BI81CH22-Marletta ARI 3 May 2012 12:17 Structure and Regulation of Soluble Guanylate Cyclase Emily R. Derbyshire1 and Michael A. Marletta2 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 2Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037; email: [email protected] Annu. Rev. Biochem. 2012. 81:533–59 Keywords First published online as a Review in Advance on nitric oxide, heme, signaling, desensitization, nitrosation February 9, 2012 The Annual Review of Biochemistry is online at Abstract biochem.annualreviews.org Nitric oxide (NO) is an essential signaling molecule in biological sys- This article’s doi: tems. In mammals, the diatomic gas is critical to the cyclic guano- 10.1146/annurev-biochem-050410-100030 sine monophosphate (cGMP) pathway as it functions as the primary Copyright c 2012 by Annual Reviews. activator of soluble guanylate cyclase (sGC). NO is synthesized from All rights reserved L-arginine and oxygen (O2) by the enzyme nitric oxide synthase (NOS). 0066-4154/12/0707-0533$20.00 Annu. Rev. Biochem. 2012.81:533-559. Downloaded from www.annualreviews.org Once produced, NO rapidly diffuses across cell membranes and binds to the heme cofactor of sGC. sGC forms a stable complex with NO Access provided by b-on: Universidade Nova de Lisboa (UNL) on 01/18/15. For personal use only. and carbon monoxide (CO), but not with O2. The binding of NO to sGC leads to significant increases in cGMP levels. The second mes- senger then directly modulates phosphodiesterases (PDEs), ion-gated channels, or cGMP-dependent protein kinases to regulate physiological functions, including vasodilation, platelet aggregation, and neurotrans- mission. Many studies are focused on elucidating the molecular mech- anism of sGC activation and deactivation with a goal of therapeutic intervention in diseases involving the NO/cGMP-signaling pathway. This review summarizes the current understanding of sGC structure and regulation as well as recent developments in NO signaling. 533 BI81CH22-Marletta ARI 3 May 2012 12:17 INTRODUCTION Contents The nitric oxide/cyclic guanosine monophos- INTRODUCTION.................. 534 phate (NO/cGMP) pathway was discovered Enzymes Critical to the Nitric in the 1980s, but chemical modulation of the Oxide/Cyclic Guanosine pathway for the treatment of angina pectoris Monophosphate Pathway. 534 had been unknowingly achieved 100 years ISOLATION OF SOLUBLE earlier. This stimulation of cGMP production GUANYLATE CYCLASE . 535 occurred with the clinical administration of SOLUBLE GUANYLATE organic nitrites (isoamyl nitrite) (1) and organic CYCLASEISOFORMS........... 537 nitrates (glyceryl trinitrate; GTN) (2). These ARCHITECTURE OF SOLUBLE compounds alleviate the pain associated with GUANYLATE CYCLASE . 538 angina by relaxing the vascular smooth muscle, Heme-Nitric Oxide and Oxygen leading to vasodilation. For years, investiga- BindingDomain................ 538 tions were focused on the mechanism of smooth Per/Arnt/Sim and Coiled-Coil muscle relaxation by these molecules, and these Domains....................... 539 efforts led to the discovery that NO is a physio- CatalyticDomain.................. 539 logically relevant signaling molecule. Addition- SOLUBLE GUANYLATE ally, these efforts led to the identification of the CYCLASE HOMOLOGS . 540 enzymes that biosynthesize NO and cGMP. Eukaryotic Atypical Soluble GuanylateCyclases............. 540 Enzymes Critical to the Nitric Prokaryotic Heme-Nitric Oxide Oxide/Cyclic Guanosine and Oxygen Binding Monophosphate Pathway Proteins........................ 541 STRUCTURAL INSIGHTS FROM Early studies showed that both cytosolic and STUDIES ON SOLUBLE particulate fractions of mammalian tissue GUANYLATE CYCLASE AND exhibit guanylate cyclase activity. Within ITSHOMOLOGS................ 542 the insoluble fractions, membrane-bound LIGANDSELECTIVITY............ 545 particulate guanylate cyclases are present, REGULATION OF SOLUBLE which are activated by natriuretic peptides GUANYLATE CYCLASE BY (reviewed in References 3 and 4), whereas the NITRICOXIDE.................. 546 cytosolic fractions contain soluble guanylate Activation and Nitric Oxide cyclases (sGCs), which are activated by NO. Association..................... 546 NO-responsive guanylate cyclase activity is Deactivation and Nitric Oxide also associated with cell membranes in certain Annu. Rev. Biochem. 2012.81:533-559. Downloaded from www.annualreviews.org Dissociation.................... 547 tissues, including skeletal muscle and brain, as Desensitization.................... 548 well as in platelets (5–7). Guanylate cyclases Access provided by b-on: Universidade Nova de Lisboa (UNL) on 01/18/15. For personal use only. MODULATORS OF SOLUBLE are found in most tissues, and the distribution GUANYLATE CYCLASE of these proteins in various cells is isoform ACTIVITY....................... 549 specific. This provides an additional means to Soluble Guanylate Cyclase regulate cGMP-dependent responses because Activators...................... 549 localized pools of the signaling molecule can Soluble Guanylate Cyclase be generated within specific tissues and in Inhibitors. 550 proximity to either soluble or membrane- bound cGMP receptors. Thus, tissues can 534 Derbyshire · Marletta BI81CH22-Marletta ARI 3 May 2012 12:17 regulate cGMP levels by expression of specific phosphodiesterases (PDEs), ion-gated chan- cGMP: cyclic GC isoforms, and the isoforms have a distinct nels, and cGMP-dependent protein kinases guanosine peptide receptor or ligand activator. Addi- in the regulation of several physiological monophosphate tionally, a reciprocal communication between functions, including vasodilation, platelet GTN: glyceryl particulate guanylate cyclase and sGC has been aggregation, and neurotransmission (9–11). trinitrate observed in the regulation of human and mouse In 1998, the Nobel Prize in Physiology or Vasodilation: blood vascular homeostasis (8), and it remains likely Medicine was awarded to Robert F. Furchgott, vessel widening from that communication between these pathways Louis J. Ignarro, and Ferid Murad, in recogni- smooth muscle occurs in several cGMP-dependent processes. tion of their achievements toward the discovery relaxation Generally, in eukaryotic NO signaling, of the NO-signaling pathway. Currently, this sGC: soluble the initial event involves calcium release, pathway is actively studied because drugs mod- guanylate cyclase followed by binding of a calcium/calmodulin ulating NO-dependent processes have the po- NOS: nitric oxide complex to nitric oxide synthase (NOS), which tential to treat several maladies, including car- synthase activates the enzyme. NO is synthesized and diovascular and neurodegenerative diseases, as then diffuses into target cells, where it binds well as various airway diseases. to the heme in sGC (Figure 1). sGC is a histidine-ligated hemoprotein that binds NO and carbon monoxide (CO), but not oxygen ISOLATION OF SOLUBLE GUANYLATE CYCLASE (O2). This binding event leads to a several hundredfold increase in cGMP synthe- Despite many years of research on sGC, an ef- sis. Once formed, cGMP targets include ficient low-cost purification of the protein has Generator cellcell TargetTa cell 2+ α1 β1 Ca /CaM GTP NO L-Arg + O2 FeII Mg2+ sGC cGMP + PPi NOS L-Cit + NO cGK cGMP-gated Annu. Rev. Biochem. 2012.81:533-559. Downloaded from www.annualreviews.org PDE ion channels Access provided by b-on: Universidade Nova de Lisboa (UNL) on 01/18/15. For personal use only. Figure 1 The nitric oxide/cyclic GMP (NO/cGMP)-signaling pathway. A Ca2+/calmodulin (CaM) complex binds nitric oxide synthase (NOS). NOS catalyzes the oxidation of L-arginine (L-Arg) to L-citrulline (L-Cit) and nitric oxide (NO). NO binds to the FeII heme of α1β1 soluble guanylate cyclase (sGC) at a diffusion- controlled rate. This binding event leads to significant increases in cGMP and pyrophosphate (PPi). cGMP then binds to and activates cGMP-dependent protein kinases (cGKs), phosphodiesterases (PDEs) and ion-gated channels. Abbreviations: α1andβ1, soluble guanylate cyclase subunits; CaM, calmodulin. www.annualreviews.org • Structure and Regulation of SGC 535 BI81CH22-Marletta ARI 3 May 2012 12:17 remained elusive, but several methods have sGC. A clear advantage of the Sf9/baculovirus been developed that yield low microgram expression system is that it enables in vitro amounts of homogeneous protein. Initial char- characterization, including the generation GTP: guanosine 5-triphosphate acterization of sGC was carried out with pro- of site-directed mutants. However, both the tein obtained from rat and bovine tissues. By COS-7 and Sf9/baculovirus expression systems the 1980s, studies were being done with pu- facilitated the biochemical characterization of rified sGC from rat lung (12) and liver (13), subunit dimerization, allowed for the gener- as well as from bovine lung (14, 15); these ation of site-directed mutants, and provided studies showed sGC to be a heterodimer. Im- larger quantities of enzyme for in vitro studies. portantly, it was observed that sGC could be The full-length mammalian α1β1het- purified with and without the heme cofac- erodimer has not yet been isolated from a bac- tor, depending on the purification protocol. terial expression system, but several truncations In short, the use of solubilizing agents or am- of sGC have been successfully obtained via ex- monium sulfate precipitation can lead to mis- pression in Escherichia coli. These proteins in-