Science - USA (2022-04-08)

164 8 APRIL 2022¥VOL 376 ISSUE 6589 science.orgSCIENCE

Fig. 1. Purification and biochemical characterization of
an active JAK1-IFNlR1 complex.(A) Schematic of a
cell-surface, ligand-induced cytokine receptor-JAK dimer.
(B) Schematic of a soluble cytokine receptor dimer mimetic
(“mini-IFNlR1”) in which the transmembrane domains of the
receptor have been replaced with a GCN4-zipper and the
intracellular tail has been trucated after Box1/Box2. (C) Mini-
IFNlR1 expression enhances JAK1 phosphorylation when
coexpressed in insect cells. Wild type (WT) or Val^657 →Phe
(VF) JAK1 was coexpressed with mini-IFNlR1 inTrichoplusia ni
(T. ni) cells by baculovirus transduction. JAK phosphorylation
and total expression were measured 2 days after infection
by immunoblot of whole-cell lysate. Results are representative
of more than two independent experiments. (D) Schematic
of the WT EpoR/Epo complex (left) and EpoR-IFNlR1
chimera (right) showing substitution of Box1/Box2 motifs.
Cytokine-mediated dimerization of IFNlR1 Box1/Box2
results in JAK1 phosphorylation in mammalian cells. NIH 3T3
cells transiently expressing mEpoR or the mEpoR-IFNlR1
chimera were stimulated with Epo for 20 min before analysis
of JAK phosphorylation by immunoblot. Results are repre-
sentative of two independent experiments. (E) Affinity
purification of JAK1 using mini-IFNlR1 yields a stable,
nonaggregated complex. Superose 6 size exclusion
chromatography (SEC, left) and sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE, right) of the
JAK1-IFNlR1 complex. (F) Representative 2D class averages
from single-particle cryo-EM imaging of the JAK1-IFNlR1
complex. PM, plasma membrane; R, receptor; mAu,
milliabsorbance units; BiBC2 nb, tandem BC2 nanobody.

Box1

Box2

Pseudokinase

(PK)

Janus Kinase (JAK)

FERM-SH2

Tyrosine

Kinase

(TK)

A

CytokineR

PM

BCInsect Cells (T. ni):

mini-IFNλR1:

mJAK1

WT

mJAK1

VF

--++

Zipper

MW(kDa)

mini-IFNλR1

mJAK1

BiBC2 nb

10

15

20

25

37

50

75

100

150

250

0 5 10 15 20 25

0

10

20

30

40

50

60

volume (mL)

A280 (mAu)

E

Cytokine

Superose 6 SEC SDS-PAGE

D

EpoR (WT)EpoR-IFNλR1

chimera P -JAK2

P -JAK1

JAK2

NIH 3T3 cells:

mEpo:+-+ - +

mEpoR

WT

mEpoR-

λR1

JAK1

P -JAK1

JAK1

F 2D class averages

50 Å

JAK2 JAK2 JAK1 JAK1

EpoR Box1/2IFNλR1 Box1/2

Box1

Box2

y

Mini-IFNλR1

PM

FERM-SH2

PK

TK

CytokineR

Top View

Side View

FERM-SH2

PK

TK

IFNλR1

IFNλR1

FERM-SH2

FERM-SH2

IFNλR1

IFNλR1

90 o

90 o

Cytokine

IFNλR1

Bottom View

PK

TK

180 o

FERM-SH2

PK

TK

PK

TK

AB

C

D

Fig. 2. Cryo-EM structure of the active JAK1-IFNlR1

dimer.(A) Segmented density map of the JAK1-IFNlR1

dimer resolved to 3.6-Å resolution with extracellular and

transmembrane domains shown as schematic. Subse-

quent panels show top (B), side (C), and bottom (D) views

of the complex. The map threshold used in ChimeraX is

set to 0.2 (~5.2s). Individual JAK monomers are colored as

a light-to-dark gradient from the N to the C terminus.

Monomer 1: FERM-SH2, light green; PK, green; TK, dark

green. Monomer 2: FERM-SH2, pink; PK, orchid; TK,

purple. Density corresponding to IFNlR1 is colored blue.

R, receptor; PM, plasma membrane.

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