The Role of Tryptophan in π Interactions in Proteins: An Experimental Approach

Abstract

In proteins, the amino acids Phe, Tyr, and especially Trp are frequently involved in π interactions such as π-π, cation-π, and CH-π bonds. These interactions are often crucial for protein structure and protein-ligand binding. A powerful means to study these interactions is progressive fluorination of these aromatic residues to modulate the electrostatic component of the interaction. However, to date no protein expression platform is available to produce milligram amounts of proteins labeled with such fluorinated amino acids. Here, we present a Lactococcus lactis Trp auxotroph-based expression system for efficient incorporation (≥95%) of mono-, di-, tri-, and tetrafluorinated, as well as a methylated Trp analog. As a model protein we have chosen LmrR, a dimeric multidrug transcriptional repressor protein from L. lactis. LmrR binds aromatic drugs, like daunomycin and riboflavin, between Trp96 and Trp96' in the dimer interface. Progressive fluorination of Trp96 decreased the affinity for the drugs 6- to 70-fold, clearly establishing the importance of electrostatic π-π interactions for drug binding. Presteady state kinetic data of the LmrR-drug interaction support the enthalpic nature of the interaction, while high resolution crystal structures of the labeled protein-drug complexes provide for the first time a structural view of the progressive fluorination approach. The L. lactis expression system was also used to study the role of Trp68 in the binding of riboflavin by the membrane-bound riboflavin transport protein RibU from L. lactis. Progressive fluorination of Trp68 revealed a strong electrostatic component that contributed 15-20% to the total riboflavin-RibU binding energy.

Figure 1

Relationship between the number of fluorine atoms in the indole group of W96 in LmrR and the binding energy (ΔG) upon binding of (blue) RBF at 25 °C, (red) Dau at 25 °C, and (black) Dau at 10 °C. The bars represent standard deviations (n = 3).

Figure 2

Binding modes of daunomycin in the crystal structures of wild-type LmrR and the three LmrR W96 fluoro-substituted variants. (A) Wild-type LmrR-Dau complex (PDB entry 3F8F(19)), (B) 5FW-LmrR-Dau (PDB entry 7QZ6, this work), (C) 5,6diFW-LmrR-Dau (PDB entry 7QZ8, this work), (D) 5,6,7triFW-LmrR-Dau (PDB entry 7QZ7, this work). The two chains of the LmrR dimer are colored in cyan and green. Daunomycin is colored in yellow (carbons), red (oxygens), and blue (nitrogens).

Figure 3

Titration of Trp analog labeled RibU W97Y proteins with RBF. The RBF fluorescence was measured at the indicated RBF concentrations in the absence (triangles) or presence (dots) of the W97Y RibU variant (see insets). The difference between these two signals were calculated to build the titration curve. This curve was used to compute the RBF K_d (see Experimental Section in the Supporting Information). Panels from left to right present the results for RibU W97Y, labeled with 5MeW, 5FW, 5,7diFW, and 5,6,7triFW, respectively.

Figure 4

Relationship between the number of fluorine atoms in the indole group of W68 in RibU and the released binding energy (ΔG) upon binding of RBF at 20 °C. The bars represent standard deviations (n = 2–4).