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

Arch Biochem Biophys. 2000 May 15;377(2):324-33. doi: 10.1006/abbi.2000.1794.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Outer Membrane Proteins / chemistry*
  • Bacterial Outer Membrane Proteins / metabolism*
  • Bacterial Outer Membrane Proteins / physiology
  • Cell Membrane / chemistry
  • Computer Simulation
  • DNA-Directed RNA Polymerases / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / chemistry
  • Escherichia coli / metabolism
  • Escherichia coli Proteins*
  • Fatty Acid Transport Proteins
  • Myoviridae / metabolism*
  • Neural Networks, Computer
  • Peptides / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Trypsin / metabolism
  • Viral Proteins

Substances

  • Bacterial Outer Membrane Proteins
  • Escherichia coli Proteins
  • Fatty Acid Transport Proteins
  • Peptides
  • Viral Proteins
  • fadL protein, E coli
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • Trypsin