VAL60

Collection of 30063 files in PDB-format, each containing all-atom structural data for the conformation of a polyvaline molecule with 60 residues. All conformations where computer-generated by means of meta-dynamics

The Molecular Mechanism of Substrate Engagement and Immunosuppressant Inhibition of Calcineurin

<div><p>Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca²⁺/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, “LxVP.” The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that “LxVP” sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent “LxVP”-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.</p> </div

Potential interaction modes of CN substrates/regulators with CN.

<p>(A) The CN-RII peptide complex obtained by MD. Colors as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio-1001492-g002" target="_blank">Figure 2A, C</a>. CN is shown in surface representation and the RII peptide in dark green with the LxVP motif (LDVP) and phospho-Ser95 as green sticks. LDVP is bound to the LxVP binding pocket (light green), and phospho-Ser95 is bound in the CN active site (cyan). (B) Electrostatic interactions between CN and the RII peptide. The CN electrostatic surface has positively and negatively charged areas colored blue and red, respectively. The LxVP motif and residues in RII that participate in polar interactions with CN are shown as green sticks. (C) Features of selected CN substrates and regulators, including substrates tested in this work (NFAT, Crz1, and the RII peptide). PxIxIT and LxVP motifs are highlighted in yellow and green, respectively, with intervening residues in grey. Regions containing S-T residues that are dephosphorylated by CN are pink. (D) Potential modes of interaction of CN with various binding partners. CN is shown in grey, with the active site in cyan, the PxIxIT docking site in yellow, and the LxVP docking site in green. CN binding partners are shown in blue, with PxIxIT and LxVP motifs in purple and phosphorylated regions shown as red circles. The residues between the two CN docking motifs, or between one docking motif and regions dephosphorylated by CN, are represented as coils, as they are predicted to be unstructured in solution. A238L is the CN-A238L crystal structure.</p

A238L interacts with CN via an LxVP and a PxIxIT motif.

<p>(A) C-terminal residues (200–239) of A238L showing putative docking site FLCVK (aa 228–232). Underlined residues were fused to GST. (B) Recombinant CN was incubated with GST fused to 15 amino acids encoding the LxVP motif of NFATc1 or the FLCVK sequence in A238L. CN co-purifies with both motifs; this interaction is disrupted by incubation with excess peptide LxVPc1 encoding the LxVP motif from NFATc1, but not LxVPmut. CN fails to co-purify with GST fused to mutated FLCVK sequence (FLCVK mutated to AACAA). (C) β-galactosidase activity of extracts from yeast strains that harbor 2xCDRE-lacZ, a CN-dependent reporter gene, and GST or GST-A238L truncations are shown. We added 50 mM CaCl₂ to the cell culture 2.5 h before harvesting to induce CN-dependent activation of the Crz1 transcription factor (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492-Stathopoulos1" target="_blank">[22]</a>; see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492.s007" target="_blank">Text S1</a>). Error bars indicate ± s.d. from three independent experiments. (D) Secondary plot of K<i>_i</i>^app as a function of [RII] for A238L_200–239 inhibition of CN. Data show a linear dependence characteristic of competitive inhibition, with K<i>_i</i> = 0.37 nM. K<i>_i</i>^app values were obtained from the nonlinear fit of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492.s001" target="_blank">Figure S1B</a>. Points represent averages ± s.e.m. (E) Isothermal titration calorimetry confirming that purified A238L_200–239 binds to CN.</p

A238L inhibits CN by interfering with substrate docking.

<p>(A) NFAT-dependent transcription was measured in stimulated HEK293T cells co-transfected with an NFAT-luciferase reporter plasmid and a plasmid expressing SV5-PK-tagged A238L wild-type or motif mutant proteins, or vector alone. Data are means ± s.e.m. from three independent experiments. (B) Transiently transfected SV5-PK-tagged A238L protein expression levels were analyzed by immunoblotting. The increased electrophoretic mobility of A238L_PKIIITmut relative to wt-A238L may be due to changes in a posttranslational modification that is known to affect the electrophoretic mobility of A238L <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492-Tait1" target="_blank">[55]</a>.</p

CN-A238L interactions: The LxVP substrate binding site.

<p>(A) Close-up view of the CN ΦLxVP substrate binding site. (B) Electrostatic surface potential of the A238L-CN complex, highlighting the hydrophobic nature of the ΦLxVP binding groove. (C) CN residues that make up the Leu229 hydrophobic binding pocket (left) and the Val231 hydrophobic binding pocket (right). (D) Electrostatic intermolecular interactions between CN and A238L and intramolecular interactions at the A238L kink, which coordinate A238L for ΦLxVP binding. (E) Secondary plot of <i>K</i>_i^app as a function of [RII] for inhibition of CN by A238L_PKIIITmut, which retains the FLCVK site. Data show a linear dependence characteristic of competitive inhibition, with <i>K</i>_i = 15 nM. Points represent averages ± s.e.m. from three independent experiments.</p

The CN active site is fully accessible in the CN-A238L complex.

<p>(A) Overview of the CN-A238L complex. CNA (gray, surface representation) interacts via helix α14 with CNB (beige, surface representation). The CNA active site is highlighted in cyan, with PxIxIT and LxVP binding pockets shown in yellow and green, respectively. A238L is shown as a cartoon representation (purple), with the PxIxIT (PKIIIT) and LxVP (LCVK) substrate binding motifs highlighted as sticks. (B) Cartoon representation of the CN-A238L complex. Structure is rotated 120° about the <i>x</i>-axis relative to (A); all colors as in (A); blue spheres representing four Ca²⁺ atoms in CNB. CNA β-sheet1 is shown in dark grey. The N-terminal β-strand formed by the A238L PxIxIT motif complements β-strand14 of CNA and thus extends β-sheet1. (C, left) CN-A238L illustrated as in (A), with the distances between the CN active site and the PxIxIT and LxVP binding grooves indicated by arrows (∼32 Å and ∼31 Å, respectively). (C, right) Overlap of the catalytic residues from the CN-A238L complex (black), apo-CN (cyan, PDBID 1AUI), CN-AKAPpeptide (dark blue, PDBID 3LL8), CN-PVIVITpeptide (green, PDBID 2P6B), CN-Cyclosporin (light blue, PDBID 1M63), and CN-FK506 (pink, PDBID 1TCO). Catalytic residues of CNA are shown as sticks and labeled (Asp90, His92, Asp118, Asn150, His199, His281). Fe³⁺ and Zn²⁺ ions from 1AUI are shown as spheres. (D) The rate of pNpp hydrolysis was measured in the presence of A238L (grey), A238L_PKIIITmut (yellow), or A238L_FLCVKmut (green). Assays were performed in triplicate using 10 mM pNpp, and error bars indicate one s.d.</p

Thermodynamic parameters and dissociation (K_D) and inhibition (K_i) constants for CN_{A1–391/B1–170} with A238L_200–239 wild-type, and A238L PxIxIT and LxVP mutants, derived from ITC experiments at 25°C or enzyme assays performed with RII at 37°C.

<p>Thermodynamic and dissociation constant data represent mean values ± one s.d. for triplicate measurements except A238L_WT, which was performed 5 times. Inhibition constants are mean values ± one s.e.m. from three independent experiments.</p