PDBsum entry: 2k31

Hydrolase

PDB id: 2k31

Contents

Protein chain

149 a.a. *

Ligands

35G

* Residue conservation analysis

PDB id:

2k31

Name:

Hydrolase

Title:

Solution structure of cgmp-binding gaf domain of phosphodiesterase 5

Structure:

Phosphodiesterase 5a, cgmp-specific. Chain: a. Fragment: cgmp-binding gaf domain (unp residues 154-320). Engineered: yes. Mutation: yes

Source:

Mus musculus. Mouse. Gene: pde5a. Expressed in: escherichia coli.

NMR struc:

20 models

Authors:

C.C.Heikaus,J.R.Stout,M.R.Sekharan,C.M.Eakin,P.Rajagopal, P.S.Brzovic,J.A.Beavo,R.E.Klevit

Key ref:

C.C.Heikaus et al. (2008). Solution structure of the cGMP binding GAF domain from phosphodiesterase 5: insights into nucleotide specificity, dimerization, and cGMP-dependent conformational change. J Biol Chem, 283, 22749-22759. PubMed id: 18534985 DOI: 10.1074/jbc.M801577200

Date:

16-Apr-08 Release date: 03-Jun-08

Protein chain

Q8CG03  (PDE5A_MOUSE) -  cGMP-specific 3',5'-cyclic phosphodiesterase

Seq: 865 a.a.

Struc: 149 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.4.35 - 3',5'-cyclic-GMP phosphodiesterase.
• Reaction: Guanosine 3',5'-cyclic phosphate + H₂O = guanosine 5'-phosphate

Guanosine 3',5'-cyclic phosphate (Bound ligand (Het Group name = 35G) corresponds exactly) + H(2)O = guanosine 5'-phosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.M801577200

J Biol Chem 283:22749-22759 (2008)

PubMed id: 18534985

Solution structure of the cGMP binding GAF domain from phosphodiesterase 5: insights into nucleotide specificity, dimerization, and cGMP-dependent conformational change.

C.C.Heikaus, J.R.Stout, M.R.Sekharan, C.M.Eakin, P.Rajagopal, P.S.Brzovic, J.A.Beavo, R.E.Klevit.

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.

Selected figure(s)

Figure 1.
FIGURE 1. A, domain organization of mouse PDE5A1. Construct borders for GAF A and GAF B are shown. S92 indicates the phosphorylation site. B,[^1H,^15N]HSQC spectrum of cGMP-bound PDE5 GAFA[154-320] recorded in 90% H[2]O/10% D[2]O(black) and 5 days after dissolution of lyophilized GAFA[154-320] in 99.9% D[2]O(red). Approximately 40 unexchanged backbone amide proton resonances were detected. Assignments of selected unexchanged backbone amides are shown. Residue numbers correspond to the full-length mouse PDE5A1. The majority of the non-exchanging amides are located in the anti-parallel β-sheet, with some in helices 3, 4, and 5 (supplemental Fig. S1). C, ribbon diagram of PDE5A GAF A in the presence of cGMP. -Helices are shown in red, β-strands in blue, and loops in gray. cGMP is shown in yellow sticks. Note: The N-terminal -helix observed in the structure is named " 2" with analogy to other GAF domain structures. D, backbone traces of the 20 lowest energy structures of PDE5 GAF A.

Figure 2.
FIGURE 2. A, comparison of cNMP-binding GAF domains from PDE5A, PDE2A, and cyaB2. The structures of PDE5A GAF A, PDE2A GAF B (1mc0), and cyaB2 GAF B (1ykd) were aligned using PyMOL.^4 The r.m.s.d. of the -carbons of the aligned region is 2.26 Å (PDE5-PDE2) and 2.00 Å (PDE5-cyaB2). -Helices are shown in red, β-strands in blue, and loops in gray. B, sequence alignment of mouse PDE5A GAF A, mouse PDE2A GAF B, and cyaB2 GAF B. Identical residues are highlighted in red, strongly similar residues in green, and weakly similar residues in blue. Residues within the cNMP binding pocket are highlighted in yellow. Alignment was performed with ClustalW (40). Asterisks highlight PDE5 residues that were involved in measured intermolecular NOEs. Secondary structure elements from PDE5A are indicated. C, overlay of cGMP and binding residues from the 20 lowest energy structures of PDE5A GAF A after alignment of residues 157-277 and 285-302. D, PDE5A GAF A binding pocket. Binding residues are displayed as sticks with carbon atoms in cyan. Hydrogen bonds are shown as black dashed lines.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 22749-22759) copyright 2008.

Figures were selected by the author.