THE BINDING OF SUGARS TO THE L-ARABINOSE-BINDING PROTEIN FROM ESCHERICHIA COLI
NEWCOMER, MARCIA ELIZABETH
Doctor of Philosophy
The sugar-binding site of the L-arabinose binding protein, an essential component of the high-affinity L-arabinose uptake system in Escherichia coli, is located deep in a cleft formed by the two domains of the protein. 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 structure might have been solved with bound L-arabinose necessitated the synthesis of the heavy-atom analog, its structure consistent with the sugar-binding specificity of the protein. Difference Fourier maps showed a peak which was partially coincident with the "extraneous" density found in the native map. This "extraneous" density was previously attributed to a bound L-arabinose molecule, and its presence accounts for early failures of difference Fourier analyses of crystals soaked in or co-crystallized with L-arabinose to locate the binding site. The location of a C6 substituent by difference Fourier analysis of crystals soaked in solutions of the C6 analog D-galactose and a fit of an L-arabinose molecule to the "extraneous" electron density allowed for a model for L-arabinose binding to the L-arabinose binding protein to be proposed. The L-arabinose molecule binds in a chair conformation with the OH(1) axial ((beta)-anomer). The anomeric hydroxyl is able to hydrogen bond to the main chain oxygen of Asp-89 and to Lys-10, the latter amino acid also forms a hydrogen bond with the hydroxyl at C2. Glu-14 and Asn-205 hydrogen bond with OH(3), and OH(4) hydrogen bonds to Asn-205 and Asn-232. Small angle X-ray scattering experiments indicate that the L-arabinose-binding protein undergoes a substantial conformational change upon the binding of substrate: the radius of gyration of the protein molecule decreases by (TURN)1 A with the addition of L-arabinose. It is proposed that this change in the radius of gyration is due to movement of one domain with respect to the other about a "hinge" deep in the base of the cleft separating the two domains of the bilobal protein.