinkers. Each of these MS applications is capable of revealing structural information for proteins when used either with or without other typical high resolution techniques, including NMR and X-ray crystallography.

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

Cited by 17 publications

References 73 publications

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

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

Activation of Phosphorylase Kinase by Physiological Temperature

Protein Structural Analysis via Mass Spectrometry-Based Proteomics

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