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Laboratory of Molecular Biophysics
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Phosphorylase kinase (PhK) is a hexadecameric holoenzyme made up of four
different subunits in the arrangement
(
ß
)4
and has a total molecular mass
of 1.3MDa. The , ß and subunits
regulate the activity of the subunit
that comprises a protein kinase domain and a calmodulin binding regulatory
domain. The 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 subunit, an intrinsic calmodulin molecule
(CaM), and through activation by extrinsic calmodulin. The subunit
interacts tightly with the subunit and the
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 chain have been hampered by the insolubility of
this protein, and all structural studies to date have used a truncated version
of the 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 on its own, co-expressing the
subunit with CaM is being explored.
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 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 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 and 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 as the first gene to form a GST fusion and CaM
as the second gene.
The C-terminal domain of the 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 -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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