Structural Biology and Molecular Biophysics

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

Shristi Pawnikar
Brenda S Magenheimer
Keya Joshi
Ericka Nevarez Munoz
Allan Haldane
Robin L Maser author has email address
Yinglong Miao author has email address

Center for Computational Biology and Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66047
Clinical Laboratory Sciences
The Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160
Department of Pharmacology and Computational Medicine Program, University of North Carolina – Chapel Hill, Chapel Hill, NC 27599
Dept of Physics, and Center for Biophysics and Computational Biology, Temple University, Philadelphia, PA 19122
Departments of Biochemistry and Molecular Biology

https://doi.org/10.7554/eLife.95992.2

Open access
Copyright information

Figures and data

Synthetic peptides derived from the stalk sequence of PC1 can stimulate signaling of stalkless PC1 CTF.
(A) Alignment of CTF stalk sequences from human (h) and mouse (m) PC1. CTF^Δst has a 21-residue deletion from the N-terminal end of the stalk region. Arrow, GPS cleavage site. Non-identical residues shown in bolded blue. (B) Activation of the NFAT-luc reporter by transfected mCTF or mCTF^Δst expression constructs shown relative to empty expression vector (ev) as means (+ standard deviation, SD) of 3 wells/construct from each of 7 independent experiments. (C) Representative Western blot of total cell lysates from one of the experiments in (B), probed with antisera A19 against mouse PC1 C-tail. ns, non-specific. (D) Summary of the total expression levels (means +SD) of CTF^Δst relative to CTF from the experiments in (B). (E) Stalk peptide treatment of ev-or mCTF^Δst-transfected cells. Sequences of stalk-derived peptides p7-p21 are shown. Graph represents the fold NFAT-luc activation for both eV- (gray bars) and CTF^Δst- (blue bars) transfected cells relative to the CTF^Δst control after 24 hr treatment with or without peptide. Results are the means (+SD) of 3 separate experiments, each with 3 wells/condition. *, p < 0.05; ***, p = 0.0001; ****, p < 0.0001. Analysis by 1-way ANOVA with Tukey-Kramer post-test.

(A) Free energy profile of the p9-bound ΔStalk CTF regarding the number of atom contacts between p9 and extracellular domains of CTF and the distance between the CZ atom of R3848 and the CD atom of R4078 in CTF calculated from Pep-GaMD simulations. (B-C) Comparison of HPEPDOCK docking (cyan) and Pep-GaMD refined (magenta) conformations of peptide p9 in 𝞓Stalk CTF. (D) Polar interactions between peptide-protein residues observed in the top-ranked representative conformations of p9. Peptide residues are numbered relative to the N terminus of the stalk with the peptide starting from 1, while residues within Δstalk CTF are numbered according to the standard PC1 residue number. (E) Distance between TOP domain residue R3848 and PL residue E4078 observed in p9-bound ΔStalk CTF.

(A) Free energy profile of the p17-bound ΔStalk CTF regarding the number of atom contacts between p17 and extracellular domains of CTF and the distance between the CZ atom of R3848 and the CD atom of R4078 in CTF calculated from Pep-GaMD simulations. (B-C) Comparison of HPEPDOCK docking (cyan) and Pep-GaMD refined (magenta) conformations of peptide p17 in 𝞓Stalk CTF. Hydrophobic interactions (red dashed lines) between peptide-protein residues observed in the (D) “Bound” and (F) “Intermediate” low-energy conformations of p17-bound ΔStalk CTF. Distance between TOP domain residue R3848 and PL residue E4078 observed in the (E) “Bound” and (G) “Intermediate” low-energy conformations of p17-bound ΔStalk CTF.

(A) Free energy profile of the p21-bound ΔStalk CTF regarding the number of atom contacts between p21 and extracellular domains of CTF and the distance between the CZ atom of R3848 and the CD atom of R4078 in CTF calculated from Pep-GaMD simulations. (B-C) Comparison of HPEPDOCK docking (cyan) and Pep-GaMD refined (magenta) conformations of peptide p21 in 𝞓Stalk CTF. (D) Polar interactions between peptide-protein residues observed in the top-ranked representative conformations of p21. (E) Distance between TOP domain residue R3848 and PL residue E4078 observed in p21-bound ΔStalk CTF.

(A) Potts interaction map based on the PKD1 multiple-sequence-alignment illustrated in Figure S4, showing interactions with the stalk. Gray dots are shown for residue position-pairs with Potts covariation scores above a threshold, colored darker for higher scores, and selected interacting pairs are annotated with the stalk residue (horizontal, numbered from the stalk N-terminus) and other residue (vertical, standard PKD1 numbering) with the PKD1 residue at each position. The secondary structure as a function of position is annotated along the axes. (B) Cartoon showing the subset of PC1 included in the Potts covariation analysis colored as in the secondary structure in panel A, using a structure predicted by AlphaFold. Gray regions were excluded from the Potts model. (C) Residue Covariation scores for selected position-pairs. The scores reflect the percentage excess frequency of the residue-pair relative to the null expected frequency if the MSA columns were uncorrelated, with blue values reflecting excess and red dearth. Only the most common residue types are shown.

Structural dynamic models are presented for binding of novel synthetic, soluble peptide agonists and associated activation of Polycystin-1 through a combination of complementary cellular signaling assays, accelerated molecular simulations and sequence coevolutionary analysis. The p17 peptide derived from the first 17 residues of the protein stalk tethered agonist is shown here.

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