The structure of the sc D-maltose-binding protein, an essential component of the Escherichia coli high affinity osmotic-shock sensitive transport and chemotaxis systems for 1-$\alpha$-4 linked glucose oligosaccharides, has been determined at a resolution of 2.3A by x-ray crystallography. The R-factor is 25% for 15,162 reflections by a restrained least squares method. The maltose-binding protein is ellipsoidal with dimensions of 30 A $\times$ 40 A $\times$ 65 A. The secondary structure is folded from a single polypeptide chain of 370 residues into two domains connected by three segments. The N-domain is made up of a five strand $\beta$ sheet (with the fourth strand antiparallel) flanked by two $\alpha$ helices on one side and three on the other. The C-domain is arranged similarly with the addition of a pair of antiparallel $\alpha$ helices that span the cleft. These helices act to extend the length of the cleft. The antiparallel strand is the first element after the initial crossover into either domain. The three crossovers are located in close physical proximity, although widely separated in sequence. The first two crossovers form a short $\beta$ sheet. The crossovers serve as the base for the cleft. The maltose-binding protein consists of 40% $\alpha$ helix and 20% $\beta$ sheet. The principle folding pattern is of alternating $\beta$ sheet and $\alpha$ helix.
The binding site(s) for maltose and maltotriose were determined to lie in the cleft formed by the two domains. The reducing end of both sugars was found to occupy the same site in MBP. Hydrogen bonds formed between side chain residues of MBP and hydroxyl groups of the sugar are the main stabilizing force in the binding of substrate. Maltose is almost entirely buried by the protein. The binding site is rich in aromatic residues.
The location of site specific mutations in MBP that are defective in chemotaxis, but not in transport have been found to lie in exterior portions of MBP in different domains. Other mutations affecting a region on the opposite side of the $\beta$ sheet from the chemotactic mutant in the C-domain eliminate transport but do not effect substrate binding or chemotaxis. These findings support the theory of recognition of ligand-bound binding protein by differential separation of two sites on opposite sides of the cleft due to conformational change upon ligand binding. The structural evidence also supports the existence of separate recognition sites for transport and chemotaxis.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16190 |
| Date | January 1988 |
| Creators | Spurlino, John Curtis |
| Contributors | Quiocho, Florante A. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 162 p., application/pdf |
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