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

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’

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)

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

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

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

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

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

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).