Structural basis for the interaction of the fluorescence probe 8-anilino-1-naphthalene sulfonate (ANS) with the antibiotic target MurA.
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.
Schonbrunn
E
E
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA. eschoenb@eagle.cc.ukans.edu
Eschenburg
S
S
Luger
K
K
Kabsch
W
W
Amrhein
N
N
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001665:Binding Sites; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase