LMP299Y1 Lecture Notes - Anthrax Toxin, Antxr2, Ion

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6 May 2014
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LMP402 - Fall 2013 - Anthrax
PA - ANTXR - PA20 and PA63 - EF/LF
CHO-K1 -> CHO-R1.1 cells
These cells have no ANTXR receptors
Do not bind PA, resistant to toxin
PA binding restored by ANTXR1 cDNA to CHO-R1.1
cDNA also restores toxin sensitivity
Anthrax toxin receptors
1. ANTXR1
2. ANTXR2
Both are ubiquitously expressed, bind extracellular components, and are type I membrane
proteins
I domain present in both - AKA VWA domain
ANTXR2-/- in macrophages - resistant to toxin, suggesting macrophages only express ANTXR2
Myeloid-only ANTXR2-/2 in mice - resistant to infection; sensitive to toxins EF and LF
Complete loss of ANTXR2 in mice - resistant to EF and LF
ANTXR1 splice variants
SV1 - longer cytosolic tail
SV2 - binds more PA than SV1
PA binds the I domain of ANTXR1/2 with divalent cation between
D683 is an amino acid in PA; aspartic acid that binds directly to divalent cation; if mutate D683,
PA cannot bind to ANTXR1/2
I domain
1. open conformation - C terminal alpha helix DOWN
2. closed conformation - C terminal alpha helix up
- phenylalanine ring moves along with alpha helix
- alpha helix shifting changes affinity for ligand
- around the MIDAS motif, from open to closed
1. T209 moves away from Mg2+
2. D242 takes T209's position
3. Mg2+ less electrophilic therefore will not bind to acidic side chain of E314, so lower ligand
affinity
T118 binds Mg in open conformation, keeps I domain open
Integrins have F302 and T209
They correspond to ANTXR I domain's F203 and T118
F302 important in modulating equilibrium between open and closed conformations
Closed - F302 (hydrophobic) into hydrophobic pocket, stabilizing closed conformation
Open - F302 hangs out, no stabilization
For ANTXR2, F203 sticks out, indicating porbably open conformation
I domains have hydrophobic pockets that stabilize conformation
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