Ranjan Chakraborty

Crystal structure of the outer membrane active transporter FepA from Escherichia coli.

Integral outer membrane receptors for iron chelates and vitamin B 12 carry out specific ligand transport against a concentration gradient. Energy for active transport is obtained from the proton–motive force of the inner membrane through physical interaction with TonB–ExbB–ExbD, an inner membrane complex. Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 Å resolution. Two distinct functional domains are revealed: (i) a 22–stranded β–barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N–terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding. These loops could provide a signaling pathway between the processes of ligand recognition and TonB–mediated transport. The blockage of the pore suggests that the N–terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm.

Molecular mechanism of ferricsiderophore passage through the outer membrane receptor proteins of Escherichia coli

Iron is an essential nutrient for all microorganisms with a few exceptions. Microorganisms use a variety of systems to acquire iron from the surrounding environment. One such system includes production of an organic molecule known as a siderophore by many bacteria and fungi. Siderophores have the capacity to specifically chelate ferric ions. The ferricsiderophore complex is then transported into the cell via a specific receptor protein located in the outer membrane. This is an energy dependent process and is the subject of investigation in many research laboratories. The crystal structures of three outer membrane ferricsiderophore receptor proteins FepA, FhuA and FecA from Escherichia coli and two FpvA and FptA from Pseudomonas aeruginosa have recently been solved. Four of them, FhuA, FecA, FpvA and FptA have been solved in ligand-bound forms, which gave insight into the residues involved in ligand binding. The structures are similar and show the presence of similar domains; for example, all of them consist of a 22 strand-β-barrel formed by approximately 600 C-terminal residues while approximately 150 N-terminal residues fold inside the barrel to form a plug domain. The plug domain obstructs the passage through the barrel; therefore our research focuses on the mechanism through which the ferricsiderophore complex is transported across the receptor into the periplasm. There are two possibilities, one in which the plug domain is expelled into the periplasm making way for the ferricsiderophore complex and the second in which the plug domain undergoes structural rearrangement to form a channel through which the complex slides into the periplasm. Multiple alignment studies involving protein sequences of a large number of outer membrane receptor proteins that transport ferricsiderophores have identified several conserved residues. All of the conserved residues are located within the plug and barrel domain below the ligand binding site. We have substituted a number of these residues in FepA and FhuA with either alanine or glutamine resulting in substantial changes in the chemical properties of the residues. This was done to study the effect of the substitutions on the transport of ferricsiderophores. Another strategy used was to create a disulfide bond between the residues located on two adjacent β-strands of the plug domain or between the residues of the plug domain and the β-barrel in FhuA by substituting appropriate residues with cysteine. We have looked for the variants where the transport is affected without altering the binding. The data suggest a distinct role of these residues in the mechanism of transport. Our data also indicate that these transporters share a common mechanism of transport and that the plug remains within the barrel and possibly undergoes rearrangement to form a channel to transport the ferricsiderophore from the binding site to the periplasm.

Identification and mutational studies of conserved amino acids in the outer membrane receptor protein, FepA, which affect transport but not binding of ferric-enterobactin in Escherichia coli.

Many gram-negative bacteria produce and excrete siderophores, which complex iron with high affinity in the environment. The ferric siderophore complexes are transported across the outer membrane by receptor proteins. This process requires energy and is TonB dependent and must involve conformational changes in the receptor proteins to allow the transport of the ferric siderophores from the extracellular binding site to the periplasm. There is a large variety in the structures, molecular weights and charges among the siderophores. It was therefore realized that when the sequences of the many different receptor proteins were compared, simultaneously, all identities and close similarities, found in this manner, could only be due to residues involved in the conformational changes and transport mechanism, common to all the proteins, and not be due to the specificity of ligand recognition. Once the crystal structures of FepA, FhuA and FecA became available, it was immediately clear that the sequence similarities which were found in the simultaneous alignment, were all localized in a few structural domains, which are identical in the three structures and can therefore be expected to be maintained in all the proteins in this family. One of these domains, tentatively named the lock region, consists of 10 residues with a central quadrupole formed by two arginines and two glutamates, from the plug region and the beta barrel. We mutated several of these residues in FepA. All showed normal binding in quantitative binding studies. Some showed normal transport as well, however, the majority showed moderate to severe defective transport with ferric enterobactin. The results therefore show the validity of the hypothesis that the simultaneous sequence alignment will select the residues involved in the transport function of the receptor proteins. In addition the results allow to relate the severity of the transport deficiency to be correlated with the structure of the lock region while it is also possible to propose a function of this region in the conformational changes of the protein during the transport of the ligand from the binding site to the periplasm.

Isolation and structural identification of the trihydroxamate siderophore vicibactin and its degradative products from Rhizobium leguminosarum ATCC 14479 bv. trifolii

The Rhizobia are a group of free-living soil bacteria known for their ability to symbiotically infect the roots of specific host plants as well as to produce siderophores in order to compete with other microorganisms for the limited availability of iron in the rhizosphere. In this study, Rhizobium leguminosarum ATCC 14479, which preferentially infects the red clover Trifolium pratense, was found to produce the trihydroxamate siderophore vicibactin (C33H55N6O15) under iron restricted conditions. In addition, two other iron-binding, siderophore-like compounds: C20H36N4O10, C31H55N6O15, were isolated and purified from the culture media. Due to the structural similarity of the latter compounds to vicibactin based on electrospray-mass spectrometry and nuclear magnetic resonance data, these heretofore unreported molecules are thought to be either modified or degraded products of vicibactin. Although vicibactin has previously been found to be commonly produced by other rhizobial strains, this is the first time it has been chemically characterized from a clover infecting strain of R. leguminosarum.

Ferric Siderophore Transport via Outer Membrane Receptors of Escherichia coli: Structural Advancement and A Tribute to Dr. Dick van der Helm—an ‘Ironman’ of Siderophore Biology

In aerobic environments, iron exists as an insoluble ferric-oxihydroxide polymer with a concentration of soluble iron in a 10−16–10−18 M range. In the human body iron remains complexed with iron binding proteins. Both of these conditions create iron restricted environments for the growth of microorganisms. Iron is an essential element for the growth of a majority of microorganisms since iron acts as a cofactor for several important enzymes, and cytochromes involved in energy generation. Microorganisms employ many different strategies to acquire iron, among them siderophore-mediated iron transport, as the most common one. These types of transport systems are mostly found in Gram-negative bacteria, where they consist of outer membrane proteins, periplasmic binding proteins, inner membrane transport proteins, and energy transducing inner membrane protein complex TonB-ExbB-ExbD. The crystal structures of the outer membrane receptors FepA and FhuA from Escherichia coli were solved in late 1990s, but to date the mechanism of transport and energy transduction is not completely known. Enormous amounts of structural and biochemical data have been published in the past decade. This chapter pays tribute to the contributions of Dr. Dick van der Helm of the University of Oklahoma to the field of siderophore biology and discusses the structural advancement of the components involved in the siderophore mediated transport systems in E. coli.

Structural basis of gating by the outer membrane transporter FecA

Siderophore-mediated acquisition systems facilitate iron uptake. We present the crystallographic structure of the integral outer membrane receptor FecA from Escherichia coli with and without ferric citrate at 2.5 and 2.0 angstrom resolution. FecA is composed of three distinct domains: the barrel, plug, and NH2-terminal extension. Binding of ferric citrate triggers a conformational change of the extracellular loops that close the external pocket of FecA. Ligand-induced allosteric transitions are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. These data establish the structural basis of gating for receptors dependent on the cytoplasmic membrane protein TonB. By compiling available data for this family of receptors, we propose a mechanism for the energy-dependent transport of siderophores.