Structural basis for the interaction of the fluorescence probe 8-anilino-1-naphthalene sulfonate (ANS) with the antibiotic target MurA

Abstract

The extrinsic fluorescence dye 8-anilino-1-naphthalene sulfonate (ANS) is widely used for probing conformational changes in proteins, yet no detailed structure of ANS bound to any protein has been reported so far. ANS has been successfully used to monitor the induced-fit mechanism of MurA [UDPGlcNAc enolpyruvyltransferase (EC )], an essential enzyme for bacterial cell wall biosynthesis. We have solved the crystal structure of the ANS small middle dotMurA complex at 1.7-A resolution. ANS binds at an originally solvent-exposed region near Pro-112 and induces a major restructuring of the loop Pro-112-Pro-121, such that a specific binding site emerges. The fluorescence probe is sandwiched between the strictly conserved residues Arg-91, Pro-112, and Gly-113. Substrate binding to MurA is accompanied by large movements especially of the loop and Arg-91, which explains why ANS is an excellent sensor of conformational changes during catalysis of this pharmaceutically important enzyme.

Figure 1

ANS-dependent fluorescence response in MurA and EPSP synthase. (A) Comparison of the fluorescence spectra of MurA and EPSP synthase at 75 μM ANS. The protein concentration was 3 μM. (B) Fluorescence signals of MurA as a function of the ANS concentration. The dissociation constant of ANS in its reaction with free MurA was determined by fitting the data to y = y_max* [ANS]/(K_d + [ANS]).

Figure 2

Comparison of the overall structures of free and ANS liganded MurA. Stereo representation of the optimally superimposed α-carbon chains of unliganded MurA_type2 (dotted line) (11) and ANS-liganded enzyme (solid line). Figs. 2 and 4 were drawn by using molscript (21).

Figure 3

The ANS binding site in MurA. (Upper) Representation of the F_o−F_c difference density contoured at 3 σ in the region of the ANS binding site. ANS was omitted in the refinement and the calculation of the Fourier synthesis. (Lower) The final 2F_o−F_c electron density contoured at 1 σ. ANS was included in the refinement and the calculation of the Fourier synthesis. Carbon atoms are colored yellow, nitrogen atoms blue, oxygen atoms red, sulfur atoms green. Solvent molecules are shown as red spheres. Thick dashed lines represent hydrogen bonds. To designate hydrophobic interactions, distances between ring centers and closest carbon atoms are given along thin dashed lines. The figure was drawn with bobscript (22).

Figure 4

Conformational changes in the loop around Cys-115. Stereo representations of the loop in unliganded MurA_type2 (11) (A), ANS-liganded MurA (B), and the closed state of MurA (7) (C). Arg-91 (yellow), the ANS molecule (orange), and the main chain of the loop Pro-112-Gly-Gly-Cys-Ala-Ile-Gly-Ala-Arg-Pro-121 together with the side chains of Pro-112, Cys-115, and Pro-121 (magenta) are represented as ball and stick. Noncarbon atoms are color-coded as in Fig. 3. Turquoise spheres and dashed lines designate water molecules and hydrogen bonds, respectively. The surrounding protein is shown as a gray α-carbon trace. The view corresponds to a zoom of Fig. 2. The structures were aligned through residues Asp-231, Asp-305, Arg-371, and Arg-331 of the bottom domain of MurA.