Cryo-EM density map fitting driven in-silico structure of human soluble guanylate cyclase (hsGC) reveals functional aspects of inter-domain cross talk upon NO binding

Abstract

The human soluble Guanylate Cyclase (hsGC) is a heterodimeric heme-containing enzyme which regulates many important physiological processes. In eukaryotes, hsGC is the only known receptor for nitric oxide (NO) signaling. Improper NO signaling results in various disease conditions such as neurodegeneration, hypertension, stroke and erectile dysfunction. To understand the mechanisms of these diseases, structure determination of the hsGC dimer complex is crucial. However, so far all the attempts for the experimental structure determination of the protein were unsuccessful. The current study explores the possibility to model the quaternary structure of hsGC using a hybrid approach that combines state-of-the-art protein structure prediction tools with cryo-EM experimental data. The resultant 3D model shows close consistency with structural and functional insights extracted from biochemistry experiment data. Overall, the atomic-level complex structure determination of hsGC helps to unveil the inter-domain communication upon NO binding, which should be of important usefulness for elucidating the biological function of this important enzyme and for developing new treatments against the hsGC associated human diseases.

Keywords: Cryo-EM density map fitting; Homology modelling; Multi-domain assembly; Protein-protein docking; Single/multiple-chain threading.

Fig. 1:

Domain architecture of Soluble Guanylate Cyclase (sGC) heterodimer. It consists of two subunits, α and β. Both subunits contains a PAS domain, a coiled-coil domain CC and a C-terminal cyclase (catalytic domain). The N-terminal of the α subunit has pseudo-HNOX domain while β subunit contains a heme-binding domain called HNOX. Length of each subunit and domain boundaries are also mentioned on the upper and lower arrows.

Fig. 2.

Sequence alignments between the hsGC β subunit domains and their homology modeling templates; HNOX (a), PAS (b), coiled-coil (c) and α subunit PAS (d), coiled-coil domain (e) with their respective homologous PDB templates. Red color boxes represent sequence identity whereas conservation bar plots, below sequence alignments, represent percentage identity at a specific residue position.

Fig. 3.

Dimeric domains orientations of αβ PAS (a), αβ coiled-coil (b), αβ cyclase (c) and βHNOX-βPAS (d) predicted through template based single chain threading approach (SPRING & COTH). In dimeric model of αβ PAS (a), αβ coiled-coil (b), αβ cyclase (c), individual domain models of hsGCα subunits are shown in cyan color while domain models of hsGCβ chains are shown in green color The while neighboring domains of same subunit βHNOX-PAS dimeric model, individual models of βHNOX and βPAS are shown in green and cyan respectively. Residues lying at the interface of dimeric domains are labelled and highlighted as sticks (red).

Fig. 4a:

Three domain model of αβ-PAS dimer with the βHNOX domain. 4b: N-terminal four domain cluster (α-pseudo-HNOX- αPAS- βPAS- βHNOX) of hsGCαβ dimer. All α subunit domains are showed in green color and β subunit domains are shown in cyan color. Experimentally known functionally critical hotspots areas on the interface are highlighted in red. Prosthetic group of β HNOX is also rendered in red.

Fig. 5.

SPRING and COTH predicted dimeric models docked into the electron density map of R. norvegicus sGC. Cyan and green color represents the dimeric structures of α and β H-NOX-PAS respectively. While blue and red describes the relative conformation of αβ coiled-coil and αβ cyclase dimers

Fig. 6

a). Flexible cryo-EM map fitting of atomic models inside the cryo-EM density Map with 180 degree rotation are shown (b). The alpha and beta subunits of hsGC are represented by wheat and cyan colors respectively.

Fig. 7.

Homology model of entire hsG heterodimer in complex with NO-HEME moiety at regulatory (βHNOX) domain while GTP and Mg ²⁺ ion at catalytic region (cyclase dimer). In the predicted hsGC heterodimer, α chain is highlighted in green and hsGCβ is shown in cyan color. Salmon color regions throughout the structure illustrated the dimeric interface critical regions such as signaling Helix-F, Loop β, H105, D106, T110 (βHNOX-βPAS), F285, Q368 (α-PAS), F217, Q309 (β-PAS), K363,K365 (α-coiled-coil), K364 (β-coiled-coil), D487,D531 (α-cyclase) and N548,R552 (β-cyclase).