Sandbox Reserved 1127

Function

PDE5 is 5th isoform of the 11 known phosphodiesterases. This enzyme family is responsible for cyclic AMP and cyclic GMP (cGMP) hydrolysis into AMP and GMP. PDE5 specifically catalyzes the hydrolysis of cGMP into 5'GMP. cGMP is an usual second messager in cell signalling pathways, like NO synthesis. By degradating such nucleotide, this phosphodiesterase of class I regulates nucletotic concentration of the cell, through an allosteric regulation system. PDE5 function affects the smooth muscles, blood vessels or penis, uterus and intestines.

Structural highlights

^{[1] [2] [3] [4]}

PDE5 is a homodimeric protein of 875 amino acids long.

This protein is composed of several important domains^[5]:

• • pol-GLY (10-24 amino acids) in Ntermini (structural importance).
  • GAF A (164-314 amino acids)
  • GAF B (346-503 amino acids)
  • Catalytic domain (588-853 amino acids) in Ctermini involved in cGMP hydrolysis.

The full structure of PDE5 has not been crystallized contrary to this of isolated domains of the protein. GAF A and GAF B are homologous domains and allosteric binding sites of cGMP. The of the catalytic domain allows us to see that this domain is mostly constituted by alpha helix and turns.

Catalytic activity

PDE5 is a phosphodiesterase. cGMP could bind the catalytic domain thanks to hydrogen bonds made with Gln775 and Gln817^[6] and coordination bonds made with Zn2+ and Mg2+ cations in the catalytic site. In order to see these interactions, report to Sildenafil in Inhibitors and medical application. cGMP is hydrolyzed in GMP. The catalytic chemical reaction deals with breaking a phosphodiester bound in cGMP between the 3'OP of the cyclic guanoside and the phosphate of cGMP: cGMP + H20 => GMP.

The catalytic domain is able to bind ligands thanks to an (hydrophilic domain). Moreover with this it appears the cGMP binding site in the catalytic domain is more conserved than the the rest of the domain that could be variable. Sildenafil is in yellow.

Some residues could be affected by post traductional modifications:

• phosphorylation : S60, S86, S92, S102, S104, S108, T111, T127, T137, S869.
• acylation : K364.
• ubiquitinylation : K714.

The ubiquitinylation) of and the phosphorylation of are present in the catalytic site and the phosphorylation of S819 is involved in cGMP binding.

Inhibitors and medical application

Inhibitors of PDE5 prevent cGMP from binding the catalytic site by competititve inhibition. They have more affinity for this domain than cGMP. Sildenafil (active substance of Viagra) binds to PDE5 catalytic domain through:

• • made between Gln775 and Gln817 of PDE5 and Sildenafil
  • Mg2+: coordination bond made with in catalytic site
  • Zn2+: coordination bond made with in catalytic site

Allosteric regulation

Positive regulation

PDE5 contains several cGMP binding sites that do not have the same effect on its activity. These allosteric domains are GAF A and GAF B play a role in PDE5 activity. Especially GAF A (see the picture below) binds cGMP, by establishing hydrogen bonds between cGMP and the mainly polar following residues^[7]: Tyr236, Asp242, Ile219, Ser176, Asp296 colored in the picture below (obtained from the modified 2FK3 PDB file^[8]by using pymol).

cGMP binding to GAF A of the PDE5 may trigger an allosteric modification that lead to the seperation of the dimeric catalytic site of the enzyme, so that it frees cGMP access to the catalytic site. This model was proposed considering the homology between PDE5 and PDE2^[9] This cGMP binding to allosteric sites increases the enzyme affinity to cGMP. Plus, the phosphorylation of the Ser92 by the PKG kinase^[10] leads to increase the catalytic activity of the enzyme.

Negative regulation

In large excess of cGMP, cGMP is sequestred by the allosteric sites and can no longer reach the catalytic site^[11]. An other model including both PKG and the myosine phosphatase^[12] shows that PKG realises an inhibitive phosphorylation of PDE5. In this way, the cell can regulate cGMP concentration by a negative feedback.

References

1. ↑ http://www.phosphosite.org/proteinAction?id=1026&showAllSites=true
2. ↑ http://www.rcsb.org/pdb/explore/explore.do?pdbId=1TBF
3. ↑ http://www.uniprot.org/uniprot/O76074#structure
4. ↑ http://www.ebi.ac.uk/interpro/sequencesearch/iprscan5-S20160124-100328-0067-2702087-es
5. ↑ Francis SH, Blount MA, Corbin JD. Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions. Physiol Rev. 2011 Apr;91(2):651-90. doi: 10.1152/physrev.00030.2010. PMID:21527734 doi:http://dx.doi.org/10.1152/physrev.00030.2010
6. ↑ Zoraghi R, Corbin JD, Francis SH. Phosphodiesterase-5 Gln817 is critical for cGMP, vardenafil, or sildenafil affinity: its orientation impacts cGMP but not cAMP affinity. J Biol Chem. 2006 Mar 3;281(9):5553-8. Epub 2006 Jan 5. PMID:16407275 doi:http://dx.doi.org/10.1074/jbc.M510372200
7. ↑ Heikaus CC, Stout JR, Sekharan MR, Eakin CM, Rajagopal P, Brzovic PS, Beavo JA, Klevit RE. Solution structure of the cGMP binding GAF domain from phosphodiesterase 5: insights into nucleotide specificity, dimerization, and cGMP-dependent conformational change. J Biol Chem. 2008 Aug 15;283(33):22749-59. Epub 2008 Jun 4. PMID:18534985 doi:10.1074/jbc.M801577200
8. ↑ http://www.rcsb.org/pdb/explore/explore.do?structureId=2K31
9. ↑ Jager R, Schwede F, Genieser HG, Koesling D, Russwurm M. Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5. Br J Pharmacol. 2010 Dec;161(7):1645-60. doi: 10.1111/j.1476-5381.2010.00977.x. PMID:20698857 doi:http://dx.doi.org/10.1111/j.1476-5381.2010.00977.x
10. ↑ Rybalkin SD, Rybalkina IG, Shimizu-Albergine M, Tang XB, Beavo JA. PDE5 is converted to an activated state upon cGMP binding to the GAF A domain. EMBO J. 2003 Feb 3;22(3):469-78. PMID:12554648 doi:http://dx.doi.org/10.1093/emboj/cdg051
11. ↑ Gopal VK, Francis SH, Corbin JD. Allosteric sites of phosphodiesterase-5 (PDE5). A potential role in negative feedback regulation of cGMP signaling in corpus cavernosum. Eur J Biochem. 2001 Jun;268(11):3304-12. PMID:11389733
12. ↑ Rybalkin SD, Rybalkina IG, Feil R, Hofmann F, Beavo JA. Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. J Biol Chem. 2002 Feb 1;277(5):3310-7. Epub 2001 Nov 26. PMID:11723116 doi:10.1074/jbc.M106562200