Science - USA (2020-09-04)

and the molecular basis for affinity enhance-
ment can be rationalized from the RBD-bound
ACE2 cryo–electron microscopy (EM) struc-
ture (Fig. 1E) ( 17 ): Hydrophobic substitutions
of ACE2-T27 increase hydrophobic packing
with aromatic residues of S, ACE2-D30E ex-
tends an acidic side chain to reach S-K417,
and aromatic substitutions of ACE2-K31 con-
tribute to an interfacial cluster of aromatics.
A search for affinity-enhancing mutations in
ACE2 using targeted mutagenesis recently
identified D30E ( 43 ), providing independent
confirmation of this result.

There are also enriched mutations in the

second shell and farther from the interface

that do not directly contact S but instead have

putative structural roles. For example, proline

substitutions were enriched at five library posi-

tions (S19, L91, T92, T324, and Q325) where

they might entropically stabilize the first

turns of helices. Proline was also enriched at

H34 where it may enforce the central bulge

ina1, and multiple mutations were enriched

at buried positions where they will change

local packing (e.g., A25V, L29F, W69V, F72Y,

and L351F). The selection of ACE2 variants

for high binding signal therefore reports not

only on affinity, but also on presentation at the

membrane of folded structure recognized

by SARS-CoV-2 S. Whether these mutations

selectively stabilize a virus-recognized local

structure in ACE2 versus the global protein

fold is unclear.

Thirty single–amino acid substitutions highly

enriched in the nCoV-S-High sorted cells were

validated by targeted mutagenesis (fig. S3).

Binding of RBD-sfGFP to full-length ACE2

mutants measured by dual-color flow cytom-

etry (fig. S3) increased compared with that of

Chanet al.,Science 369 , 1261–1265 (2020) 4 September 2020 2of5

Fig. 1. Sequence preferences

of ACE2 residues for high

binding to the RBD of SARS-

CoV-2 S.(A) Log 2 enrichment

ratios from the nCoV-S-High

sorts are plotted from depleted

or deleterious (orange) to

enriched (dark blue). ACE2

primary structure is shown on

the vertical axis, amino acid

substitutions are indicated on

the horizontal axis. Wild-type

amino acids are in black. Aster-

isk (*) denotes stop codon.

(B) Conservation scores are mapped

to the structure (Protein Data

Bank 6M17) of RBD (green

ribbon)–bound protease domain

(surface), oriented with the

substrate-binding cavity facing

the reader. Residues conserved

for RBD binding are shown in

orange; mutationally tolerant

residues are in pale colors;

residues that are hot spots for

enriched mutations are in blue;

and residues maintained as wild

type in the ACE2 library are in

gray. Glycans are depicted as

dark red sticks. (C) Viewed

looking down on to the RBD

interaction surface. (D) Average

hydrophobicity-weighted enrich-

ment ratios are mapped to the

structure, with residues tolerant

of polar substitutions in blue and

residues that prefer hydrophobics

in yellow. (E) A magnified view

of the ACE2–RBD interface

[colored as in (B) and (C)]. Heat-

map plots log 2 enrichment ratios

from the nCoV-S-High sort. Ab-

breviations 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.

MLIHQNTSAV DEGRK W*FY CP

S 19

Q 24E 23

E 22I 21

T 20

F 28L 29

T 27K 26

A 25

D 38E 37

E 35H 34

N 33K 31

D30

A 46L 45

Q 42Y 41

F 40L 39

E 56I 54

N 51Y 50

N 49W 48

N 64N 63

M 62N 61

Q 60V 59

E 57

F 72A 71

W 69K 68

D 67A 65

M 82L 79

Q 76E 75

K 74L 73

T 92L 91

N 90Q 89

P 84Y 83

R 273Q 102

Q 98Q 96

V 93L 95

E 329G 326

Q 325T 324

N 290N 277

T 276F 274

W 349T 347

P 346H 345

V 343N 330

G 354K 353

G 352L 351

D 350

M 366R 357

F 356D 355

H 374T 371

L 370D 367

A 386M 383

D 382H 378

Amino Acid Substitution

ACE2 residue position

R 393F 390

P 389Q 388

A 387

E 402H 401

E 398N 394

K 441F 438

S 409E 406

F 504I 446

T 445Q 442

S 511Y 510

D 509H 505

R 518Y 515

R 514F 512

S-RBD

ACE2

N90-glycan

B

C

T27D30K31

WT

WT

WT

K31

T27

E

D30

K417

L455

Y489

Y453

Q493 H34

E35

F456F456F456

M

L

I

V

A

S

T

N

Q

D

E

K

R

H

W

Y

F

P

G

C

*

Under selectionto change Conserved Tolerates polaramino acids hydrophobicsPrefers

A

D

-3 (depleted)

+3 (enriched)

Log Enrichment Ratio

2

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