Solution Structure of the cGMP Binding GAF Domain from Phosphodiesterase 5: INSIGHTS INTO NUCLEOTIDE SPECIFICITY, DIMERIZATION, AND cGMP-DEPENDENT CONFORMATIONAL CHANGE

Clemens C Heikaus,Joseph R Stout,Monica R Sekharan,Catherine M Eakin,Ponni Rajagopal,Peter S Brzovic,Joseph A Beavo,Rachel E Klevit

doi:10.1074/jbc.m801577200

Abstract

Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded beta-sheet and four alpha-helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of approximately 20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.

Highlights

Intracellular concentrations of the second messengers cAMP and cGMP are tightly regulated by the rate of synthesis through cyclases and hydrolysis through cyclic nucleotide phosphodies
There is no experimental evidence that Phosphodiesterase 5 (PDE5) GAF B binds cGMP
Detailed structural information of the involved elements at high resolution is necessary to understand the complexity of the allosteric regulation and the corresponding conformational changes

Summary

Introduction

Intracellular concentrations of the second messengers cAMP and cGMP are tightly regulated by the rate of synthesis through cyclases and hydrolysis through cyclic nucleotide phosphodies-. All mammalian PDE families are dimeric and contain a homologous, but distinct C-terminal catalytic domain, whereas they differ more in their family-specific N-terminal regulatory domains. Though homologous in sequence and topologically similar, not all PDE GAF domains bind cyclic nucleotides and may instead be involved in dimerization or play other unknown regulatory roles. The question of how events that involve the regulatory N-terminal region of PDE5 regulate the catalytic domain and its activity remains to be answered. Structural information on the PDE5 holoenzyme is limited to electron microscopy images at 28 Å resolution [23] These suggest an extended dimeric structure with distinct domains, in which the subunits are aligned in a head-to-head fashion. No structure of an unliganded cyclic nucleotide binding GAF domain has been reported, and little is known about the cyclic nucleotide-dependent conformational change and mechanism of binding

Methods

Results

Conclusion