NMR Analysis of Site-Specific Ligand Binding in Oligomeric Proteins. Dynamic Studies on the Interaction of Riboflavin Synthase with Trifluoromethyl-Substituted Intermediates

Abstract

The binding of small ligands to symmetrical oligomeric proteins may lead to a number of different partially ligated intermediates but should finally yield a symmetrical fully ligated enzyme/ligand complex. In the case of the trimeric protein, riboflavin synthase, some ligands form an unexpected protein/ligand complex, even in the presence of a large excess of ligand. Three different bound forms were observed by 19F NMR spectroscopy, and Scatchard-type analysis suggested binding sites of similar affinities. NOESY analysis of the kinetic network revealed that the three bound states exchange with free ligand, but not with each other, thus suggesting that the trimeric enzyme could be asymmetrical. This information permits appropriate precautions to be taken during X-ray structure analysis of riboflavin synthase, which is in progress. Quantitative analysis of the NOESY spectra yielded different rate constants for the different binding sites. For comparison, the monomeric lumazine protein was investigated as an example of a case with simple two-site exchange. For such systems, all kinetic parameters including kon and the dissociation constant can be determined from the NOESY spectrum. The data show that NMR spectroscopy can produce qualitative and quantitative information in cases of nonequivalent binding sites in oligomeric proteins if isolated NMR signals of the different forms can be observed. The technique is not limited to 19F as reporter nucleus.

Riboflavin Synthase of Escherichia coli

“…6, and 14). The dismutation reaction involves the transfer of a 4-carbon unit between the two identical substrate molecules and requires the simultaneous presence of two 6,7-dimethyl-8-ribityllumazine molecules at the active site of the enzyme (15)(16)(17). This is well in line with the hypothesis of two topologically similar folding domains and with the experimentally observed binding of two substrate molecules per enzyme subunit.…”

“…Protein Perturbation Experiments-In the absence of protein structural data, we decided to perform protein perturbation studies to characterize the interaction of the mutant proteins with a ligand that binds to the catalytic site of riboflavin synthase. The fluorinated Compounds 5 and 6 have been used extensively in ligand binding studies with riboflavin synthase and lumazine synthase of Bacillus subtilis (17,20,28,29). Fluorine substitution favors the covalent hydration of the pteridine ring system to such an extent that the diasteromeric Compounds 5 and 6 are not subject to epimerization.…”

“…Riboflavin synthase of B. subtilis has been shown to bind Compound 5 at a molar ratio of 1:1 per subunit (17,20). Binding of Compound 5 to the enzyme was accompanied by downfield shifts of the 19 F NMR signals of both trifluoromethyl groups.…”

Structure of Lumazine Protein, an Optical Transponder of Luminescent Bacteria

“…Notably, up to 18 residues can be deleted from the C-terminus of the B. subtilis enzyme without loss of activity, which indicates that only the proximal part of the C-terminal α-helix is mandatory for trimerization. 21 By comparison with the trimeric riboflavin synthase, the most important factor for the monomeric state of lumazine protein may be the absence of the C-terminal α-helix. Moreover, nine residues in the intramolecular domain linker are not visible in the lumazine protein electron density (residue Lys88 to Glu96), probably due to their mobility, whereas this segment is clearly visible even in the monomeric riboflavin synthase structure of S. pombe.…”

“…21,22 Recently, the single binding site was localized to the N-terminal domain using site-specific mutagenesis in conjunction with 13 C NMR analysis. 18 Here, we report for the first time the 3-D structure of lumazine protein of P. leiognathi solved by X-ray crystallography.…”