Effect of loop deletions on the binding and transport of ferric enterobactin by FepA.
The siderophore ferric enterobactin enters Escherichia coli through the outer membrane (OM) porin FepA, which contains an aqueous transmembrane channel that is normally occluded by other parts of the protein. After binding the siderophore at a site within the surface loops, FepA undergoes conformational changes that promote ligand internalization. We assessed the participation of different loops in ligand recognition and uptake by creating and analysing a series of deletions. We genetically engineered 26 mutations that removed 9-75 amino acids from nine loops and two buried regions of the OM protein. The mutations had various effects on the uptake reaction, which we discerned by comparing the substrate concentrations of half-maximal binding (Kd) and uptake (Km): every loop deletion affected siderophore transport kinetics, decreasing or eliminating binding affinity and transport efficiency. We classified the mutations in three groups on the basis of their slight, strong or complete inhibition of the rate of ferric enterobactin transport across the OM. Finally, characterization of the FepA mutants revealed that prior experiments underestimated the affinity of FepA for ferric enterobactin: the interaction between the protein and the ferric siderophore is so avid (Kd < 0.2 nM) that FepA tolerated the large reductions in affinity that some loop deletions caused without loss of uptake functionality. That is, like other porins, many of the loops of FepA are superficially dispensable: ferric enterobactin transport occurred without them, at levels that allowed bacterial growth.
Newton
S M
SM
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019, USA.
Igo
J D
JD
Scott
D C
DC
Klebba
P E
PE
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D003087:Colicins / Q000378:metabolism; D004758:Enterobactin / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017384:Sequence Deletion
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003087:Colicins; D011956:Receptors, Cell Surface; C029489:enterobactin receptor; D004758:Enterobactin