Binding events of ligands to receptors are the key for an understanding of biological processes. Gaining insight into protein-protein and protein-ligand interactions in solution has recently become possible on an atomic level by new NMR spectroscopic techniques. These experiments identify binding events either by looking at the resonance signals of the ligand or the protein. Ideally, both techniques together deliver a complete picture of ligand binding to a receptor. The approaches discussed in this review allow screening of compound libraries as well as a detailed identification of the groups involved in the binding events. Also, characterization of the binding strength and kinetics is possible, competitive binding as well as allosteric effects can be identified, and it has even been possible to identify ligand binding to intact viruses and membrane-bound proteins.

NMR Spectroscopy Techniques for Screening and Identifying Ligand Binding to Protein Receptors

Physiological processes are mainly controlled by intermolecular recognition mechanisms involving protein–protein and protein–ligand (low molecular weight molecules) interactions. One of the most important tools for probing these interactions is high-field solution nuclear magnetic resonance (NMR) through protein-observed and ligand-observed experiments, where the protein receptor or the organic compounds are selectively detected. NMR binding experiments rely on comparison of NMR parameters of the free and bound states of the molecules. Ligand-observed methods are not limited by the protein molecular size and therefore have great applicability for analysing protein–ligand interactions. The use of these NMR techniques has considerably expanded in recent years, both in chemical biology and in drug discovery. We review here three major ligand-observed NMR methods that depend on the nuclear Overhauser effect—transferred nuclear Overhauser effect spectroscopy, saturation transfer difference spectroscopy and water–ligand interactions observed via gradient spectroscopy experiments—with the aim of reporting recent developments and applications for the characterization of protein–ligand complexes, including affinity measurements and structural determination.

NMR-based analysis of protein–ligand interactions

Erkenntnisse uber Bindungsvorgange zwischen Liganden und Rezeptoren sind der Schlussel zu einem Verstandnis biologischer Prozesse. Seit kurzem ist es moglich, durch neue NMR-spektroskopische Techniken Protein-Protein- und Protein-Ligand-Wechselwirkungen auf atomarer Ebene zu verstehen. Diese Experimente charakterisieren die Bindungsvorgange entweder von der Seite des Liganden oder von der des Proteins. Idealerweise werden beide Techniken gemeinsam angewendet und liefern dann ein komplettes Bild der Ligandenbindung an einen Rezeptor. Die in diesem Aufsatz vorgestellten Ansatze erlauben das Screening von Verbindungsbibliotheken genauso wie eine detaillierte Charakterisierung von Bindungsepitopen und einzelnen funktionellen Gruppen, die in die Bindungsvorgange involviert sind. Die Bestimmung der Bindungsstarke und der Bindungskinetik ist ebenso moglich wie die Untersuchung kompetitiver Bindung, allosterischer Effekte oder der Bindung von Liganden an intakte Viren und membranstandige Proteine.

NMR-Techniken zum Screening und zur Identifizierung der Bindung von Liganden an Proteinrezeptoren

Nowadays, the molecular basis of interaction between low molecular weight compounds and biological macromolecules is thesubject of numerous investigations aimed at the rational design of molecules with specific therapeutic applications. In the last decades, ithas been demonstrated that DNA quadruplexes play a critical role in several biological processes both at telomeric and gene promotinglevels thus providing a great stride in the discovery of ligands able to interact with such a biologically relevant DNA conformation. Sofar, a number of experimental and computational approaches have been successfully employed in order to identify new ligands and tocharacterize their binding to the DNA. The main focus of this review is the description of these methodologies, placing a particular emphasison computational methods, isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR),circular dichroism (CD) and fluorescence spectroscopies.

State-of-the-Art Methodologies for the Discovery and Characterization of DNA G-Quadruplex Binders

Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR·DHF·NADP+ catalytic complex, resolved to 1.26 A. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3−O4 amide function on the pteridine ring. This set of interact...

/pdf/crystal-structure-of-a-type-ii-dihydrofolate-reductase-1wcwpgowj0.pdf

Crystal structure of a type II dihydrofolate reductase catalytic ternary complex.

R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to the increased clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The type II enzymes contain four identical subunits which form a homotetramer containing a single active site pore accessible from either end. Although the crystal structure of the complex of R67 DHFR with folate has been reported [Narayana et al. (1995) Nat. Struct. Biol. 2, 1018], the nature of the ternary complex which must form with substrate and cofactor is unclear. We have performed transferred NOE and interligand NOE (ILOE) studies to analyze the ternary complexes formed from NADP+ and folate in order to probe the structure of the ternary complex. Consistent with previous studies of the binary complex formed from another type II DHFR, the ribonicotinamide bond of NADP+ was found to adopt a syn conformation, while the adenosine moiety adopts an ...

Interligand Overhauser effects in type II dihydrofolate reductase.

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Interligand Overhauser effects in type II dihydrofolate reductase.