Location of the sugar-binding site of L-arabinose-binding protein. Sugar derivative syntheses, sugar binding specificity, and difference Fourier analyses.
Abstract
The sugar-binding site of the L-arabinose-binding protein, an essential component of the high affinity L-arabinose uptake system in Escherchia coli, is located deep in a cleft formed by the asymmetric contributions from both of the two similar domains. The site was unambiguously identified with the electron-rich substrate analog 6-bromo-6-deoxy-D-galactose in a difference Fourier analysis. The observation that the original native structure might have been solved with bound L-arabinose necessitated the synthesis of a heavy atom analog, its structure consistent with the known sugar-binding specificity of the protein. Difference Fourier maps (3.5 A) of crystals soaked in 46 mM analog showed a peak 3.5 times background, which is attributed to the -CH2Br moiety of the analog. Superposition of a difference map onto a 2.8-A native electron density map indicated that the difference peak is 6 to 7 A from the reactive single cysteine (Cys-64) and partially coincident with an "extraneous" density found in the native map. This "extraneous" peak was previously attributed to a bound L-arabinose molecule, and its presence accounts for the early failures of difference Fourier analyses of crystals soaked in or co-crystallized with L-arabinose to locate the sugar-binding site.