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 form a long-chain fatty acid specific channel. Proteolysis of FadL within whole cells, total membranes, and isolated outer membranes identified two trypsin-sensitive sites, both predicted to be in externally exposed loops of FadL. Amino acid sequence analysis of the proteolytic fragments determined that the first followed R93 and yielded a peptide beginning with 94S-L-K-A-D-N-I-A-P-T-A104 while the second followed R384 and yielded a peptide beginning with 385S-I-S-I-P-D-Q-D-R-F-W395. Proteolysis using trypsin eliminated the bacteriophage T2 binding activity associated with FadL, suggesting the T2 binding domain within FadL requires elements within one of these extracellular loops. A peptide corresponding to the amino-terminal region of FadL (FadL28-160) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the amino-proximal extracellular loop of the protein confers T2 binding activity. Using an artificial neural network (NN) topology prediction method in combination with Gibbs motif sampling, a predicted topology of FadL within the outer membrane was developed. According to this model, FadL spans the outer membrane 20 times as antiparallel beta-strands. The 20 antiparallel beta-strands are presumed to form a beta-barrel specific for long-chain fatty acids. On the basis of our previous studies evaluating the function of FadL using site-specific mutagenesis of the fadL gene, proteolysis of FadL within outer membranes, and studies using the FadL28-160 peptide, the predicted extracellular 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-chain fatty acid and bacteriophage T2 binding. The first trypsin-sensitive site (R93) 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 is predicted to contain 13 antiparallel beta-strands and contribute to the long-chain fatty acid specific channel.
Cristalli
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Center for Cardiovascular Sciences, The Albany Medical College, New York 12208, USA.
DiRusso
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CC
Black
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D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D003198:Computer Simulation; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D050612:Fatty Acid Transport Proteins; D017900:Myoviridae / Q000378:metabolism*; D016571:Neural Networks, Computer; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D050612:Fatty Acid Transport Proteins; D010455:Peptides; D014764:Viral Proteins; C065691:fadL protein, E coli; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin