Biomedical subjects

Phillip E Klebba

Publications and source records attributed to Phillip E Klebba.

6 recordsLinked to original sources

PubMed2003

Three paradoxes of ferric enterobactin uptake.

Bacteria elaborate iron chelators that scavenge iron from the environment, including their human and animal hosts, and iron acquisition is a determinant of pathogenicity. One such iron chelate, the siderophore ferric enterobactin, enters Gram-negative bacteria through the FepA protein of the outer membrane. The ferric enterobactin transport process is a high-affinity, multi-specific, multi-component, energy dependent reaction, that is a paradigm of ligand-gated transport: FeEnt binding activates FepA to transport competency. On the basis of the FepA, FhuA, FecA and BtuB crystal structures, and in light of recent molecular biological, biochemical, and biophysical findings, this review considers the mechanism of ferric enterobactin uptake. The discussion focuses on three preeminent questions about the transport reaction: the function of the N-terminal globular domain that resides within the FepA channel, the mechanistic contributions of TonB to the activities of ligand-gated porins, and the energy dependence of metal transport reactions through the OM bilayer. Available data points to the idea that the N-terminal globular domains of these receptor proteins dynamically exit their pores during transport, creating a suction-force that pulls ligands through the surface loops into the periplasm. The functions of TonB and energy in these processes remain unknown.

Amino Acid Sequence↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2002

Spectroscopic observations of ferric enterobactin transport.

We characterized the uptake of ferric enterobactin (FeEnt), the native Escherichia coli ferric siderophore, through its cognate outer membrane receptor protein, FepA, using a site-directed fluorescence methodology. The experiments first defined locations in FepA that were accessible to covalent modification with fluorescein maleimide (FM) in vivo; among 10 sites that we tested by substituting single Cys residues, FM labeled W101C, S271C, F329C, and S397C, and all these exist within surface-exposed loops of the outer membrane protein. FeEnt normally adsorbed to the fluoresceinated S271C and S397C mutant FepA proteins in vivo, which we observed as quenching of fluorescence intensity, but the ferric siderophore did not bind to the FM-modified derivatives of W101C or F329C. These in vivo fluorescence determinations showed, for the first time, consistency with radioisotopic measurements of the affinity of the FeEnt-FepA interaction; K(d) was 0.2 nm by both methods. Analysis of the FepA mutants with AlexaFluor(680), a fluorescein derivative with red-shifted absorption and emission spectra that do not overlap the absorbance spectrum of FeEnt, refuted the possibility that the fluorescence quenching resulted from resonance energy transfer. These and other data instead indicated that the quenching originated from changes in the environment of the fluor as a result of loop conformational changes during ligand binding and transport. We used the fluorescence system to monitor FeEnt uptake by live bacteria and determined its dependence on ligand concentration, temperature, pH, and carbon sources and its susceptibility to inhibition by the metabolic poisons. Unlike cyanocobalamin transport through the outer membrane, FeEnt uptake was sensitive to inhibitors of electron transport and phosphorylation, in addition to its sensitivity to proton motive force depletion.

Biological Transport↗

PubMed2002

Mechanism of maltodextrin transport through LamB.

The Gram-negative bacterial outer membrane contains several independent, biochemically distinct transport systems for the acquisition of solutes from the environment. Three or more different classes of membrane proteins exist within the porin superfamily, that facilitate the uptake of sugars, amino acids, nucleotides, vitamins and metals. In spite of crystallographic descriptions of these protein transporters over the past decade, the mechanisms by which porins catalyze solute internalization are controversial, and in some cases still obscure. For many years the research of Maurice Hofnung endeavored to explain the transport of maltose and maltodextrins by LamB, also known as maltoporin. In the shadow of recent crystal structures, his work helped outline a different picture of outer membrane transport physiology, that is a tribute to the powerful genetic approaches Maurice pioneered. These data suggest that the principal determinant of maltodextrin recognition by maltoporin derives from the configuration of aromatic amino acids in its surface loops.

Bacteria↗

PubMed2002

Surface loop motion in FepA.

Using a lysine-specific cleavable cross-linking reagent ethylene glycolbis(sulfosuccimidylsuccinate) (Sulfo-EGS), we studied conformational motion in the surface loops of Escherichia coli FepA during its transport of the siderophore ferric enterobactin. Site-directed mutagenesis determined that Sulfo-EGS reacted with two lysines, K332 and K483, and at least two other unidentified Lys residues in the surface loops of the outer membrane protein. The reagent cross-linked K483 in FepA L7 to either K332 in L5, forming a product that we designated band 1, or to the major outer membrane proteins OmpF, OmpC, and OmpA, forming band 2. Ferric enterobactin binding to FepA did not prevent modification of K483 by Sulfo-EGS but blocked its cross-linking to OmpF/C and OmpA and reduced its coupling to K332. These data show that the loops of FepA undergo conformational changes in vivo, with an approximate magnitude of 15 A, from a ligand-free open state to a ligand-bound closed state. The coupling of FepA L7 to OmpF, OmpC, or OmpA was TonB independent and was unaffected by the uncouplers CCCP (carbonyl cyanide m-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) but completely inhibited by cyanide.

Bacterial Outer Membrane Proteins↗

PubMedDate not supplied

Mechanisms of colicin binding and transport through outer membrane porins.

To kill Escherichia coli, toxic proteins, called colicins, pass through the permeability barrier created by the outer membrane (OM) of the bacterial cell envelope. We consider a variety of different colicins, including A, B, D, E1, E3, Ia, M and N, that penetrate through the porins OmpF, FepA, BtuB, Cir and FhuA, to subsequently interact with a few targets in the periplasm, including TolA, TolB, TolC and TonB. We review the mechanisms, demonstrated and postulated, by which such toxins enter bacterial cells, from the initial binding stage on the cell surface to the internalization reaction through the OM bilayer. Our discussions endeavor to answer two main questions: what is the origin of colicin-binding affinity and specificity, and after adsorption to OM porins, do colicin polypeptides translocate through porin channels, or enter by another, currently unknown pathway?

Bacterial Outer Membrane Proteins↗