NMR reveals novel mechanisms of protein activity regulation

Abstract

NMR spectroscopy is one of the most powerful tools for the characterization of biomolecular systems. A unique aspect of NMR is its capacity to provide an integrated insight into both the structure and intrinsic dynamics of biomolecules. In addition, NMR can provide site-resolved information about the conformation entropy of binding, as well as about energetically excited conformational states. Recent advances have enabled the application of NMR for the characterization of supramolecular systems. A summary of mechanisms underpinning protein activity regulation revealed by the application of NMR spectroscopy in a number of biological systems studied in the lab is provided.

Figure 1

Effect of the anticooperative binding of cAMP to CBD of CAP (CAP^N). Binding of the first cAMP changes the structure of the liganded subunit but has no effect on the mean structure of the unliganded subunit. In contrast, the slow motions (s-ms) of the unliganded subunit are stimulated (denoted by the thicker red line of the cAMP-binding site). Binding of the second cAMP suppresses the fast motions (ps-ns) on both subunits (denoted by the blue color). As a result, binding of the second cAMP incurs an unfavorable conformational entropy change, which is the source of the negative cooperativity (from Ref. 31).

Figure 2

Reaction pathways for cAMP-mediated CAP activation and DNA binding. (A) cAMP binding to WT-CAP elicits the active conformation so that DBD becomes structurally poised to interact favorably with DNA. Complex formation is strongly enthalpically favored and entropically unfavorable. (B) cAMP binding to CAP-S62F stabilizes only marginally the active conformation, which is poorly populated (∼2%). Because DNA binds to the active conformation of CAP with many orders of magnitude stronger affinity than to the inactive conformation, DNA will bind selectively to the active, low-populated DBD state and shift the population from the inactive DBD to the active DBD conformation. DNA binding to CAP-S62F-cAMP is entirely driven by entropy, which is dominated by favorable conformational entropy change upon DNA binding (from Ref. 55).

Figure 3

Mechanistic basis for the regulation of Crk activity by a proline switch. Crk adopts predominantly (∼90%) the closed, autoinhibited conformation, but a minor population (∼10%) adopts the open, uninhibited conformation in which the PPII-binding site on SH3^N is accessible for binding by ligands such as the Abl kinase. The open conformation exists as an equilibrium between the cis and the trans isomer, but only the cis version forms the closed conformation. The rates of the cis–trans interconversion are regulated by the action of CypA, which accelerates the interconversion by four orders of magnitude (from Ref. 63).

Figure 4

Signal sequence binding to SecA. SecA consists of several domains: the nucleotide-binding domain (NBD), the PBD, the intramolecular regulator of ATP hydrolysis 2 domain (IRA2), a long a-helical scaffold domain (SD), the IRA1 hairpin, the winged domain (WD), and the C-tail. (A) The lowest-energy structure of SecA bound to the signal peptide is shown. SecA is displayed as a semi-transparent solvent-accessible surface and the signal peptide is shown in yellow. A ribbon model is displayed below the surface. (B) Closer view of the groove bound to the signal peptide. Green and red surface indicates hydrophobic and acidic residues, respectively. Peptide is shown as a ribbon ball-and-stick representation and most of its residues are numbered. (C,–D) The signal sequence binds to a groove formed at the interface between PDB and IRA1. The groove is sterically inhibited by the C-tail and signal sequence binding results in C-tail displacement. SecA exists in an equilibrium between a compact and a more loose conformational state, with the latter being favored by temperature increase and SecEYG binding (from Ref. 101).