Structural insights into single-pass transmembrane receptor GC-A activation by distinct antihypertensive antibodies

Figures

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  Fig. 1. Stimulation of GC-A by the two antibodies XX16 and REGN5308 in cellular signaling assays. a Domain organization of GC-A and the cellular signaling events. The receptor comprises an N-terminal extracellular domain (ECD), a juxtamembrane linker (JL), a single-pass transmembrane helix (TM), and an intracellular domain (ICD) that includes a protein kinase-homology domain (KHD), an amphipathic α-helical region (forming a parallel coiled-coil (CC) in a receptor dimer), and a C-terminal guanylyl cyclase catalytic domain (GCD). b Cellular cGMP assays for the activation of full-length human GC-A expressed in HEK293 cells by the two antibodies without or with sub-saturating concentrations (40 or 75 pM) of ANP. Data are represented as mean ± SD of three experiments.

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  Fig. 2. Structures of XX16-bound GC-A without and with ANP. a, b cartoon representation of GC-A-XX16 in the absence (a) or presence (b) of ANP. c, d The front (c) and side (d) views of two complexes overlayed. e The key amino acid interactions between the CDRs from XX16 heavy chain and the top region of ECD dimer of GC-A. f Comparison of the active GC-A–XX16 complex conformation with the inactive state of GC-A.

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  Fig. 3. Structures of REGN5308-bound GC-A without and with ANP. a–c Cartoon representation of GC-A–REGN5308 in the absence (a) or presence (b, c) of ANP. d The key amino acid interactions between the CDRs from REGN5308 and the top region of a ECD monomer of GC-A. e and g Comparisons of the active GC-A–REGN5308 structures with the inactive state of GC-A. f and h Top views of GC-A–REGN5308 in the absence (f) or presence (h) of ANP reveal the mechanism of steric clash across the symmetry axis of ECDs.

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  Fig. 4. Simulations of ANP dissociation from XX16 or REGN5308-bound GC-A complexes. Ligand Gaussian accelerated Molecular Dynamics (LiGaMD3) was applied to simulate ANP dissociation from GC-A in the absence and presence of activating antibodies. Three independent 800 ns LiGaMD3 simulations were carried out for the systems without antibody (a), REGN5308-bound (b), and XX16-bound (c). Representative intermediate conformational states of ANP during dissociation were obtained for the control (no antibody) (d), REGN5308-bound (e), and XX16-bound (f) GC-A + ANP systems.

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  Fig. 5. GC-A activation by an antibody agonist: the compaction-twist model. Left: A representative conformation of apo GC-A (the top #1 pose in the GaMD simulations). Right: A representative conformation of XX16-bound GC-A (the top #1 pose in the GaMD simulations). Monomer A in XX16-bound GC-A is selected to align with the entire complex of apo GC-A in PyMol. Arrows indicate the movements of monomer B from apo GC-A to XX16-bound GC-A.

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  Fig. 6. Lowering blood pressure by XX16 in mice. a C57BL/6 male mice at 6–7 weeks of age were fed with HFD (60% fat; #D12492i, Research diet) for 6 months, followed by radiotelemetry implantation for BP measurements (Created with BioRender.com). Representative graphs showing changes in (b) Systolic blood pressure (SBP) in HFD mice after the administration of the Fab fragment of XX16. BP was measured for 30 min (basal) before XX16 administration and for the following 180 min. c SBP, d diastolic BP (DBP), and e mean BP (MBP) represent the averages of 3 h consecutive recording measured by radiotelemetry. Data were collected from wild-type (WT) mice fed a high-fat diet (HFD) for 6 months (n = 3), both before and after drug administration. Data are presented as mean ± SEM. Statistical analysis was performed with paired two-sided t-test (Prism 10) (* c: p = 0.0339; d: p = 0.0349; e p = 0.0371). Source data are provided as a Source Data file.