Periplasmic binding protein structure and function: Refined X-ray structures of the leucine/isoleucine/valine-binding protein and its complex with leucine

Abstract

The three-dimensional structure of the native unliganded form of the Leu/Ile/Val-binding protein ( M r = 36,700), an essential component of the high-affinity active transport system for the branched aliphatic amino acids in Escherichia coli, has been determined and further refined to a crystallographic R-factor of 0·17 at 2·4 Å resolution. The entire structure consists of 2710 non-hydrogen atoms from the complete sequence of 344 residues and 121 ordered water molecules. Bond lengths and angle distances in the refined model have root-mean-square deviations from ideal values of 0·05 Å and 0·10 Å, respectively. The overall shape of the protein is a prolate ellipsoid with dimensions of 35 Å × 40 Å × 70 Å. The protein consists of two distinct globular domains linked by three short peptide segments which, though widely separated in the sequence, are proximal in the tertiary structure and form the base of the deep cleft between the two domains. Although each domain is built from polypeptide segments located in both the amino (N) and the carboxy (C) terminal halves, both domains exhibit very similar supersecondary structures, consisting of a central β-sheet of seven strands flanked on either side by two or three helices. The two domains are far apart from each other, leaving the cleft wide open by about 18 Å. The cleft has a depth of about 15 Å and a base of about 14 Å × 16 Å. Refining independently the structure of native Leu/Ile/Val-binding protein crystals soaked in a solution containing l-leucine at 2.8 Å resolution ( R-factor = 0.15), we have been able to locate and characterize an initial, major portion of the substrate-binding site of the Leu/Ile/Val-binding protein. The binding of the l-leucine substrate does not alter the native crystal structure, and the l-leucine is lodged in a crevice on the wall of the N-domain, which is in the inter-domain cleft. The l-leucine is held in place primarily by hydrogen-bonding of its α-ammonium and α-carboxylate groups with main-chain peptide units and hydroxyl side-chain groups; there are no salt-linkages. The charges on the leucine zwitterion are stabilized by hydrogen-bond dipoles. The side-chain of the l-leucine substrate lies in a depression lined with non-polar residues, including Leu77, which confers specificity to the site by stacking with the side-chain of the leucine substrate. So far in our high-resolution crystallographic studies of several binding proteins (including those with specificities for l-arabinose, d-galactose, and sulfate), we have observed three structural forms that are related by flexible domains: an unliganded form with a wide open substrate-binding site cleft between the two domains, an “open” structure with the substrate bound to one domain, and a “closed” form with the substrate bound in the cleft and completely entrapped between the domains. The function of binding proteins in active transport is discussed in light of the existence of the various forms and the other common structural and ligand-binding features of these proteins.