Structure-guided discovery of a single-domain antibody agonist against human apelin receptor

Abstract

Developing antibody agonists targeting the human apelin receptor (APJ) is a promising therapeutic approach for the treatment of chronic heart failure. Here, we report the structure-guided discovery of a single-domain antibody (sdAb) agonist JN241-9, based on the cocrystal structure of APJ with an sdAb antagonist JN241, the first cocrystal structure of a class A G protein-coupled receptor (GPCR) with a functional antibody. As revealed by the structure, JN241 binds to the extracellular side of APJ, makes critical contacts with the second extracellular loop, and inserts the CDR3 into the ligand-binding pocket. We converted JN241 into a full agonist JN241-9 by inserting a tyrosine into the CDR3. Modeling and molecular dynamics simulation shed light on JN241-9-stimulated receptor activation, providing structural insights for finding agonistic antibodies against class A GPCRs.

Fig. 1. Potent APJ competitive sdAb JN241 antagonist and its cocrystal structure with human APJ.

(A and B) Antagonistic activity of JN241 in cAMP (A) and β-arrestin (B) assays. (C) Competition binding of JN241 to APJ by displacement of ¹²⁵I–Apelin 13 in radioligand-binding assay. (D) Side view of APJ (orange) and JN241 (blue) complex, parallel to the cell membrane. Transparent surface shows the close contact between APJ and JN241. (E) The electrostatic potential surfaces of APJ and JN241. The extensive positively charged surface of the CDR2 and negatively charged surface of the ECL2, contributing to the complex formation, are marked up by white lines. RLU, relative luminescence units; CPM, counts per minute.

Fig. 2. The close views of APJ-JN241 complex interfaces.

(A) Side view of APJ (orange) and JN241 (blue) complex. (B to D) Close views of the complex interfaces between APJ and JN241 CDR1 (C), CDR2 (B), and CDR3 (D). The interfaces were stabilized by hydrogen bonds (blue dashed lines) and hydrophobic interactions between several key residues (sticks) (see the main text).

Fig. 3. Structure-guided rational design and experimental validation for the conversion of sdAb JN241 antagonist into agonist.

(A and B) Binding mode of peptide agonist AMG3054 in APJ-AMG3054 cocrystal structure (A) in comparison to that of JN241 CDR3 in APJ-JN241 cocrystal structure (B) at the orthostric site. AMG3054-bound APJ and AMG3054 are colored in cyan and magenta, respectively. JN241-bound APJ and JN241 are colored in light orange and sky blue, respectively. Hydrogen bonds are shown as black dashed lines. Key residues in the C-terminal AMG3054 (Nle15, Pro16, and 4-Cl-Phe17) and in JN241 CDR3 (I103, E104, and S105) contributing to the polar interactions and hydrophobic interactions in APJ orthosteric site are shown in sticks. (C) Surface overlay of the orthosteric site in APJ-JN241 and APJ-AMG3054 complex crystal structures. Three JN241 mutants, JN241-7, JN241-8, and JN241-9 with F, W, and Y insertion, respectively, between E104 and S105 in CDR3 were shown in the embedded box. (D and E) Functional test of JN241-7, JN241-8, and JN241-9 in the absence (D) or presence (E) of Apelin 13 (AP-13) by cAMP assay in comparison to parental sdAb JN241. (F) Functional test of JN241-7, JN241-8, and JN241-9 for agonistic activities by β-arrestin assay in comparison to AP-13. The function assays were repeated three times, and representative data are shown.

Fig. 4. Molecular modeling of WT APJ in complex with JN241 or JN241-9.

(A and B) Comparison of JN241 (A) and JN241-9 (B) in binding mode at the APJ orthosteric site: (A) model of WT APJ (in light orange) in complex with JN241 (in sky blue) that was built on the basis of APJ-JN241 cocrystal structure and (B) model of WT APJ (in light orange) in complex with JN241-9 (in magenta). (C and D) Comparison of conformational changes of WT APJ-JN241 (C) and WT APJ–JN241-9 (D) complex models before and after 240-ns MD simulation: (C) WT APJ-JN241 complex model before (in light orange and sky blue) and after (in cyan and light blue) simulation and (D) WT APJ–JN241-9 complex model before (in light orange and magenta) and after (in cyan and light pink) simulation. The proteins are shown as cartoons. Residues E104 and S105 in JN241 and residues E104, inserted tyrosine [Y], and S105 in JN241-9 are shown as sticks. APJ residues are shown as lines. The movement of Y299^7.43 and TM6 of APJ is labeled with arrows. (E and F) Cartoon illustrations of JN241-mediated APJ antagonism (E) and JN241-9–mediated APJ agonism (F).