Abstract
Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, β, γ, and δ). The catalytic protein kinase subunit within this complex is γ, 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 β subunit, the predominant subunit responsible for PhK's activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple β subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αβγδ 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.
Highlights
From the ‡Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK; §Department of Chemistry, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK; ¶Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160; ʈDepartment of Chemistry, the Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QZ, UK
The intact complex has proved to be refractory to high resolution x-ray crystallographic or NMR techniques; low resolution structures of the nonactivated and Ca2ϩ-saturated conformers of phosphorylase kinase (PhK) have been deduced through modeling [3] and solved by means of three-dimensional electron microscopic (EM) reconstruction (4 –7), and they show that the complex is a bilobal structure with interconnecting bridges
MS Analyses of the Intact 1.3 MDa Complex of Nonactivated PhK—To compare subunit interactions in the two PhK conformers, we first established conditions for measuring the intact (␣␥␦)4 complexes via MS from nondenaturing solutions [41, 44]. nES MS of native, nonactivated PhK revealed a predominant and well-resolved charge state series centered on a ϩ83 charge state, with an experimental mass of 1,305,393 Ϯ 244 Da, which corresponds to the partially solvated (␣␥␦)4 hexadecameric form of the complex (Fig. 1A)
Summary
Proteins—Nonactivated PhK was purified from the psoas muscle of female New Zealand White rabbits as described elsewhere [43]. Nanoelectrospray MS—Nondenaturing nanoelectrospray (nES) mass spectra were acquired on a Q-ToF 2 mass spectrometer (Micromass/Waters, Milford, MA), modified for high mass detection [42], or on a Qstar XL (MDS Sciex, Applied Biosystems, Carlsbad, CA) using a previously described protocol optimized for the transmission of noncovalent protein complexes [41]. The following experimental parameters were applied in positive ion mode for the analysis of intact phospho-PhK on the Q-time-offlight 2 instrument: capillary voltage, 1.7 kV; sample cone, 194 V; extraction cone, 5 V; collision energy, 200 V; collision cell pressure, 20 mbar; hexapole ion guide pressure, 42 mbar; analyzer pressure, 1.5 ϫ 10Ϫ4 mbar; backing pressure, 1.0 mbar; and ToF pressure, 2.1 ϫ 10Ϫ6 mbar. Subcomplex compositions were determined using the iterative search algorithm SUMMIT [45]
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