Conformational Recognition of an Intrinsically Disordered Protein

Abstract

There is a growing interest in understanding the properties of intrinsically disordered proteins (IDPs); however, the characterization of these states remains an open challenge. IDPs appear to have functional roles that diverge from those of folded proteins and revolve around their ability to act as hubs for protein-protein interactions. To gain a better understanding of the modes of binding of IDPs, we combined statistical mechanics, calorimetry, and NMR spectroscopy to investigate the recognition and binding of a fragment from the disordered protein Gab2 by the growth factor receptor-bound protein 2 (Grb2), a key interaction for normal cell signaling and cancer development. Structural ensemble refinement by NMR chemical shifts, thermodynamics measurements, and analysis of point mutations indicated that the population of preexisting bound conformations in the free-state ensemble of Gab2 is an essential determinant for recognition and binding by Grb2. A key role was found for transient polyproline II (PPII) structures and extended conformations. Our findings are likely to have very general implications for the biological behavior of IDPs in light of the evidence that a large fraction of these proteins possess a specific propensity to form PPII and to adopt conformations that are more extended than the typical random-coil states.

Molecular determinants of the interaction of EGCG with ordered and disordered proteins

“…We therefore measured the backbone resonances, including 13 Cα, 13 Cβ, 13 CO, 1 Hα, 1 HN, and 13 N of Aβ40 and α‐synuclein in the presence and absence of three molar excess of EGCG (Figure B, Supporting Information Figure S1A), and analyzed the chemical shifts of the resonances that were previously assigned to assess the effects of the interaction with EGCG on the conformational properties of the monomeric states of the two proteins. Backbone chemical shifts of 13 Cα or 13 Cβ in particular are extremely sensitive probes of the secondary structure content of disordered proteins, and their values can be used in methods such as δ2D to obtain accurate information on the nature of the residual structure and of the dynamical properties of the residues in the sequence …”

Structural Ensembles of Membrane-bound α-Synuclein Reveal the Molecular Determinants of Synaptic Vesicle Affinity

“…The structural refinement of this N-terminal region of the protein, denoted as αS 1-30 , has been obtained by using experimental chemical shifts from ssNMR as restraints in ensemble-averaged molecular dynamics simulations3334, employing an established protocol based on four replicas35 that evolve simultaneously starting from random conformations. Samplings were carried out for 1 μs until convergence was observed for four parameters, namely the root mean square deviations (RMSDs) in the C α Cartesian coordinates and in the backbone dihedral angles, the radius of gyration and the solvent accessible surface area (Fig.…”

“…The restrained molecular dynamics simulations were performed by averaging chemical shift restraints over four replicas, as previously described35. The calculations were made using an implementation of the GROMACS package54 that allows the simulations to be restrained using the CamShift program33.…”

Conserved Helix-Flanking Prolines Modulate Intrinsically Disordered Protein:Target Affinity by Altering the Lifetime of the Bound Complex

“…4,8 However, kinetic information is required to justify such mechanistic conclusions. 9,10 Here we determine the mechanism behind the increase in the affinity of p53 for MDM2 upon proline to alanine mutation (PtoA).…”