Elucidating Subunit Interactions within the Hexadecameric Phosphorylase Kinase Complex through Chemical Crosslinking, Mass Spectrometry and Biophysical Techniques

Presented by Dr. Owen Nadeau, Kansas University Medical Center

Charlotte

Seminar will be held Friday, April 1 at 1:30 PM in Bioinformatics 105.

ABSTRACT:

Phosphorylase kinase (PhK) is a 1.3 MDa (αβγδ)4 complex that regulates glycogenolysis in skeletal muscle. The activity of the catalytic γ subunit in the complex is regulated by allosteric activators that target the regulatory α, β, and δ subunits. The approximate locations of the subunits have been mapped in 3D electron microscopic reconstructions of PhK, showing it to comprise two large lobes that associate with D2 symmetry through interconnecting bridges. Within the lobes, the α, β, γ, and δ subunits have been shown to pack head to head as tetramers to form (αβγδ)2 dimers; however, little else is known about the interactions of these subunits within the context of the PhK complex. Most of the structural information regarding known subunit interactions in the complex has been obtained using bottom-up approaches involving the identification by M.S. analyses of peptide conjugates obtained from digests of chemically crosslinked PhK. To complement our cross-linking approach, we have recently employed top-down M.S. methods designed to study the organization of large protein complexes. We show for the first time mass spectra of intact nonactivated PhK, as well as its phosphorylated counterpart. Aside from the mass differences due to phosphorylation, native and phosphorylated complexes exhibit different dissociation patterns, forming αγδ and γδ subcomplexes with the former and a potential β4 subcomplex with the latter. The first two subcomplexes have been previously observed by crosslinking and other techniques, thus validating this approach. The presence of the previously unobserved β4 subcomplex supports our previous hypothesis that the PhK complex packs as a bridged core of four β subunits upon which four αγδ trimers are arrayed. Our initial results demonstrate that this approach should prove invaluable in further comparing subunit interactions in nonactivated and activated forms of PhK.

Owen Nadeau, Ph.D.
Research Assistant Professor, Department of Biochemistry and Molecular Biology
University of Kansas Medical Center