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

Abstract

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...

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…The conformational changes when GTP and cGMP are bound to the CAT domain of human sGC were studied using molecular dynamics simulations, and the results suggest that structural changes in the CAT domain lock and stabilize GTP within a closed pocket for cyclization. On the other hand, binding of cGMP causes a loss of global structure compactness, likely allowing for product release after cGMP is formed during turnover …”

Probing the Structural Basis of Citrus Phytochrome B using Computational Modelling and Molecular Dynamics Simulation Approaches

“…Since the active/holo PSM modules of A. thaliana (PDB ID: 4OUR) and G. max (PDB ID: 6TL4) (Nagano et al, 2020) are available, four different systems, including these two as references and Citrus PSM-PhyB modules in apo and holo states were explored with 100 ns MD simulations using Amber16 (Pearlman et al, 1995). The parameterisation of systems was performed with the ff14SB force field (Maier et al, 2015) and system neutralisation along with addition of missing hydrogen atoms was done by tleap module (Khalid et al, 2020). The systems were immersed in orthogonal boxes with TIP3P-model water molecules and periodic boundary conditions (Mark and Nilsson, 2001).…”

“…Therefore, its particular characteristics and action mechanism can be studied, which is of great interest. Hence, in the current study, the full-length structure of Citrus PhyB in apo/holo states has been explored employing protein-structure modelling along with molecular docking and MD simulation (Tahir Ul Qamar and Khan, 2017; Maryam et al, 2019; Khalid et al, 2020; Maryam et al, 2020). Initially, the three-dimensional (3D) structure of Citrus PhyB was predicted and docking of phytochromobilin ligand was evaluated with its GAF domain.…”

Probing the structural basis of Citrus phytochrome B using computational modelling and molecular dynamics simulation approaches

“…Since the active/holo PSM modules of A. thaliana (PDB ID: 4OUR) and G. max (PDB ID: 6TL4) (Nagano et al, 2020) are available, four different systems, including these two as references and Citrus PSM-PhyB modules in apo and holo states were explored with 100 ns MD simulations using Amber16 (Pearlman et al, 1995). The parameterisation of systems was performed with the ff14SB force field (Maier et al, 2015) and system neutralisation along with addition of missing hydrogen atoms was done by tleap module (Khalid et al, 2020). The systems were immersed in orthogonal boxes with TIP3P-model water molecules and periodic boundary conditions (Mark and Nilsson, 2001).…”

“…Therefore, its particular characteristics and action mechanism can be studied, which is of great interest. Hence, in the current study, the full-length structure of Citrus PhyB in apo/holo states has been explored employing proteinstructure modelling along with molecular docking and MD simulation (Tahir Ul Qamar and Khan, 2017;Maryam et al, 2019;Khalid et al, 2020;Maryam et al, 2020). Initially, the threedimensional (3D) structure of Citrus PhyB was predicted and docking of phytochromobilin ligand was evaluated with its GAF domain.…”