Activation of Polycystin-1 Signaling by Binding of Stalk-derived Peptide Agonists

Abstract

Polycystin-1 (PC1) is the membrane protein product of the PKD1 gene whose mutation is responsible for 85% of the cases of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is primarily characterized by the formation of renal cysts and potential kidney failure. PC1 is an atypical G protein-coupled receptor (GPCR) consisting of 11 transmembrane helices and an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal (NTF) and membrane-embedded C-terminal (CTF) fragments. Recently, signaling activation of the PC1 CTF was shown to be regulated by a stalk tethered agonist (TA), a distinct mechanism observed in the adhesion GPCR family. A novel allosteric activation pathway was elucidated for the PC1 CTF through a combination of Gaussian accelerated molecular dynamics (GaMD), mutagenesis and cellular signaling experiments. Here, we show that synthetic, soluble peptides with 7 to 21 residues derived from the stalk TA, in particular, peptides including the first 9 residues (p9), 17 residues (p17) and 21 residues (p21) exhibited the ability to re-activate signaling by a stalkless PC1 CTF mutant in cellular assays. To reveal molecular mechanisms of stalk peptide-mediated signaling activation, we have applied a novel Peptide GaMD (Pep-GaMD) algorithm to elucidate binding conformations of selected stalk peptide agonists p9, p17 and p21 to the stalkless PC1 CTF. The simulations revealed multiple specific binding regions of the stalk peptide agonists to the PC1 protein including an "intermediate" bound yet inactive state. Our Pep-GaMD simulation findings were consistent with the cellular assay experimental data. Binding of peptide agonists to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of the PC1 CTF signaling activation mechanism. Using sequence covariation analysis of PC1 homologs, we further showed that the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. Therefore, structural dynamic insights into the mechanisms of PC1 activation by stalk-derived peptide agonists have enabled an in-depth understanding of PC1 signaling. They will form a foundation for development of PC1 as a therapeutic target for the treatment of ADPKD.