The amino-terminal region of the long-chain fatty acid transport protein FadL contains an externally exposed domain required for bacteriophage T2 binding

The fatty acid transport protein FadL from Escherichia coli is predicted to be rich in beta-structure and span the outer membrane multiple times to fo rm a long-chain fatty acid specific channel. Proteolysis of FadL within who le cells, total membranes, and isolated outer membranes identified two tryp sin-sensitive sites, both predicted to be in externally exposed loops of Fa dL. Amino acid sequence analysis of the proteolytic fragments determined th at the first followed R-93 and yielded a peptide beginning with S-94-L-K-A- D-N-I-A-P-T-A(104) while the second followed R-384 and yielded a peptide be ginning with S-385-I-S-I-P-D-Q-D-R-F-W-395. Proteolysis using trypsin elimi nated the bacteriophage T2 binding activity associated with FadL, suggestin g the T2 binding domain within FadL requires elements within one of these e xtracellular loops. A peptide corresponding to the amino-terminal region of FadL (FadL(28-160)) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the ami no-proximal extracellular loop of the protein confers T2 binding activity. Using an artificial neural network (NN) topology prediction method in combi nation with Gibbs motif sampling, a predicted topology of FadL within the o uter membrane was developed. According to this model, FadL spans the outer membrane 20 times as antiparallel beta-strands. The 20 antiparallel beta-st rands are presumed to form a beta-barrel specific for long-chain fatty acid s. On the basis of our previous studies evaluating the function of FadL usi ng site-specific mutagenesis of the fadL gene, proteolysis of FadL within o uter membranes, and studies using the FadL(28-160) peptide, the predicted e xtracellular regions between beta-strands 1 and 2 and beta-strands 3 and 4 are expected to contribute to a domain of the protein required for long cha in fatty acid and bacteriophage T2 binding. The first trypsin-sensitive sit e (R-93) lies between predicted beta-strands 3 and 4 while the second (R384 ) is between beta-strands 17 and 18. The trypsin-resistant region of FadL i s predicted to contain 13 antiparallel beta-strands and contribute to the l ong-chain fatty acid specific channel. (C) 2000 Academic Press.