Ligand-Induced Conformational Change of Plasmodium falciparum AMA1 Detected Using ¹⁹F NMR

Abstract

We established an efficient means of probing ligand-induced conformational change in the malaria drug target AMA1 using 19F NMR. AMA1 was labeled with 5-fluorotryptophan (5F-Trp), and the resulting 5F-Trp resonances were assigned by mutagenesis of the native Trp residues. By introducing additional Trp residues at strategic sites within a ligand-responsive loop, we detected distinct conformational consequences when various peptide and small-molecule ligands bound AMA1. Our results demonstrate an increase in flexibility in this loop caused by the native ligand, as inferred from, but not directly observed in, crystal structures. In addition, we found evidence for long-range allosteric changes in AMA1 that are not observed crystallographically. This method will be valuable in ongoing efforts to identify and characterize therapeutically relevant inhibitors of protein-protein interactions involving AMA1 and is generalizable to the study of ligand-induced conformational change in a wide range of other drug targets.

Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules

“…We and others have also described PrOF NMR experiments yielding both slow and fast exchange binding kinetics, demonstrating the ability to detect molecules with dissociation constants in the low nanomolar up to millimolar range. 13,14,16,17,27 Finally, binding site position can be estimated by which resonance is perturbed. In most cases, the W81 resonance of Brd4 located in the WPF shelf is the most dramatically perturbed by a binding interaction in the histone recognition pocket, while W120 on the other side of the protein remains unperturbed.…”

Identification of the Binding Site of Apical Membrane Antigen 1 (AMA1) Inhibitors Using a Paramagnetic Probe

“…[20] Moreover,c omputational dockingw ithout experimental support does not provide reliable binding information because of the dynamic nature of AMA1 and the plasticity of the protein-protein interaction surface. [21] As ar esult, the process of SAR-guided lead optimisation of these fragments hasbeen hindered.…”

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen

“…21,28–31 PrOF NMR utilizes 19 F NMR spectra of fluorine-labeled proteins, in this case 5-fluorotryptophan (5FW) labeled Brd4(1) and BrdT(1). Because of the high environmental sensitivity of the 19 F protein resonances, perturbations to the 19 F NMR spectrum of the protein, such as resonance broadening or shifting in the presence of ligand, correlate to ligand binding.…”