Transmembrane topology of the two FhuB domains representing the hydrophobic components of bacterial ABC transporters involved in the uptake of siderophores, haem and vitamin B

Abstract

Transport of siderophores of the hydroxamate type across the Escherichia coli cytoplasmic membrane depends on a periplasmic binding-protein-dependent (PBT) system. This uptake system consists of the binding protein FhUD, the membrane-associated putative ATP-hydrolase FhuC and the integral membrane protein FhuB. The two halves of FhuB [FhuB(N) and FhuB(C)], both essential for transport, are similar with respect to structure and function. Regions were identified in FhuB(N) and FhuB(C) which are presumably involved in the interaction of the two FhuB halves with each other or with other components of the uptake system, or with the different substrates. To determine the topology of the membrane-embedded polypeptide chain, FhuB'-'/?-lactamase protein fusions were analysed. The experimental data suggest that each half of the FhuB is able to fold autonomously into the lipid bilayer, which is a prerequisite for the assembly of both halves into a transport-competent formation. The hydrophobic components from PBT systems involved in the uptake of siderophores, haem and vitamin B, , define a subclass of polytopic integral membrane proteins. The topology of these 'siderophore family' proteins differs from that of the equivalent components of other PBT systems in that each polypeptide (and each half of FhuB) consists of 10 membranespanning regions, with the N-and C-termini located in the cytoplasm. The conserved region a t a distance of about 90 amino acids from the C-terminus, typical of all hydrophobic PBT proteins, is also oriented to the cytoplasm. However, in the siderophore family' proteins this putative ATPase interaction loop is followed by four instead of two transmembrane spans.

Identification and Characterization of a Membrane Permease Involved in Iron-Hydroxamate Transport in Staphylococcus aureus

“…Of note, FhuB proteins from S. aureus and B. subtilis are approximately half the size of the E. coli FhuB protein (32). In E. coli and B. subtilis, it has been shown that FhuB acts as an integral membrane permease, allowing for the passage of iron-hydroxamate complexes across the cytoplasmic membrane (3,18,31). According to data currently available in the databases, the FhuB protein in S. aureus would appear, at first glance, to be significantly smaller (by 43 amino acids and 4.7 kDa) than its homolog in B. subtilis.…”

Identification of rhtX and fptX , Novel Genes Encoding Proteins That Show Homology and Function in the Utilization of the Siderophores Rhizobactin 1021 by Sinorhizobium meliloti and Pyochelin by Pseudomonas aeruginosa , Respectively

“…The ferric hydroxamate uptake (Fhu) system of Escherichia coli (23) facilitates the transport of different hydroxamate siderophores, including ferrichrome, coprogen, and aerobactin, each of which requires its cognate receptor at the outer membrane. The Fhu system is representative of an ABC transporter, composed of the cytoplasmic membrane integral protein FhuB, which resembles a heterodimer and has each half associated with the ATPase FhuC (16). In addition, the periplasmic protein FhuD binds the ferrisiderophore (24) and chaperones it across the periplasm to the cytoplasmic membrane FhuBC complex.…”

The Haemophilus influenzae hFbpABC Fe 3+ Transporter: Analysis of the Membrane Permease and Development of a Gallium-Based Screen for Mutants

“…In accordance with the primary sequence, there is twofold internal homology between the first and latter halves of the protein (helices 1 to 6 and 7 to 12). This twofold homology within a single fused permease subunit is similar to that of the FhuB permease from the ferric hydroxymate ABC transporter, which is composed of a single permease subunit with 20 putative transmembrane segments (25). These permeases stand in contrast to the majority of ABC transporter permeases, which are typically composed of two smaller, similar, or identical subunits that together form a functional dimer (4,5,28).…”

“…The BtuC permease homodimer consists of 20 transmembrane ␣-helices arranged with pseudo-twofold symmetry around a central pore and periplasmically exposed substrate vestibule. Although this number of transmembrane helices is substantially more than the 12 helices predicted for the canonical ABC transporter, the increased helical content may be required to stabilize a central channel associated with B 12 translocation that is larger than that of smaller substrates (21,25). Consistent with this, biochemical evidence suggests that other well-characterized permeases, including the maltose (MalFG) and histidine (HisQM) permeases, possess fewer total numbers of membrane-spanning domains (12 [MalFG] and 10 [HisQM] transmembrane helices) (20,29).…”