a3 p2 a3 p2 a2 5’ a2 5’ a1 p3 a1 p3 2-5a · wtp + 2-5a 0.75 ± 0.06 y310a + 2-5a 0.54 ± 0.05...
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Supplementary Figure 1
B
AMP-PNP AMP-PNP
RNAse L + 2-5A + AMP-PNP RNase L + 2-5A
A1A2
A3
P1
P2
P35’
2’
2-5A
A1A2
A3
P1
P2
P35’
2’
2-5A
RNase L + 2-5A + AMP-PNP RNase L + 2-5AA
B C
D
RNase L crystal contact 1 RNase L crystal contact 2
Yeast IRE1 crystal contact
Linear translation Linear translation
Helical translation
Supplemental Text and Figures
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αAR1 αAR2 αAR3
αAR4 αAR6αAR5 αAR7 αAR8
αAR8’ αAR10αAR9 αAR11 αAR12
αAR13 αAR15αAR14 αAR16
αAR17 αAR18 αA
αC
αEαD
αGαF αGH
αH αI α1
α3 α3’ α4α2
α6 α7α5 α8
αEF αFactivation segment αEF-αF loop
catalytic loop
β1 β2 β3 β4 β5
β6β6’
β8 β9
β7
Self-domain dimerization ANK-PK interaction 2-5A interaction AMP-PNP/Mg2+ interaction
AN
K d
omai
nPK
dom
ain
RN
ase
dom
ain
β9
non-canonical β9-αF loop
Supplementary Figure 2
α1’
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D371K368
E365
E374
Y378
R391
I363D364
R353
F362
S393
Q400E394
S426
S423
I351
G395
C427
G425
I162
R308E235
R269G236
R201
M199
Q159
K164
E55
W56
D158
R161
W352Y310R319
S312K316
R307
R282
R135Y133
D68
M67W58
E33
Q66
Q32
Q29
I99
Supplementary Figure 3
Q337
P730
K493
N461K726
E457E587
N494
K722F724
R475A411
E472D413
T417R410S408
Q407Y419Q385E383
K358
Y382
G420
S421E422
F497
E599S600
N598 K603 R675H680
E683N682
R591
D595
R594
E679
N682
100ο
Self-domain dimer interface
ANK-PK dimer interface (in trans)
2-5A binding surface
AMP-PNP:Mg2+ binding surface
ANK-PK dimer interface (in cis)
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Prot
ein
Kina
se d
omai
nR
Nas
e do
mai
n
AMP-PNPAMP-PNP
C-lobe
N-lobe
C-lobe
N-lobe
RNase L
Prot
ein
Kina
se d
omai
nR
Nas
e do
mai
n
ADPADP
C-lobe
N-lobe
C-lobe
N-lobe
IRE1
Ankyrin repeat
domain
Kinase
domain
RNase
domain
Protomer 1
N-lobe
C-lobe
A
2-5A
W352
R308
A
B
AMP-PNP
F583
W586
W584
Y712
M709
F299
K302
W300
Y415
I412
90 90
I
F
I
FH
H
1
8
7
6
1
8
7
6
2
2
A
AR17
AR18
R1
R6
R5
R4
R3
R2
R8
R7
R9 R9
Supplementary Figure 4
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2-5A (porcine RNaseL)2-5A (human RNaseL)
A1
A2
A3
P1
P2
P3
5’
2’
ANK domain (porcine RNaseL)ANK domain (human RNaseL)
Supplementary Figure 5
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0 2 4 6 8 10 120.0
0.5
1.0
1.5
0 2 4 6 8 10 120.00
0.05
0.10
0.15
0 2 4 6 8 10 120.00
0.01
0.02
0.03
0.04
0.05
0 2 4 6 8 10 120.00
0.05
0.10
0.15
0 2 4 6 8 10 120.00
0.01
0.02
0.03
0.04
0.05
0 2 4 6 8 10 120.0
0.5
1.0
1.5
2.0
0 2 4 6 8 10 120.0
0.1
0.2
0.3
0.4
0.5
0 2 4 6 8 10 120.00
0.05
0.10
0.15
0 2 4 6 8 10 120.00
0.02
0.04
0.06
0.08
0.10
0 2 4 6 8 10 120.0
0.5
1.0
1.5
2.0
2.5
0 2 4 6 8 10 120.0
0.2
0.4
0.6
0.8
0 2 4 6 8 10 120.0
0.1
0.2
0.3
0 2 4 6 8 10 120.00
0.05
0.10
0.15[RNAse L]
0.5 μM
1 μM
3 μM
10 μM
c(S)
c(S)
c(S)
Sedimentation Coefficient (s)
c(S)
APO
Monomer Dimer
0 2 4 6 8 10 120.0
0.5
1.0
1.5
2.0
Sedimentation Coefficient (s)
0 2 4 6 8 10 120.0
0.1
0.2
0.3
0.4
0.5
40 μM 2-5A
Monomer Dimer
Sedimentation Coefficient (s)
250 μM AMP-PNP
Monomer Dimer
Sedimentation Coefficient (s)
0 2 4 6 8 10 120.0
0.1
0.2
0.3
250 μM AMP-PNP + 40 μM 2-5A
Monomer Dimer
0 2 4 6 8 10 120
500
1000
1500
2000
Sedimentation Coefficient (s)
c(s)
0 2 4 6 8 10 120
100
200
300
400
500
c(s)
0 2 4 6 8 10 120
50
100
150
200
c(s)
0 2 4 6 8 10 120
500
1000
1500
2000
Sedimentation Coefficient (s)
c(s)
0 2 4 6 8 10 120
100
200
300
400
500
c(s)
0 2 4 6 8 10 120
50
100
150
200
250
c(s)
[RNase L]
3 nM
10 nM
30 nM
2 μM 2-5A 2 μM 2-5A & 12.5 μM AMP-PNP
Monomer Dimer Monomer Dimer
A
B
Supplementary Figure 6
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0 10 20 300
1.0
2.0
3.0
4.0
WT+RNA+2-5A+ADPN487A+RNA+2-5A+ADPD482A+RNA+2-5A+ADPE402A+RNA+2-5A+ADPD500A+RNA+2-5A+ADPK390A+RNA+2-5A+ADPL432A+RNA+2-5A+ADPA388F+RNA+2-5A+ADPRNA alone
Time (min)
Fluo
resc
ence
Inte
nsity
(x10
8 )
C
A
B
2-5A binding to full length RNase Lin the presence of 20 μM ATP
A binding to full
2-5A binding to full length RNase Lin the presence of 20 μM ATP
0 200 400 600 800 10000
50
100
[Full length RNase L] nM
Perc
enta
ge R
espo
nse
Uni
ts
Kd (FL RNase L) = 104 nM ± 18 n = 2
0 5 10 150
50
100
150
200
WT 0.63 ± 0.06Y310A 0.47 ± 0.04crystal 0.33 ± 0.02WT + 2-5A 0.75 ± 0.06Y310A + 2-5A 0.54 ± 0.05crystal + 2-5A 0.36 ± 0.03
[RNase L] μM
mP,
arb
itrar
y un
its
Kd
Supplementary Figure 7
ATP binding to full length RNase L
ATP binding site mutants
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SUPPLEMENTARY FIGURE LEGENDS
Figure S1. Representative electron density maps and crystal contacts. Related to Figure 1. (A) Unbiased Fo-Fc electron density maps (contoured at 2.5σ) centered on the 2-5A (top panels) and AMP-PNP (bottom panels) ligand binding sites. Maps reflect an atomic model prior to introduction of ligands into the refinement process. Ligands from the final model are shown for comparison. In the absence of electron density for ADP in the ADP free RNase L crystal structure, ADP was not modeled. (B & C) Packing contacts of RNase L dimers present in the crystal lattice. Protomers within each 2-5A induced dimer are coloured blue and green or red and beige with RNase active sites highlighted by black circle. Inter-dimer contact area = 603 Å2 and 1718 Å2 for (B) and (C), respectively. (D) Crystal packing contacts of yeast IRE1 dimers implicated in polymer formation (PDBID 3FBV). Protomers within each back-to-back dimer are coloured blue and green or red and beige with RNase active sites highlighted by black circle. Inter-dimer contact area = 2714 Å2. Figure S2. Sequence alignment of RNase L orthologues and Ire1. Related to Figures 1, 2 and 3. Structure based multiple sequence alignment of RNase L sequences from Sus scrofa (porcine), H. sapiens (human), M. musculus (mouse), B. taurus (cattle), G. gallus (chicken) and the dual PK domain – RNase domain module of S. cerevisiae (yeast) Ire1 (PDBID 2RIO). The ANK domain, PK domain, and RNase domain regions are highlighted by blue, tan and pink backgrounds. Residues invariant or highly conserved across the RNase L orthologues are shaded black and grey respectively. The secondary structure of S. scrofa RNase L is drawn above the alignment. Disordered regions are depicted as dashed lines. Non-canonical features of the pseudokinase domain are highlighted by coloring the secondary structure in red. Conserved RNase domain catalytic residues are colored in purple and key protein kinase catalytic motifs are indicated by magenta-bordered boxes. 2-5A—interacting residues are indicated by stars and AMP-PNP—interacting residues are indicated by grey triangles. Cross-protomer contacts (calculated by AREAIMOL) are indicated by filled boxes colored in green (ANK-PK contacts) and in orange (ANK-ANK, PK-PK or RNase-RNase contacts) boxes above the alignment. Dashed lines represent not modeled disordered regions of the crystal structure. Figure S3. Contact surfaces between two RNase L protomers. Related to Figure 3. Surface representation of a single RNase L protomer with dimer interface residues highlighted. Surfaces of atom pairs closer than 5.9 Å are colored as indicated. AMP-PNP and 2-5A are shown as sticks. Figure S4. Structure analysis of RNase L. Related to Figure 4. (A) Ribbons comparison of the back-to-back dimer configuration of the dual pseudo PK domain – RNase domain module of RNase L and IRE1. A detailed representation of the
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contiguous hydrophobic core linking the protein kinase and RNase domains is shown in the zoom in views below. (B) Ribbons representation of the tenuous contact between ANK and PK domains within each protomer of a higher order dimer. A detailed representation of interacting residues is shown in the zoom in view to the right. Figure S5. Structure comparison of ANK domains and 2-5A ligands from isolated ANK and full length RNase L structures. Related to Figure 4. Cartoon representation of superimposed ANK domains of porcine RNase L and isolated human ANK domain structure (PDBID 4G8L) with bound 2-5A. Bound 2-5A ligands are shown in the zoom in view at top. 2-5A bound to human RNase L (ANK domain alone) is depicted as sticks with grey colored carbons and cyan colored phosphates. 2-5A bound to porcine RNase L is depicted as sticks with magenta colored carbons and orange colored phosphates. Figure S6. Influence of ligand binding on the oligomerization status of WT porcine RNase L. Related to Figure 5. Analytical ultracentrifugation analyses were performed on (A) WT RNase L using refractive index detection or (B) fluorescein labeled WT RNase L using fluorescence detection at the indicated protein concentrations with the indicated concentrations of ligands. Figure S7. In vitro functional characterization of RNase L. Related to Figure 5. (A) Binding of 2-5A to full length RNase L in the presence of 20 µM ADP assessed by surface plasmon resonance. Normalized response levels for specific binding of RNase L to immobilized biotinylated 2-5A were plotted against increasing protein concentration. Reported Kd is the average of two independent experiments (+/- s.e.m). Displayed results are representative of two independent experiments. (B) RNase activity profiles for wild type RNase L and the indicated ATP binding site mutants monitored in the presence of 2-5A activator and 50 µM ATP using a FRET pair labeled RNA substrate. Displayed results are representative of two independent experiments measured in duplicate (+/- s.e.m.). (C) Binding of the fluorescently labeled ATP analogue (BODIPY FL ATP-γ-S) to full length RNase L, a Tyr310Ala mutant, and the crystallization construct (crystal) in the presence and absence of 3 µM 2-5A activator assessed by monitoring the fluorescence polarization signal in the presence of increasing concentrations of protein. Displayed results are representative of two independent experiments measured in duplicate (+/- s.e.m).
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