Pre-Anchoring of Pin1 to Unphosphorylated c-Myc in a Fuzzy Complex Regulates c-Myc Activity

Abstract

SUMMARY Hierarchic phosphorylation and concomitant Pin1-mediated proline isomerization of the oncoprotein c-Myc controls its cellular stability and activity. However, the molecular basis for Pin1 recognition and catalysis of c-Myc and other multisite, disordered substrates in cell regulation and disease is unclear. By nuclear magnetic resonance, surface plasmon resonance, and molecular modeling, we show that Pin1 subdomains jointly pre-anchor unphosphorylated c-Myc1–88 in the Pin1 interdomain cleft in a disordered, or “fuzzy”, complex at the herein named Myc Box 0 (MB0) conserved region N-terminal to the highly conserved Myc Box I (MBI). Ser62 phosphorylation in MBI intensifies previously transient MBI-Pin1 interactions in c-Myc1–88 binding, and increasingly engages Pin1PPIase and its catalytic region with maintained MB0 interactions. In cellular assays, MB0 mutated c-Myc shows decreased Pin1 interaction, increased protein half-life, but lowered rates of Myc-driven transcription and cell proliferation. We propose that dynamic Pin1 recognition of MB0 contributes to the regulation of c-Myc activity in cells.

On protein structure, function and modularity from an evolutionary perspective

“…axioms). A bound sub-ensemble model of the c-MYC IDPs [134] MoRFs-like [136] MB0 in fuzzy complex [131] with PIN1 [66]. Tryptophan 35 in PIN1 is indicated with red spheres, PIN1 bound PO 4 3.1 Co-evolution, co-adaptation, evolutionary couplings and direct contacts are all gauged as variants of analysis on co-occurrence of amino acid types in columns of multiple sequence alignments.…”

“…[132,108] Furthermore, since also IDPs seem to contain small functional modular elements, it is a tantalizing idea that they also could be supported by the protein prime model. Evidence exists that IDPs contain functional short molecular recognition features (MoRFs) [136] and transiently structured islets that form fuzzy complexes upon interaction [10,134,66]. These phenomenons are similar by two analogies to that of the foldons of the foldon theory [49].…”

“…For an example, experimental data from differential HSQC NMR experiments express that, upon binding of a ligand, the chemical environments of certain residues in both receptor and ligand are being perturbed by the interaction. [66] These perturbations can then be translated into plausible ambiguous interaction constraints between perturbed ligand and receptor residues. One unbiased method of generating such ambiguous constraints are by assuming equal probability of interaction between any one of the perturbed interactants, forming a tuple set of interactions from the Cartesian product between the set of perturbed residues in the ligand and receptor respectively.…”

“…From NMR experiments, HNCO titration ratios can be measured, which reveals patterns of interactions through chemical shift perturbations (CSP) in NMR spectra influenced by factors such as nearness to specific chemical groups (amino acid side chains, surrounding water or other ligands) as well as the local orientation (phi/psi angles) of the protein backbone. [66] By assuming these indirect HNCO perturbations are due to direct interactions, between ligand and receptor in NMR titration experiments, one can transform these CSP into signals of interac-3. METHOD tion that can be used in weighing constraints in simulation.…”

“…The delta-CSP's are transformed into signals by first filtering them, using a standard approach [117], and then rescaling them into appropriate weights [66]. The rescaling is done relative to the strengths of the signals observed in experiment, yielding weights between 1 and 0, i.e.…”