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The three-dimensional structure of the native unliganded form of the
Leu/Ile/Val-binding protein (Mr = 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 A 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 A and 0.10 A,
respectively. The overall shape of the protein is a prolate ellipsoid with
dimensions of 35 A x 40 A x 70 A. 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 beta-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 A. The cleft has a depth of about 15 A and a base of about 14 A
x 16 A. Refining independently the structure of native Leu/Ile/Val-binding
protein crystals soaked in a solution containing L-leucine at 2.8 A 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 alpha-ammonium and alpha-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.(ABSTRACT TRUNCATED AT 400 WORDS)
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