Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter

Biochemistry. 2001 Nov 20;40(46):13964-71. doi: 10.1021/bi015602p.

Abstract

The bacterial outer membrane transporter for vitamin B(12), BtuB, derives its energy for transport by interacting with the trans-periplasmic membrane protein TonB. This interaction with TonB occurs in part through an N-terminal segment in the BtuB sequence called the Ton box. In the present study, site-directed spin labeling of intact outer membrane preparations was used to investigate the conformation of the Ton box in wild-type BtuB and in two transport-defective mutants, L8P and V10P. In the wild-type protein, the Ton box is folded into the barrel of the transporter. The conformation of this segment is dramatically different in the transport-defective mutants L8P and V10P, where the Ton box is found to be flexible, and undocked from the transporter barrel with a greater exposure to the periplasm. In the wild-type protein, vitamin B(12) induces an undocking of the Ton box, but its addition to these transport defective mutants produces little or no change in the conformation of the Ton box. Proline substitutions at positions that do not alter transport do not alter the wild-type conformation of the Ton box; thus, the effect of substituting proline at positions 8 and 10 on the docked state of the Ton box appears to be unique. The failure of these mutants to execute the B(12) transport cycle may be a result of the altered conformation of the Ton box.

Publication types

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

MeSH terms

  • Amino Acid Motifs / genetics
  • Amino Acid Substitution / genetics
  • Bacterial Outer Membrane Proteins / chemistry*
  • Bacterial Outer Membrane Proteins / genetics*
  • Bacterial Outer Membrane Proteins / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism*
  • Bacterial Proteins / physiology
  • Biological Transport / genetics
  • Electron Spin Resonance Spectroscopy
  • Energy Metabolism / genetics
  • Energy Transfer / genetics*
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / metabolism
  • Leucine / genetics
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism*
  • Membrane Proteins / physiology
  • Membrane Transport Proteins
  • Mutagenesis, Site-Directed*
  • Proline / genetics
  • Protein Conformation
  • Receptors, Peptide / genetics*
  • Receptors, Peptide / metabolism
  • Spin Labels
  • Valine / genetics
  • Vitamin B 12 / metabolism

Substances

  • Bacterial Outer Membrane Proteins
  • Bacterial Proteins
  • BtuB protein, E coli
  • Escherichia coli Proteins
  • Membrane Proteins
  • Membrane Transport Proteins
  • Receptors, Peptide
  • Spin Labels
  • tonB protein, Bacteria
  • tonB protein, E coli
  • Proline
  • Leucine
  • Valine
  • Vitamin B 12