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September 11, 2002 Structural Basis of Gating by the Outer Membrane Transporter FecAA.D. Ferguson1, B.S. Smith1, J. Deisenhofer1, R. Chakraborty2, L. Esser3, and D. van der Helm4 Scientists working at the NSLS and Argonne National Laboratory’s Advanced Photon Source (APS) have determined the crystallographic structure of the outer membrane receptor FecA from Escherichia coli with and without ferric citrate – an essential nutrient of bacteria – at 2.5 and 2.0 angstrom resolution. This study establishes the structural basis of gating for receptors dependent on the cytoplasmic membrane protein TonB.
Bacteria survive by using highly selective mechanisms designed to actively pump iron across the cell envelope. These mechanisms, responsive to both the internal and external iron concentration, control the transcription of genes involved in iron uptake. The ferric citrate uptake (fec) genes are responsible for the transport of ferric citrate from the external medium into the cell. Embedded within the outer membrane is FecA, a receptor that binds and transports ferric citrate, and is required to initiate transcription of the fec uptake genes. We have determined the architecture of FecA from Escherichia coli and its gating mechanism by x-ray crystallography. Using mixed detergent-protein micelles, FecA crystals with and without ferric citrate were grown. Data was collected to 2.0 Å from unliganded FecA at NSLS beamline X12C; and to 2.5 Å from liganded FecA at the Argonne National Laboratory’s Advanced Photon Source. FecA is composed of three domains (Figure 1): (i) a 22-stranded antiparallel β-barrel embedded within the outer membrane, with long extracellular loops and short periplasmic turns; (ii) a “plug,” consisting of a mixed four-stranded β-sheet with short interspersed helices, and extracellular and periplasmic pockets, located above and below the plug; (iii) a disordered third domain, the NH2-terminal extension, which resides entirely within the periplasm and is required for the initiation of transcription.
Ferric citrate binding affects the conformation of the barrel and the plug domain of FecA by causing a dramatic change in the spatial arrangement and conformations of primarily the seventh and eighth extracellular loops, as shown in Figure 2.
From our structural observations, we propose the following transport mechanism. Stage 1: Ferric citrate is adsorbed from the medium primarily by aromatic residues found within the upper portion of the external pocket of FecA. Stage 2: Ferric citrate is transferred to its high-affinity binding site, causing an allosteric transition within the plug that signals the occupancy of FecA in the periplasm. Stage 3: Multiple extracellular loops of the barrel change their relative conformation and position, thereby closing the external pocket of the barrel. Stage 4: Transitions that modify the conformation of the plug domain and/or barrel are needed for transport to occur, and are mediated by physical interactions between FecA and the cytoplasmic membrane protein TonB. Our findings clarify the current understanding of energy-dependent siderophore uptake across the bacterial outer membrane, and establish the structural basis of gating for TonB-dependent receptors. Further genetic and biophysical studies are needed to establish the molecular basis of energy-dependent transport. FUNDING PUBLICATION FOR MORE INFORMATION |
The National Synchrotron Light Source at Brookhaven National Laboratory in New York, is a national user research
facility funded by the U.S. Department of Energy's Office of Basic Energy Science. The NSLS operates two electron
storage rings: an X-Ray ring and a Vacuum UltraViolet (VUV) ring which provide intense light spanning the electromagnetic
spectrum from the infrared through x-rays. Each year over 2300 scientists from universities, industries and government
labs perform research at the NSLS.
Despite its relative abundance in Earth’s crust, iron is
biologically unavailable in the atmosphere. So, in response to iron
deficiency, most microbes secrete organic chelators – compounds
having a central metallic ion attached to two or more nonmetallic
atoms – called siderophores, which are designed to sequester ferric
iron.
