Stability Studies of FhuA, a Two-Domain Outer Membrane Protein from Escherichia coli

Abstract

FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein. To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork. Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively. Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C. Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome. FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

“…In addition, ITC data indicated that, in vitro, MccJ25 binds to one single class of sites, with a stoichiometry consistent with two ligands per receptor (n = 1.9). By comparing the thermal denaturation profile of the MccJ25-FhuA complex with that of FhuA, which presents two transitions as characterized previously [20,28], we showed that MccJ25 binding affected only the external loops and plug (first domain) of FhuA, and not the β-barrel (second domain). Indeed, unfolding of the first domain required a much higher enthalpy when MccJ25 was associated with FhuA, indicating that stabilization of the first domain results from binding of MccJ25 to FhuA.…”

“…FhuA displays two well resolved thermal transitions, corresponding to the unfolding of the loops and plug, and to the unfolding of the β-barrel [20,28]. Under our experimental conditions, these were observed at 63 and 70 • C respectively ( Figure 5B).…”

“…Bacterial strains used for MccJ25 and FhuA expression were E. coli MC4100 carrying the pTUC202 plasmid [19] (a gift from Professor Felipe Moreno, Hospital Ramón y Cajal, Madrid, Spain) and E. coli HO830fhuA carrying the pHX405 plasmid [20] respectively. Bioassays were performed with the bacterial strains described previously [21,22], as well as with S. enterica Paratyphi SL369 [23], S. enterica Typhimurium LT2 [23], P. agglomerans K4 [24] (generously provided by Professor Volkmar Braun, University of Tübingen, Germany) and Pseudomonas aeruginosa A.T.C.C.…”

The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable β-Sheet Domain

“…(i) ␤-Barrel proteins are known for their remarkable stability. For instance, the ␤-barrels of FhuA of E. coli (22 ␤-strands) or Omp21 from D. acidovorans (8 ␤-strands) are stable up to 75°C and Ͼ80°C (40,41). It is interesting to note in this regard that the stability of the ␤-barrel against denaturation is not coupled to localization of those proteins in the outer membrane, since also soluble ␤-barrel proteins such as the lipocalins or green fluorescent protein show very high transition temperatures of 61 and 82°C, respectively (42,43).…”

Dimerization of TonB Is Not Essential for Its Binding to the Outer Membrane Siderophore Receptor FhuA of Escherichia coli

“…Stability studies using differential scanning calorimetry experiments have shown the autonomous behavior of the cork and the ␤-barrel that unfold at different temperatures (12). The interactions between the cork domain and the ␤-barrel consist of nine salt bridges and more than 60 hydrogen bonds (11).…”