University of Oxford Crest

Laboratory of Molecular Biophysics
Laboratory Journal 2001
Prof. L. N. Johnson

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Atlanta Cook and Ed Lowe

Regulation of Phosphorylase Kinase

Phosphorylase kinase (PhK) is a hexadecameric holoenzyme made up of four different subunits in the arrangement (alphaßgammadelta)4 and has a total molecular mass of 1.3MDa. The alpha, ß and delta subunits regulate the activity of the gamma subunit that comprises a protein kinase domain and a calmodulin binding regulatory domain. The alpha and ß chains (139 and 125 kDa respectively) are targets for cAPK phosphorylation and metabolite binding. The calcium dependency of enzyme activity is conferred by the delta subunit, an intrinsic calmodulin molecule (CaM), and through activation by extrinsic calmodulin. The delta subunit interacts tightly with the gamma subunit and the gammadelta complex can be recovered from the holoenzyme after treatment with detergents. A further CaM binding site has been identified on the ß subunit through sequence prediction and has also been shown to bind tightly to CaM. Electron microscope studies (described in section 11.2 C. Vénien-Bryan) have resulted in a 3D model from image reconstruction methods of the phosphorylase kinase holoenzyme at 22Å resolution. Decoration of the particle with glycogen phosphorylase has further shown the binding site for the substrate. In this section we focus on work designed to elucidate the molecular basis of control by calcium.

Studies on the full-length gamma chain have been hampered by the insolubility of this protein, and all structural studies to date have used a truncated version of the gamma subunit (residues 1-298). This truncation produces a constitutively active kinase by removing the CaM binding sites in the C-terminal part of the chain. As it is not possible to express soluble and correctly folded full length gamma on its own, co-expressing the gamma subunit with CaM is being explored.

gammadelta Co-expression

Initial attempts at coexpression made use of the bacterial dual vector PRO expression system. This system consists of two expression vectors that can be controlled independently by two different promoters. The first vector, PROTet.E, was used to clone and express CaM from an anhydrotetracycline inducible promoter. The full-length gamma gene was cloned into the second vector, PROLar.A, as a number of constructs including a N- terminal HIS-tagged construct and as a GST fusion. The promoter in this vector is IPTG inducible, formed by a Plac/ara fusion. The constructs with the PROLar A vector did not produce detectable levels of gamma expression under a number of different expression conditions.

Although a dual vector system has a number of advantages (independent expression control being one of them), there are also a number of problems with such a system. The main difficulty is that in order to have two compatible vectors present in the same cell it is necessary to have one high copy number plasmid and one low copy number plasmid. This makes it difficult to express the two proteins in a 1:1 ratio because one gene is always present at a much lower copy number than the other. The current approach to co- expressing gamma and delta is to use a polycistronic strategy designed by Jane Endicott and Nicole Schüller to drive soluble coexpression of the Skp1/Skp2 complex. The first gene is cloned into the pGEX vector to form a GST-fusion and then a copy of the second gene is inserted after the stop codon of the GST fusion, complete with an independent ribosome-binding site. Cloning of this construct is currently underway, using gamma as the first gene to form a GST fusion and CaM as the second gene.

Calmodulin interactions with PhK Peptides.

The C-terminal domain of the gamma chain contains two binding sites for CaM. These were identified as 25 residue peptides, known as PHK5 and PHK13 and have binding constants for CaM in the nanomolar range. Spectroscopic analysis of the binding of these two peptides to CaM indicate that the PHK13 peptide is likely to bind to as a ß-hairpin while the PHK5 peptide is more likely to bind as an alpha-helix, as seen in other CaM peptide structures (Trewella et al. 1990).

The PROTet/CaM vector that was created as part of our initial co-expression strategy expresses CaM in large quantities and CaM can be purified using hydrophobic interaction chromatography (Hayashi et al. 1998). At present we have the PHK5 and PHK13 peptides in complex with CaM in crystallisation trials.

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