Characterization of conformational states of the homodimeric enzyme fluoroacetate dehalogenase by 19F-13C two-dimensional NMR

Abstract

Tryptophan plays a critical role in proteins by contributing to stability, allostery, and catalysis. Using fluorine (¹⁹F) nuclear magnetic resonance (NMR), protein conformational dynamics and structure-activity relationships (SARs) can be studied via fluorotryptophan reporters. Tryptophan analogs such as 4-, 5-, 6-, or 7-fluorotryptophan can be routinely incorporated into proteins during heterologous expression by arresting endogenous tryptophan biosynthesis. Building upon the large ¹⁹F chemical shift dispersion associated with 5-fluorotryptophan, we introduce an approach to the incorporation of ¹³C-enriched 5-fluorotryptophan using a direct biosynthetic precursor, 5-fluoroanthranilic acid-(phenyl-¹³C₆). The homodimeric enzyme fluoroacetate dehalogenase (FAcD), a thermophilic alpha/beta hydrolase responsible for the hydrolysis of a C-F bond in fluoroacetate, was expressed and biosynthetically labeled with (phenyl-¹³C₆) 5-fluorotryptophan. The resulting two-dimensional ¹⁹F-¹³C (transverse relaxation optimized spectroscopy) TROSY heteronuclear correlation spectra provide complete resolution of all 9 tryptophan residues in the apo enzyme and FAcD saturated with the substrate analog bromoacetate. The (¹⁹F,¹³C) correlation spectra also reveal a multitude of minor resonances in the apo sample. The role of each tryptophan residue in allosteric communication was validated with computational rigidity transmission allostery analysis, which in this case explores the relative interprotomer communication between all possible tryptophan pairs.

Scheme 1. Synthesis of 5-fluoroanthranilic acid-(phenyl-¹³C₆) from 4-fluoronitrobenzene-¹³C₆.

Fig. 1. Structure of the FAc-bound FAcD dimer (5SWN) illustrating the Trp side chains. Of note, W156, W185, and W142 lie in or around the active site pocket.

Fig. 2. (¹⁹F,¹³C) TROSY HSQC spectra of apo FAcD (red) and BrAc-bound FAcD (blue). (A) TROSY spectrum of apo FAcD at 60 °C. Partial assignments were obtained by mutagenesis. A prominent minor peak, designated as ‘m’, is also shown in the 1D and 2D spectra. (B) TROSY spectra of apo FAcD (red) and BrAc-bound FAcD (blue) at 50 °C. The ¹⁹F 1D NMR spectra at the top were acquired separately and are added as a visual comparison.

Fig. 3. RTA interprotomer heat map showing allosteric coupling between each Trp residue in the empty protomer to the Trp residues in the BrAc-bound protomer. Interprotomer allostery is considerably weaker in the apo structure.

Fig. 4. RTA maps predicting the interprotomer allosteric network resulting from perturbing each Trp residue in the opposite protomer, using the BrAc-bound FAcD crystal structure. Perturbation of W142, W156, and W185 results in similar and intense responses in allosteric transmission in large regions of the opposite protomer, while residues that are not a part of the allosteric network (e.g., W47) show a very weak overall response.