NMR insights into protein allostery

Abstract

Allosterism is one of nature's principal methods for regulating protein function. Allosterism utilizes ligand binding at one site to regulate the function of the protein by modulating the structure and dynamics of a distant binding site. In this review, we first survey solution NMR techniques and how they may be applied to the study of allostery. Subsequently, we describe several examples of application of NMR to protein allostery and highlight the unique insight provided by this experimental technique.

Figure 1

General depiction of the shift of a two-dimensional NMR resonance as the protein is progressively saturated with ligand. The resonance shifts from red (apo) to purple (fully saturated).

Figure 2

Mechanism of intramolecular allostery in the Hsp70 NBD. (A) Mapping of allosteric hot spots onto the structure of the DnaK NBD: Residues with large and significant chemical shift differences are shown in red and yellow, respectively. Cyan and gray indicate significant changes and residues with no data, respectively. (B) Structural model for two way coupling pathway between the nucleotide-binding site and the interdomain linker: superposition of the IIA β-sheet and the crossing α-helices for the ATP- (yellow) and ADP- (green) bound conformations of the NBD. The ATP γ-phosphate and the linker are in red, and residues involved in nucleotide binding are shown as spheres.

Figure 3

Summary of ms motions. (A) Venn-type diagram illustrating the relation between flexible residues from ILV relaxation dispersion experiments and the enzyme complex in which they occur for apo (black), acivicin (green), PRFAR (blue), and ternary (red). Residues common to all four are shown in a larger font size. (B) Residues exhibiting ILV methyl ¹³C¹H₃ MQ dispersion in the PRFAR bound and ternary states are shown mapped onto IGPS. Residues with dispersion in only the PRFAR bound state are shown in blue spheres, while those with dispersion in only the ternary complex are shown in red. Residues with dispersion in both states are shown in magenta.

Figure 4

CAP-S62F-cAMP₂ visits an active, low-populated conformational state. (A) Representative relaxation dispersion data of ¹⁵N backbone amides of CAP-S62F-cAMP₂ DBD residues. (B) Backbone ¹⁵N chemical shift differences of the DBD residues between the apo and cAMP₂-bound WT-CAP mapped on the structure. (C) Conformational exchange dynamics of CAP-S62F-cAMP₂ on the μs – ms timescale as indicated by R_ex, are mapped on the structure. (D) Plot of ¹⁵N static Δδ values (blue), R_ex values (orange), and dynamic Δδ (green).

Figure 5

Backbone dynamics of PKA-C in different ternary complexes. Mapping of (A) fast and (B) slow backbone dynamics show that upon inhibition with PKI_5-24 and with high Mg²⁺, a decrease of picosecond to millisecond dynamics occurs throughout the backbone. For the comparison, the dynamics of PKA-C with the substrate PLN_1-20 is shown.

Figure 6

Measurement of methyl dipolar (D_methyl) coupling values. Top (bottom) panels of each pair are spectra obtained in the absence (presence) of alignment from which the values of J_methyl (J_methyl+D_methyl) are measured.