Heme Uptake Receptor

Prosthetic group with an iron atom in the middle of a porphyrin ring

Ring contains N, alkenes, and carboxylate groups

Commonly recognized as parts of hemoglobin (4 per)

Iron is responsible for binding oxygen in order to distribute to the rest of the body

Uptake of heme is one way the cell can bring in iron

What is Heme?

http://omlc.ogi.edu/spectra/hemoglobin/hemestruct/index.html

Heme with iron atom bound in the middle of the porphyrin ring

Intracellular◦ Cytochromes (involved in cellular respiration) ◦ Proteins involved in DNA synthesis and cell

division

◦ Extracellular Hemoglobin Myoglobin Connective tissue, nervous system, immune system

Uses of Iron

http://sandwalk.blogspot.com/2007/08/heme-groups.html

Soluble Fe3+ (ferric iron) – retrieved by compounds called siderophores

Ferriproteins *Heme Hemoproteins (Hb and Mb) *Hemophores (proteins with high affinity for

heme)*discussed in this presentation

Other sources of Iron

Entry into cells must be regulated because too much can be toxic

Requires a membrane protein because cannot naturally diffuse

Active transport mechanism◦ Energy is derived from proton gradient◦ Proton gradient formed and energy derived is

transduced by proteins Ton B or TonB – related proteins

Iron Transport

Escherichia Coli

Test Organism

Carrier protein that brings heme to receptor Serratia marcescens hemophore: HasA 188-residue protein Very high affinity for heme Beta sheet layer and 4 alpha helices Heme iron is bound by coordination of His-

32 and Tyr-75 on opposing loops

Hemophore

http://www.pasteur.fr/recherche/unites/Mbbact/mbbact-en/research-01.html

HasR Can internalize both free heme or that

bound to hemophore into periplasm Has a weaker affinity for heme than HasA Binds heme via 2 histidine residues Uses energy derived from proton gradient to

move heme to interior of cell

Receptor

HasA= hemophore (carrier protein that brings heme to receptor)

HasR = heme transport receptor TonB/HasB = protein complex involved in

transduction of energy from proton motive force

holoHasA = HasA with heme attached apoHasA = HasA without heme

Abbreviations

HasA receives heme Migrates to and docks onto receptor (HasR) Heme transferred from hemophore to

receptor Heme passes into periplasmic space and

enters cell Hemophore HasA dissociates and can pick

up more heme

Overview of Process

http://www.pasteur.fr/recherche/RAR/RAR2003/Mbbact-en.html

HasR can form tight complexes with both hemophores with heme (holoHasA) and those without (apoHasA)

Since HasA has high heme affinity, iron uptake can be very high at low [heme]

Good because too much reduced iron in the body is harmful

Efficiency of Process

When holoHasA is bound to HasR, heme is spontaneously transferred to receptor (no energy is required here)

Energy from proton motive force required for entry of heme into cell and apoHasA dissociation from HasR

HasB (paralog of TonB) transduces energy◦ Signaling stimulus due to transcriptional

autoregulation when HasA and heme bound to receptor

Energy requirments

http://chemistry.gsu.edu/Dixon.php

Determine function of entire heme transport system

2 ternary complexes: HasA-HasR-heme (WT and mutant HasR)

Binary complex: HasA-HasR Resolutions: 2.7 angstroms for ternary

complexes and 2.8-angstrom for binary complex

Crystal structures

WT ternary solved by MAD (multiwavelength anomalous diffraction)

Other two done by difference Fourier methods

Final residue counts:◦ HasR= 752 residues◦ HasA= 161 residues

Solving the structures

HasR contains 22 antiparallel beta-strands like other TonB-dependent receptors

Unlike others in the family, HasR has elongated extracellular loops (L2, L6, L9) – bind HasA

L7 and C apex used to attain heme

Analysis of Structures

http://strucbio.biologie.uni-konstanz.de/strucbio/

HasA-HasR-heme complex

L6L9

Heme (green) bound to L7

Initially, heme-binding site of HasA oriented to face extracellular loops of HasR

Heme then binds to the two His residues of HasR (transferred about 9.2 angstroms)◦ His-603 from L7 and His-189 from apex C of a

plug that is common in these receptors◦ Mutants of these two residues show no heme

binding

Process

http://www.pnas.org/content/106/4/1045.full

Superposition of the heme groups attached to holoHasA(blue) and to HasA-HasR-heme (red)

Spontaneous transfer from HasA to HasR Transfer is endergonic (non-spont.) Coupling of HasA and HasR is exergonic and

exothermic (spontaneous) Latter overrides former

Transfer of Heme

During complex formation, heme is not lost to solution

HasR-Ile-671 in L8 clashes with heme on holoHasA (Figure A)

Without the Ile, heme transport is not possible because the heme rotates to face HasA

Mutant with Glycine-671 used (Figure B)

Transfer of Heme

http://www.pnas.org/content/106/4/1045.full

L7 and L8 of HasR displace the loop with HasA-His-32 causing break in coordination between residue and heme

Heme and HasA-Tyr-75 (stronger connection) still persists◦ Stablized by deprotonation of phenol that H-bonds

with HasA-His-83

Process

Later, the His-83 may get protonated and so the coordination is lost

Ile-671 displaces heme from HasA Rotation of His-83 side chain prevents

sliding back of heme to hemophore

Shows that free heme can bind to HasR with apoHasA bound as well

There is a channel that goes from between loops 3 and 4 all the way to the heme binding site in which heme can travel

Binary complex

http://www.pnas.org/content/106/4/1045.full

Mirrors ABC transport of cargo from bacterial periplasm to inside cell

Both have cargo molecule bound to protein that binds to and spontaneously transfers cargo to cis receptor

Energy is required (heme-proton motive; ABC-ATP hydrolysis) to get cargo to trans and dissociate protein from receptor

Parallels of Heme Transport

How to get substance to protein with lower affinity?

Part of binding energy of donor to ligand is consumed when displacing the first loop (His-32)

Ligand transfer occurs when donor-acceptor come together due to steric clash (from Ile-671)

Analysis of Transfer

Refinement-Resolution, Å 49.2–2.7 (2.73–2.70) 49.4–3.0 (3.03–3.0) 39.2–2.8 (2.83–

2.80)-No. of reflections 99,334 (2,329) 77,295 (2,431) 92,482 (2,123)-Completeness, % 95.03 (71) 99.17 (93) 98.1 (71)-Rwork, % 23.7 (34.9) 21.4 (37.4) 22.6 (46.6)-Rfree*, % 27.3 (38.4) 24.3 (39.1) 26.2 (48.3)

Model composition-Protein residues 1,850 1,850 1,850-Heme atoms 86 0 86-Water molecules 58 19 13

B-factors-Protein 93.5 80.2 110.6-Heme 84.6 — 120.4

Deviation from ideal values-Bond lengths, Å 0.010 0.010 0.006-Residues with bad bond lengths†, % 0 0.05 0-Bond angles, ° 0.61 1.27 1.08-Residues with bad bond angles†, % 0.22 0.71 0.550

Ramachandran plot†-Favored regions, % 92.4 89.6 89.7-Allowed regions, % 99.2 99.5 99.1

measure of how well refined structure predicts observed data

R-factors usually range from 0.2-0.6 Smaller R-factor is better R-factors for the three structures are

0.237, .214, .226 for the WT ternary complex, mutant ternary, and binary complex, resp.

Shows well-defined structure

R factor