Tài liệu Báo cáo khoa học: Structural characterization of Ca2+/CaM in complex with the phosphorylase

Structural characterization of Ca2+/CaM in complex with

the phosphorylase kinase PhK5 peptide

Atlanta G. Cook*, Louise N. Johnson and James M. McDonnell

Laboratory of Molecular Biophysics, Department of Biochemistry, Oxford University, UK

Phosphorylase kinase (PhK) is a Ca2+-regulated pro￾tein kinase that controls the breakdown of glycogen

through phosphorylation of glycogen phosphorylase

(reviewed in [1]). The enzyme is a large, 1.3-MDa hexa￾decameric complex consisting of four copies of four

subunits, a, b, c and d. The a and b subunits are regu￾latory and are the sites of phosphorylation and meta￾bolite binding and are also regulated by the binding

of extrinsic calmodulin (CaM). The c subunit is the

catalytic subunit and the d subunit is an intrinsic mole￾cule of CaM that binds to the enzyme even in the

absence of Ca2+ [2]. The regulation of PhK through

Ca2+⁄ CaM enables the coordination of muscle contrac￾tion with the production of glucose through the action

of Ca2+ on calmodulin and troponin C [3].

PhK is related to other Ca2+⁄ CaM-dependent protein

kinases including myosin light chain kinase (MLCK),

CaM kinases I, II and IV (CaMKI, CaMKII and

CaMKIV, respectively), CaM kinase kinase (CaMKK),

titin kinase, and death associated kinase [4]. Structural

studies on CaMKI [5], titin kinase [6] and twitchin

kinase [7] have upheld the prediction that many

CaM-dependent protein kinases are regulated through

an autoinhibitory mechanism [8]. In these structures a

C-terminal extension to the protein kinase folds back

on the kinase domain and interferes with the substrate

binding sites. In the case of the titin and twitchin

kinases, the autoinhibitory sequence acts as a pseudo￾substrate, occluding ATP binding and preventing

protein substrates from binding (reviewed in [9]).

PhK shows typical traits associated with such an

autoinhibitory mechanism. The sequence of the PhKc

subunit encodes a C-terminal extension to the protein

kinase domain and treatment of the kinase with

Keywords

calmodulin; kinase regulation; protein–

protein interaction; NMR spectroscopy

Correspondence

J. M. McDonnell, Laboratory of Molecular

Biophysics, Department of Biochemistry,

Oxford University, South Parks Road,

Oxford OX1 3QU, UK

Fax: +44 1865 275182

Tel: +44 1865 275381

E-mail: [email protected]

*Present address

EMBL, Meyerhofstrasse 1, D-69117

Heidelberg, Germany

(Received 7 December 2004, revised 23

January 2005, accepted 1 February 2005)

doi:10.1111/j.1742-4658.2005.04591.x

Phosphorylase kinase (PhK) is a large hexadecameric enzyme consisting of

four copies of four subunits: (abcd)4. An intrinsic calmodulin (CaM, the d

subunit) binds directly to the c protein kinase chain. The interaction site

of CaM on c has been localized to a C-terminal extension of the kinase

domain. Two 25-mer peptides derived from this region, PhK5 and PhK13,

were identified previously as potential CaM-binding sites. Complex forma￾tion between Ca2+⁄ CaM with these two peptides was characterized using

analytical gel filtration and NMR methods. NMR chemical shift perturba￾tion studies showed that while PhK5 forms a robust complex with

Ca2+⁄ CaM, no interactions with PhK13 were observed. 15N relaxation

characteristics of Ca2+⁄ CaM and Ca2+⁄ CaM⁄ PhK5 complexes were

compared with the experimentally determined structures of several

Ca2+⁄ CaM⁄ peptide complexes. Good fits were observed between

Ca2+⁄ CaM⁄PhK5 and three structures: Ca2+⁄ CaM complexes with pep￾tides from endothelial nitric oxide synthase, with smooth muscle myosin

light chain kinase and CaM kinase I. We conclude that the PhK5 site is

likely to have a direct role in Ca2+-regulated control of PhK activity

through the formation of a classical ‘compact’ CaM complex.

Abbreviations

CaM, calmodulin; CaMK, CaM kinase; CaMKK, CaM kinase kinase; eNOS, endothelial nitric oxide synthase; MLCK, myosin light chain

kinase; PhK, phosphorylase kinase; TFA, trifluoracetic acid.

FEBS Journal 272 (2005) 1511–1522 ª 2005 FEBS 1511