Science - USA (2022-01-21)

bind human ACE2 (e.g., RaTG13 virus RBD
affinity of 3.9mM) ( 30 ). The day 146 RBD,
however, had a similar affinity (Kdof 46 nM)
for ACE2 as that of the Wuhan-Hu-1 SARS-
CoV-2 RBD (fig. S1 and table S3).
We determined the x-ray crystal structure of
the day 146 RBD bound to the human ACE2
ectodomain (Fig. 1B, fig. S2, and table S4). This
structure is similar to previously determined

structures of ACE2–SARS-CoV-2 RBD com-

plexes ( 2 , 3 ), except we observed contacts be-

tween two N-linked glycans on ACE2 (attached

to N53ACE2and N90ACE2)andtheRBD(fig.S3).

Removing the N90ACE2glycan, which interacts

with the RBD in both copies of the crystal

asymmetric unit (fig. S3), increased Wuhan-

Hu-1 SARS-CoV-2 and day 146* RBD affinity

for ACE2, although the effect was modest (fig.

S1 and table S3). This finding is consistent

with prior work implicating the N90ACE2

glycan, which is removed in a human poly-

morphism (T92IACE2), as a barrier to SARS-

CoV-2 RBD binding to ACE2 ( 31 , 32 ).

The N501YRBDsubstitution is found in mul-

tiple VOCs (Fig. 1A); once it surfaced in the

immunocompromised individual, it was re-

tained at later time points ( 14 – 16 ). As also

Nabelet al.,Science 375 , eabl6251 (2022) 21 January 2022 2 of 10

Gamma (P.1)Epsilon (B.1.429/427)Lamda (C.37)B.1.258Iota E484K (B.1.526)Iota S477N (B.1.526)Mu (B.1.621)Kappa (B.1.617.1)Delta (B.1.617.2)Delta K417N (AY.2)Delta +3Day 146*Day 152*RBM-1RBM-2RBM-3

K 417 N/T

N 439 K

R 346 K

N 440 D

L 452 R/Q

S 477 N

T 478 K

E 484 K/Q/A

F 486 I

F 490 S

Y 489 H

Q 493 K

S 494 P

N 501 Y

K 417 N/T

N 439 K

R 346 K

N 440 D

L 452 R/Q

S 477 N

T 478 K

E 484 K/Q/A

F 486 I

F 490 S

Y 489 H

Q 493 K

S 494 P

N 501 Y

RBD substitution Alpha (B.1.1.7)Beta (B.1.351)Beta L452R (B.1.351)

134 31211132236* 66675

# of RBD substitutions

day 146*

RBD

K478

S477

F486

E484

F490

H489

K493

L452

P494

K417

RBM

Core

Variable

D440 positions

R346

N439

Y501

ACE2

CD

G

E

B

A

F

N58

glycan

N343

glycan

N103

glycan

E484 E484

Y489

H489

Q493

S494 K493

P494

Y451

Y453 Y451 Y453

N501 Y501

T500 D38 T500 D38

K353 K353

Y41 Y41

E35 H34 E35

H34

K31 K31

Y83 Y83

WT RBD/ACE2 day 146*/ACE2

C1C-A3

HC

LC

T478

S477

K417

F490

R346L452

F486

Y489

E484

Q493

S494

N501

N440

N439

N343

glycan

SARS-CoV-2

RBD

Fig. 1. Structure of intrahost evolved RBD bound to human ACE2.(A) Key
RBD substitutions discussed in the text and the SARS-CoV-2 variants that
contain them. Single-letter abbreviations for the amino acid residues are as
follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu;
M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
(B) Day 146 RBD–ACE2 ectodomain x-ray crystal structure. RBD residues
that are mutated in variants discussed in the text are shown. Boxed residues are
mutated in the day 146 RBD as compared with the Wuhan-Hu-1 (wild-type)

SARS-CoV-2 RBD. The Delta +3 variant contains an additional RBD mutation

that is not shown in the schematic diagram (see table S2). (C) Wild-type

RBD–ACE2 contacts near N501RBD[Protein Data Bank (PDB) ID 6M0J] ( 2 ).

(D) Day 146* RBD contacts near Y501RBD.(E) Wild-type SARS-CoV-2 RBD–

ACE2 interactions near Q493RBD.(F) Day 146* RBD interactions near K493RBD.

(G) Cryo-EM structure of the SARS-CoV-2 RBD bound to the C1C-A3

antibody Fab. RBD residues discussed in the text are labeled. LC, light

chain; HC, heavy chain.

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