Membrane Transport

Active transport is a generalized process occurring in all living cells, yet the molecular mechanisms by which these processes take place are not understood.We are currently studying a fascinating class of proteins that function to transport rare nutrients such as iron and vitamin B12(CNCbl) across the outer membrane of Gram negative bacteria.These membrane transport proteins are termed TonB-dependent because they derive energy for transport by coupling to the inner membrane protein TonB.

TonB-dependent transport is important for a number of reasons.First, it provides a model for transport and transmembrane signaling.Ligands have been identified that bind to the exterior surface and promote conformational changes on the periplasmic surface of the transporter.Second, this system provides a model for reversible and regulated protein-protein interactions.Finally, TonB driven transport is critical to the survival of many pathogens.For example, the Gram negative meningococucs Neisseria meningitides (which leads to meningitis) is a significant cause of morbidity and mortality in both developed and developing countries.It requires and extracts iron from its human host from lactoferrin, transferrin and hemoglobin, and has no less than five outer membrane proteins specialized for these tasks.These TonB driven processes may be an excellent target for the development of a new classes of antibiotics.

Using site-directed spin labeling and EPR, we have shown that the first step in the transport process involves an undocking of the Ton box from the barrel of the transporter (see Figure below).The Ton box is an N-terminal segment that is highly conserved in these transporters.It interacts with TonB and is responsible for energy coupling between the two proteins.This unfolding or undocking event provides the signal that initiates coupling between BtuB and TonB.Shown below is a model for the this conformational change based upon EPR data and the crystal structure of the protein.

Listed below are several of the more significant observations our lab has made regarding BtuB.

  • The binding of vitamin B12 undocks the energy coupling segment of the protein (the Ton box) from the BtuB barrel. This conformational change likely represents the first step in the transport process and is a signal to the inner membrane protein (TonB) that the transporter is loaded with substrate (1,2).

  • Transport-defective mutants have a constitutively unfolded Ton box. We have examined several mutants that produce a transport defective phenotype and we have demonstrated that the Ton box in these mutants is constitutively unfolded (Coggshall et al., Biochemistry 2001).
  • Detergents alter the protein dynamics and structure. The substrate-induced conformational change in the Ton box takes place when BtuB is reconstituted into POPC bilayers, but in mixed micelles (OG:POPC) the N-terminal Ton box unfolds into the periplasm.This is also accompanied by an increase in backbone dynamics along the b-strands of the barrel.This observation may explain why the Ton box is not resolved in the crystal structures obtained from some members of this family, and it also indicates that membrane mimetic environments do not necessarily maintain membrane protein structures (3).
  • Colicin E3 stabilizes the Ton box. Colicins are bactericidal proteins that use BtuB and other transporters as receptors.The receptor domain from colicin E3 competes with vitamin B12 for binding on the external side of BtuB; however, the binding of E3 produces an opposite effect on the Ton box to that of the substrate.Whereas vitamin B12 undocks the Ton box, colicin E3 actually stabilizes the docked configuration of the Ton box.Thus, two competitive extracellular ligands transduce opposite conformations for this periplasmic segment(4,5).
  • The crystal structure of BtuB is osmotically trapped. Osmolytes alter the energetics of the docked (folded) to undocked (unfolded) substrate-induced conformational transition of BtuB.We demonstrate that this transition is blocked in the crystal structure of BtuB because osmolytes drive the protein to its least hydrated state (6).

Our long-range goal is to understand signaling in this protein and to map the molecular structural changes that accompany transport in this system.We are testing the idea that proteins in this family function in a homologous manner, and as a result we are purifying and labeling the iron transporters FecA and FhuA.We believe that Ton B-dependent systems may be an ideal target for new classes of antibiotics and we are planning to develop procedures to screen for small compounds that interfere with the TonB-Ton box interaction.

References

  1. Merianos, H.J., N. Cadieux, C.H. Lin, R. Kadner, and D.S. Cafiso, Substrate-induced exposure of an energy-coupling motif of a membrane transporter. Nat. Struct. Biol., 2000,7:205-209.
  2. Fanucci, G.E., K.A. Coggshall, N. Cadieux, M. Kim, R.J. Kadner, and D.S. Cafiso, Substrate-Induced conformational changes of the perplasmic N-terminus of an outer-membrane transporter by site-directed spin labeling. Biochemistry, 2003, 42: 1391-1400.
  3. Fanucci, G.E., J.Y. Lee, and D.S. Cafiso, Membrane mimetic environments alter the conformation of the outer membrane protein BtuB. J. Am. Chem. Soc., 2003, 125: 13932-13933.
  4. Fanucci, G.E., N. Cadieux, R. Kadner, and D.S. Cafiso, Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter. Proc. Natl. Acad. Sci. USA, 2003, 100: 11382-11387.
  5. Cadieux, N., P.G. Phan, D.S. Cafiso, and R.J. Kadner, Differential substrate-induced signaling through the TonB-dependent transporter BtuB. Proc. Natl. Acad. Sci. USA, 2003. 100, 10688-10693.
  6. Fanucci, G.E., J.Y. Lee, and D.S. Cafiso, Spectroscopic evidence that osmolytes used in crystallization buffers Inhibit a conformation change in a membrane protein. Biochemistry, 2003. 42, 13106-13112.