Probing the Molecular Mechanism of Human Soluble Guanylate Cyclase Activation by NO in vitro and in vivo

Pan, Jie; Yuan, Hong; Zhang, Xiaoxue; Zhang, Huijuan; Xu, Qiming; Zhou, Yajun; Li, Tan; Nagawa, Shingo; Huang, Zhong‐Xian; Tan, Xiangshi

doi:10.1038/srep43112

Cited by 14 publications

(11 citation statements)

References 45 publications

(94 reference statements)

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“…The structural transition of these elements away from the binding pocket suggests a role in the post-catalysis structural dynamics. These findings are compatible with the stability analysis and earlier experimental studies 7,19,22 .…”

Section: Principal Component Analysis (Pca)supporting

confidence: 93%

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Probing the Structural Dynamics of the Catalytic Domain of Human Soluble Guanylate Cyclase

Khalid

Maryam

Sezerman

et al. 2020

Sci Rep

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In the nitric oxide (NO) signaling pathway, human soluble guanylate cyclase (hsGc) synthesizes cyclic guanosine monophosphate (cGMP); responsible for the regulation of cGMP-specific protein kinases (pKGs) and phosphodiesterases (pDes). the crystal structure of the inactive hsGc cyclase dimer is known, but there is still a lack of information regarding the substrate-specific internal motions that are essential for the catalytic mechanism of the hsGC. In the current study, the hsGc cyclase heterodimer complexed with guanosine triphosphate (Gtp) and cGMp was subjected to molecular dynamics simulations, to investigate the conformational dynamics that have functional implications on the catalytic activity of hsGc. Results revealed that in the Gtp-bound complex of the hsGC heterodimer, helix 1 of subunit α (α:h1) moves slightly inwards and comes close to helix 4 of subunit β (β:h4). This conformational change brings loop 2 of subunit β (β:L2) closer to helix 2 of subunit α (α:h2). Likewise, loop 2 of subunit α (α:L2) comes closer to helix 2 of subunit β (β:h2). These structural events stabilize and lock GTP within the closed pocket for cyclization. In the cGMP-bound complex, α:L2 detaches from β:h2 and establishes interactions with β:L2, which results in the loss of global structure compactness. Furthermore, with the release of pyrophosphate, the interaction between α:h1 and β:L2 weakens, abolishing the tight packing of the binding pocket. this study discusses the conformational changes induced by the binding of Gtp and cGMp to the hsGC catalytic domain, valuable in designing new therapeutic strategies for the treatment of cardiovascular diseases. Guanylate cyclase (GC) exists as membrane-bound particulate guanylate cyclase (pGC) and cytoplasmic soluble guanylate cyclase (sGC) in mammalian tissues. Both these forms of the sGC utilize guanosine triphosphate (GTP) as a substrate and metal ions (Mg 2+ , Mn 2+) as cofactors to stimulate the release of inorganic pyrophosphate and promote cyclization at the α-phosphorus atom to form 3′,5′-cyclic guanosine monophosphate (cGMP) 1. Nitric oxide (NO) and hormones modulate the activity of sGC and pGC respectively. It is now established that cGMP binds to various downstream signal transduction effector proteins and ligand-gated ion channels to regulate a multitude of physiological processes and pathological conditions such as cerebellar motor control, cardiac failure, pulmonary hypertension, erectile dysfunction, gut peristalsis and neurodegeneration 2-5. Soluble guanylate cyclase (sGC) is a heterodimeric protein consisting of α and β subunits. In human, four different subunits (α 1 , α 2 , β 1 and β 2) exist which assemble to make α 1 β 1 , α 2 β 1 and α 1 β 2 heterodimer isozymes while α 1 α 1 & β 1 β 1 homodimers also exist. Out of all these isoforms, only two sGC isozyme, α 1 β 1 and α 2 β 1 , are well characterized in human 1,6. α 1 β 1 is an abundantly expressed cytosolic protein and a well-established member in the NO signaling cascade of mammals and insects. In contrast, t...

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Section: Principal Component Analysis (Pca)supporting

confidence: 93%

“…The structural assembly and activation of the soluble guanylate cyclase has been extensively studied by conventional biochemical studies 7,23,24 . Recently a cryo-EM-driven multi-domain quaternary structure of the human cyclase domain has been proposed 1 .…”

Section: Discussionmentioning

confidence: 99%

Probing the Structural Dynamics of the Catalytic Domain of Human Soluble Guanylate Cyclase

Khalid

Maryam

Sezerman

et al. 2020

Sci Rep

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“…Adding GTP slightly reduced FRET efficiency by ~7%, which could indicate opening of the substrate binding pocket. However, none of the in vitro or in vivo fluorescence measurements recorded by Pan et al were correlated to GC-1 activity [98]. Adding NO had no effect on FRET efficiency when using CFP-αGC-1 (N-terminally tagged) and βGC-1-YFP (C-terminally tagged), in agreement with previous studies [97].…”

Section: Protein-protein Interactions Regulating Gc-1 Activitysupporting

confidence: 79%

Structure/function of the soluble guanylyl cyclase catalytic domain

Childers

Garcin

2018

Nitric Oxide

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Soluble guanylyl cyclase (GC-1) is the primary receptor of nitric oxide (NO) in smooth muscle cells and maintains vascular function by inducing vasorelaxation in nearby blood vessels. GC-1 converts guanosine 5′-triphosphate (GTP) into cyclic guanosine 3′,5′-monophosphate (cGMP), which acts as a second messenger to improve blood flow. While much work has been done to characterize this pathway, we lack a mechanistic understanding of how NO binding to the heme domain leads to a large increase in activity at the C-terminal catalytic domain. Recent structural evidence and activity measurements from multiple groups have revealed a low-activity cyclase domain that requires additional GC-1 domains to promote a catalytically-competent conformation. How the catalytic domain structurally transitions into the active conformation requires further characterization. This review focuses on structure/function studies of the GC-1 catalytic domain and recent advances various groups have made in understanding how catalytic activity is regulated including small molecules interactions, Cys-S-NO modifications and potential interactions with the NO-sensor domain and other proteins.

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“…Once the TC motif binds the indicator dye FlAsH (FlAsH-TC-sGC␤), it becomes fluorescent and in turn undergoes quenching when heme binds. Such FlAsH-TC-sGC␤ constructs have been used to study heme content and conformational changes in sGC␤ (25,26).…”

Section: Hsp90 Interaction Is Needed For Heme Insertion Into Apo-sgc␤mentioning

confidence: 99%

Heat shock protein 90 regulates soluble guanylyl cyclase maturation by a dual mechanism

Dai

Schlanger

Haque

et al. 2019

Journal of Biological Chemistry

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Edited by Ruma Banerjee The enzyme soluble guanylyl cyclase (sGC) is a heterodimer composed of an ␣ subunit and a heme-containing ␤ subunit. It participates in signaling by generating cGMP in response to nitric oxide (NO). Heme insertion into the ␤1 subunit of sGC (sGC␤) is critical for function, and heat shock protein 90 (HSP90) associates with heme-free sGC␤ (apo-sGC␤) to drive its heme insertion. Here, we tested the accuracy and relevance of a modeled apo-sGC␤-HSP90 complex by constructing sGC␤ variants predicted to have an impaired interaction with HSP90. Using site-directed mutagenesis, purified recombinant proteins, mammalian cell expression, and fluorescence approaches, we found that (i) three regions in apo-sGC␤ predicted by the model mediate direct complex formation with HSP90 both in vitro and in mammalian cells; (ii) such HSP90 complex formation directly correlates with the extent of heme insertion into apo-sGC␤ and with cyclase activity; and (iii) apo-sGC␤ mutants possessing an HSP90-binding defect instead bind to sGC␣ in cells and form inactive, heme-free sGC heterodimers. Our findings uncover the molecular features of the cellular apo-sGC␤-HSP90 complex and reveal its dual importance in enabling heme insertion while preventing inactive heterodimer formation during sGC maturation.

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Probing the Molecular Mechanism of Human Soluble Guanylate Cyclase Activation by NO in vitro and in vivo

Cited by 14 publications

References 45 publications

Probing the Structural Dynamics of the Catalytic Domain of Human Soluble Guanylate Cyclase

Probing the Structural Dynamics of the Catalytic Domain of Human Soluble Guanylate Cyclase

Structure/function of the soluble guanylyl cyclase catalytic domain

Heat shock protein 90 regulates soluble guanylyl cyclase maturation by a dual mechanism

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