Heme Uptake Receptor
Jan 12, 2016
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