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Structural basis for regulation of human calcium-sensing receptor by magnesium ions and an unexpected tryptophan derivative co-agonist

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Science Advances  27 May 2016:
Vol. 2, no. 5, e1600241
DOI: 10.1126/sciadv.1600241
  • Fig. 1 Crystal structure of hCaSR-ECD.

    (A) Monomeric hCaSR-ECD with labeled secondary structural elements. (B) Homodimer of hCaSR-ECD. (C) Structural overlap of hCaSR-ECD with rat mGluR1 (rmGluR1) in the closed conformation (PDB ID: 1EWK).

  • Fig. 2 Structural basis for Mg2+/Ca2+ modulated CaSR activities.

    (A) CaSR-mediated [Ca2+]i responses measured by imaging of single-cell calcium oscillations with Fura-2 using HEK293 cells transfected with CaSR in the presence of various concentrations of [Ca2+]o and [Mg2+]o and fit to the Hill equation. (B) ERK1/2 activities upon stimulation by agonists were detected using Western blot and further quantified using ImageJ. The measurements were plotted against different concentrations of [Ca2+]o or [Mg2+]o and fit to the Hill equation. (C) Identified metal binding sites in the structure of hCaSR-ECD homodimer. Mg2+ and Gd3+ are depicted as hot pink and dark blue spheres, respectively. An anomalous difference map of Gd3+ (σ = 8.0) is shown in purple. W, water molecules. (D to F) Both site 1 (E) and site 3 (D) are on the “acidic patch” at the dimerization interface of subdomain 2 (fig. S7), whereas Mg2+ at site 2 in subdomain 1 (F) is primarily coordinated by the backbone carbonyl oxygen atoms. (G) Single mutations of E228I on the acidic patch significantly reduce CaSR-mediated [Ca2+]i responses in the cell population assay.

  • Fig. 3 Identification and characterization of a tryptophan derivative bound to hCaSR-ECD as a novel high-affinity co-agonist of CaSR.

    (A) Fo-Fc omit map (Fo and Fc are the observed and the calculated structure factor amplitudes, respectively) of TNCA at σ = 4.5. The protein is shown in ribbon mode, and the ligand is shown in stick mode. The residues around TNCA are labeled in the zoomed-in figure. (B) LC-ESI-MS of protein sample (top), buffer (middle), and the standard compound (bottom) in negative-ion mode. The high-resolution isotopic MS spectra of the indicated peaks are shown in the inserted figures. (C and D) A representative oscillation pattern from a single HEK293 cell stimulated with various concentrations of extracellular Ca2+ or Mg2+ in the absence (C) and presence (D) of 0.25 mM TNCA. (E) Frequency distribution of the [Ca2+]i oscillation frequency (peak/min) in HEK293 cells transfected with wild-type CaSR stimulated with metals in the presence (red bar) and absence (black bar) of TNCA. The frequency was recorded at the point when more than 50% single cells started to oscillate. Around 40 cells were analyzed and further plotted as a bar chart. (F and G) TNCA potentiates [Mg2+]o- or [Ca2+]o-evoked [Ca2+]i responses in a population assay in 5001 cells measured by Fura-2 acetoxymethyl (AM) in the absence (black square) or presence of Phe (blue triangular) or TNCA (red closed circle). (H) A maximally active concentration of 0.1 to 0.5 mM TNCA markedly reduces the EC50 for activation of [Ca2+]i signaling by [Mg2+]o in the presence of 0.5 mM [Ca2+]o. Inset: The EC50 changes of [Mg2+]o are shown over a narrow concentration range of TNCA.

  • Fig. 4 Key determinants for the molecular basis of disease-associated mutations and regulation.

    (A) Involvement of loop 1 (yellow) and loop 2 (gold) in dimerization. (B) Working model for activation occurs through a conformational change induced by ligand binding at the hinge region between subdomains 1 and 2, as well as bridging interactions provided by metal ion binding at the acidic patch at the interface between the two subdomain 2 regions of their respective protomers. Mutations at these key determinants in the ECD of CaSR cause human disorders with abnormal [Ca2+]o and [Mg2+]o homeostasis.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/5/e1600241/DC1

    table S1. Crystallographic statistics of hCaSR-ECD and hCaSR-ECD/Gd3+.

    table S2. EC50 of [Mg2+]o for stimulation of [Ca2+]i signaling in the presence of different coactivators.

    table S3. EC50 of [Mg2+]o for stimulation of [Ca2+]i signaling in cell population assay with or without TNCA.

    table S4. EC50 of [Mg2+]o for stimulation of [Ca2+]i signaling in single cell assay with or without TNCA.

    table S5. EC50 of [Mg2+]o-elicited [Ca2+]i responses in cell population assay with coapplication of various concentrations of TNCA.

    fig. S1. Structure-based sequence alignment of CaSRs and mGluRs (by PROMALS3D).

    fig. S2. Size exclusion chromatography of purified hCaSR-ECD.

    fig. S3. Comparison of CaSR and mGluR2 structures.

    fig. S4. CaSR-mediated ERK1/2 activation.

    fig. S5. [Ca2+]i responses of CaSR stimulated by increasing [Mg2+]o.

    fig. S6. Determining Mg2+ binding to hCaSR-ECD.

    fig. S7. Metal binding at the acidic patch.

    fig. S8. Identification of TNCA.

    fig. S9. Determining TNCA binding capability to hCaSR-ECD.

    fig. S10. Replacement of TNCA by phenylalanine.

    fig. S11. Structural comparison of CaSRL binding site with that of mGluR1.

    fig. S12. Disease-related mutations on CaSR ECD.

    fig. S13. Structure of the proposed calcium binding site 1.

    fig. S14. Identification of a bicarbonate anion near the ligand binding site.

    fig. S15. A positively charged pocket for loop 1 association.

    fig. S16. Identification of a potential Mg2+ binding site in the hinge region.

  • Supplementary Materials

    This PDF file includes:

    • table S1. Crystallographic statistics of hCaSR-ECD and hCaSR-ECD/Gd3+.
    • table S2. EC50 of Mg2+o for stimulation of Ca2+i signaling in the presence of different coactivators.
    • table S3. EC50 of Mg2+o for stimulation of Ca2+i signaling in cell population assay with or without TNCA.
    • table S4. EC50 of Mg2+o for stimulation of Ca2+i signaling in single cell assay with or without TNCA.
    • table S5. EC50 of Mg2+o-elicited Ca2+i responses in cell population assay with coapplication of various concentrations of TNCA.
    • fig. S1. Structure-based sequence alignment of CaSRs and mGluRs (by PROMALS3D).
    • fig. S2. Size exclusion chromatography of purified hCaSR-ECD.
    • fig. S3. Comparison of CaSR and mGluR2 structures.
    • fig. S4. CaSR-mediated ERK1/2 activation.
    • fig. S5. Ca2+i responses of CaSR stimulated by increasing Mg2+o.
    • fig. S6. Determining Mg2+ binding to hCaSR-ECD.
    • fig. S7. Metal binding at the acidic patch.
    • fig. S8. Identification of TNCA.
    • fig. S9. Determining TNCA binding capability to hCaSR-ECD.
    • fig. S10. Replacement of TNCA by phenylalanine.
    • fig. S11. Structural comparison of CaSRL binding site with that of mGluR1.
    • fig. S12. Disease-related mutations on CaSR ECD.
    • fig. S13. Structure of the proposed calcium binding site 1.
    • fig. S14. Identification of a bicarbonate anion near the ligand binding site.
    • fig. S15. A positively charged pocket for loop 1 association.
    • fig. S16. Identification of a potential Mg2+ binding site in the hinge region.

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