Fe assimilation in prokaryotes
A.-F. Miller, 2008, pg
1
Fe assimilation in prokaryotes
Fe3+ bound by siderophore
Siderophore bound by outer-membrane receptor (FecA)
Signaling to and energy from Ton system in cytoplasmic
membrane.
Transfer of Fe from periplasmic side of FecA to FecB in
periplasm.
FecB transports Fe to cytoplasmic membrane (plasma
membrane) where an ABC transporter delivers it to the
cytosol.
Fe release from siderophore upon reduction by flavoprotein
reductase.
A.-F. Miller, 2008, pg 2
Fe-Enterobactin assimilation
Last time,
as FecA
This time, as
homologous
BtuCD
A.-F. Miller, 2008, pg
3
Raymond et al. (2003) PNAS 100:3584.
ABC transporters
ATP-binding cassette transporters
An ancient family of
transmembrane transporters,
with members found in all phyla.
Transport nutrients into cells and
export toxins, or metabolic
products (drugs).
Two transmembrane domains with 6
(-11) helices each form binding site
facing the exterior for import
transporters
A.-F. Miller, 2008, pg
4
vit B12 transporter, BtuCD. Kaspar
et al. (2002) Science 296: 1091
ABC transporters
One or two ATP-binding domains
(NBD)on the cytoplasmic side and
several ABC transporter specific
sequences (‘cassettes’).
Nterm-TM-NBF-TM-NBF-Cterm
Upon ATP binding and hydrolysis,
the TM re-orient closing one end of
the translocation pathway and
opening the other.
molybdate transporter,
open conformation.
A.-F. Miller, 2008, pg
5
http://en.wikipedia.org/wiki/ATP-binding_cassette_transporter
ABC transporters
A.-F. Miller, 2008, pg
6
Kaspar et al. (2002) Science 296: 1091
Fe-enterobactin esterase structure
Kim, Raymond et al (deposited in pdb, Feb 2008, to be published.)
A.-F. Miller, 2008, pg
Fe is released from enterobactin inside cells upon
enzymatic hydrolysis of the lactone ring by the
cytoplasmic esterase, reduction of Fe3+ to Fe2+ or
7
protonation of the catechol ligands (at lower pH).
Fe in eukaryotes (humans)
4 g Fe in a 70 kg person.
0.5 - 2 mg acquired / day
Trivial losses via skin & mucosa loss and menstruation
65% - 75% of Fe is in red blood cells’ heme.
This is recycled.
Excess Fe is stored in liver: 0.5 g - 1 g.
Excessive deposits accumulate in cardiac myosytes,
pancreatic cells.
A.-F. Miller, 2008, pg
8
Transferrinmediated Fe
uptake
Transferrin uptake
and iron release in
cells by receptormediated
endocytosis.
A.-F. Miller, 2008, pg
9
Andrews (2000) Nat. Rev. Genet. 1:208-217
Transferrin
Member of a family of proteins, exemplified here by lactoferrin.
Structural and chemical bases for ability to bind, sequester and
release.
Glycoprotein, MW = 80 kDa, two lobes that are homologous
to one another (≈ 40% ID).
Kd ≈ 10-20 M , Fe3+ and CO32- bind synergistically.
Protein has two domains. In each domain there are two
subdomains that clamp down on the iron and carbonate
ions.
Hinge motion accompanies binding of Fe3+ and CO32-.
A.-F. Miller, 2008, pg
10
Fe binding by lactoferrin
Fe3+ ion and CO32in the interdomain
cleft. An α-helix
joins the two lobes.
N-lobe, C-lobe; domains 1, domains 2.
A.-F. Miller, 2008, pg
11
Baker, Anderson, and Baker, PNAS, 2003, 100, 3579.
Fe binding by lactoferrin
N-lobe, one domain reorients by 60° relative to the other
upon Fe3+ and CO32- binding.
A.-F. Miller, 2008, pg
Baker, Anderson, and Baker, PNAS, 2003, 100, 3579.
12
Fe binding by lactoferrin
Fe release from
transferrin upon
protonation of two
Lys in Tf,
protonation of
CO32- and
protonation of two
Tyr ligands.
A.-F. Miller, 2008, pg
13
Baker, Anderson, and Baker, PNAS, 2003, 100, 3579.
Expansion of the ligand repertoire
in proteins.
Lactoferrin
See Lippard & Berg text.
A.-F. Miller, 2008, pg
14
Lys* carbamate in urease,
rubisco, phosphotriesterase
Evolutionary parentage of Tf
Tf (left) vs. Bacterial periplasmic ferric binding protein (right).
Both display homology with bacterial periplasmic binding
proteins, that carry anions across periplasmic space of G-ve
23+
bacteria.
CO
3 is ancestral, Fe acquired later.
A.-F. Miller, 2008, pg
15
The Arms Race
between
pathogens and the
immune system.
Bacteria have evolved devices
for intercepting the Tf-bound
Fe and capturing it. The host
produces a siderocalin that
captures the Fe-enterobactin,
but bacteria produce
alternative siderophores such
as salmochelin S4 that are not
recognized.
A.-F. Miller, 2008, pg
16
http://www.cchem.berkeley.edu/knrgrp/fe.html
Fe must be able
to move on.
Fe release from
transferrin upon
protonation ligands and
opening of the Tf jaws.
Transport out of endosome
by divalent metal ion
transporter, and reduction
to Fe2+ . . . .
A.-F. Miller, 2008, pg
17
Andrews (2000) Nat. Rev. Genet. 1:208-217
3+
Fe
Example of an
reductase involved
in Fe uptake by erythroid cells.
Fe-Tf binds to cognate erythroid cell surface receptor.
Fe-Tf enters endosome, Fe3+ is reduced to Fe2+ and
transported across endosomal membrane by divalent metal
ion transporter 1.
The reductase is Steap3 (Six Transmembrane Epithelial Antigen of
Prostrate) : major ferri-reductase: N-terminal cytosolic
reductase domain, C-terminal heme-containing domain.
Cytoplasmic domain binds FMN, and NADPH in a FNO-like
fold (homologous to an archeal enzyme). This is to reduce a
heme bound within the transmembrane domain.
A.-F. Miller, 2008, pg
Fe emerges as Fe2+ !!!!!
18
Fe storage in cells: Ferritin
Can be loaded with
some 4,500 Fe each.
24 protein monomers.
8 nm diam. internal
cavity.
Fe2+ enters and is
oxidized in a site with
a 2-Fe-oxo core.
Exit may be via 8
pores formed upon
partial local melting of
the protein structure.
Liu & Thiel (2003) PNAS 100:3652.
A.-F. Miller, 2008, pg
19