Tài liệu Báo cáo khoa học: Changes in purine specificity in tandem GAF chimeras from cyanobacterial

Changes in purine specificity in tandem GAF chimeras

from cyanobacterial cyaB1 adenylate cyclase and rat

phosphodiesterase 2

Ju¨rgen U. Linder, Sandra Bruder, Anita Schultz and Joachim E. Schultz

Abteilung Pharmazeutische Biochemie, Fakulta¨t fu¨r Chemie und Pharmazie, Universita¨t Tu¨bingen, Germany

The second messengers cAMP and cGMP mediate

many intracellular functions. Swift signal modulation

requires highly regulated biosynthesis and degradation.

In mammals, the latter is accomplished by a family of

11 phosphodiesterase (PDE) isoforms which possess

similar C-terminal catalytic domains (20–45% identity

[1]). Different regulatory features are imparted by the

N-terminal domains. Five PDE families, 2, 5, 6, 10

and 11, have an N-terminal tandem GAF domain of

about 500 amino acids, i.e. two GAF domains, termed

A and B, are sequentially connected by a short linker.

The acronym GAF is derived from the proteins of

initial identification: mammalian cGMP-binding PDEs,

Anabaena adenylate cyclases (ACs; EC 4.6.1.1), and

Escherichia coli transcription factor FhlA. Meanwhile,

GAF domains have been identified in more than 3000

proteins. They mediate protein dimerization and can

allosterically regulate cognate enzymes [2,3]. The lig￾and for the tandem GAF domains of PDE2, PDE5,

PDE6, and PDE11 is cGMP and that for PDE10 is

cAMP. Usually ligand-binding enhances catalytic

activity [4–6]. Two ACs of the cyanobacterium Ana￾baena sp. PCC 7120, cyaB1 and cyaB2, have N-ter￾minal tandem GAF domains with sequence similarity

Keywords

adenylate cyclase; cAMP; cGMP; cyclic

nucleotide phosphodiesterase; GAF domain

Correspondence

J. E. Schultz, Abteilung Pharmazeutische

Biochemie, Fakulta¨t fu¨r Chemie und

Pharmazie, Universita¨t Tu¨bingen,

Morgenstelle 8, 72076 Tu¨bingen, Germany

Fax: +49 7071 295952

Tel: +49 7071 2974676

E-mail: [email protected]

(Received 7 November 2006, revised 18

December 2006, accepted 12 January 2007)

doi:10.1111/j.1742-4658.2007.05700.x

The C-terminal catalytic domains of the 11 mammalian phosphodiesterase

families (PDEs) are important drug targets. Five of the 11 PDE families

contain less well-characterized N-terminal GAF domains. cGMP is the lig￾and for the GAF domains in PDEs 2, 5, 6 and 11, and cAMP is the ligand

for PDE10. Structurally related tandem GAF domains signalling via cAMP

are present in the cyanobacterial adenylate cyclases cyaB1 and cyaB2.

Because current high-resolution crystal structures of the tandem GAF

domains of PDE2 and cyaB2 do not reveal how cNMP specificity is enco￾ded, we generated chimeras between the tandem GAF domains of cyaB1

and PDE2. Both bind the ligand in the GAF B subdomains. Segmental

replacements in the highly divergent b1–b3 region of the GAF B sub￾domain of cyaB1 by the corresponding PDE2 regions switched signalling

from cAMP to cGMP. Using 10 chimeric constructs, we demonstrated

that, for this switch in purine specificity, only 11% of the sequence of the

cyanobacterial GAF B needs to be replaced by PDE2 sequences. We were

unable, however, to switch the purine specificity of the PDE2 tandem GAF

domain from cGMP to cAMP in reverse constructs, i.e. by replacement of

PDE2 segments with those from the cyaB1 GAF tandem domain. The data

provide a novel view on the structure–function relationships underlying the

purine specificity of cNMP-binding GAF domains and indicate that, as

potential drug targets, they must be characterized structurally and bio￾chemically one by one.

Abbreviations

AC, adenylate cyclase; PDE, phosphodiesterase; mPDE2, mouse PDE2; rPDE2, rat PDE2.

1514 FEBS Journal 274 (2007) 1514–1523 ª 2007 The Authors Journal compilation ª 2007 FEBS