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An artiﬁcial metallolyase with pliable 2-His-1carboxylate facial triad for stereoselective Michael
addition†
Ryusei Matsumoto,a Saho Yoshioka,b Miho Yuasa,b Yoshitsugu Morita,aGenji Kurisu c and Nobutaka Fujieda *a
We repurposed the metal-binding site of a cupin superfamily protein into the 2-His-1-carboxylate facial
triad, which is one of the common motifs in natural non-heme enzymes, to construct artiﬁcial

Received 11th December 2022
Accepted 28th February 2023

metalloenzymes that can catalyze new-to-nature reactions. The Cu2+-H52A/H58E variant catalyzed thestereoselective Michael addition reaction and was found to bear a ﬂexible metal-binding site in the highresolution crystal structure. Furthermore, the H52A/H58E/F104W mutant accommodated a watermolecule, which was supported by Glu58 and Trp104 residues via hydrogen bonding, presumably

DOI: 10.1039/d2sc06809e

leading to high stereoselectivity. Thus, the 2-His-1-carboxylate facial triad was conﬁrmed to be

rsc.li/chemical-sciencea versatile and promising metal-binding motif for abiological and canonical biological reactions.

Introduction
Many natural enzymes accommodate metal ions for structural
stability and to catalyze numerous challenging reactions.1
Mononuclear non-heme metalloenzymes catalyze the chemicaltransformation of substances, such as aromatic C–H bondhydroxylation and cis-dihydroxylation of arene double bonds.2,3
Based on this competent catalytic ability, various approaches todevelop engineered metalloproteins with catalysis and articial
metalloenzymes (ArMs) have been developed. In ArMs, chemo-,
regio-, and/or enantioselectivity is endowed with the boundsynthetic metal complex by the matrix of the host protein.
Additionally, the chemical stability and solubility in water areconferred to the protein-bound metal complexes. Therefore,
ArMs are a powerful means of accelerating chemically diﬃcult
reactions under environmentally benign conditions. Several
methods, such as covalent, supramolecular anchoring, andcofactor substitution, have been established for the sitedirected anchoring of metal complexes to the protein
cavity.4–10 An alternative strategy involves the direct linking ofbare metal ions with coordinating endogenous amino acids via
dative covalent bonds.11 This type of ArM is constructed bya

Department of Applied Biological Chemistry, Graduate School of Agriculture, Osaka
Metropolitan University, 1-1 Gakuen-cho, Naka-ku, Sakai-shi, Osaka 599-8531,
Japan. E-mail: fujieda@omu.ac.jpbDepartment of Applied Life Sciences, Graduate School of Life and Environmental
Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai-shi, Osaka
599-8531, Japanc
Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka 5650871, Japan† Electronic supplementary information
https://doi.org/10.1039/d2sc06809e

3932 | Chem. Sci., 2023, 14, 3932–3937(ESI)

available.

See

DOI:

employing non-metalated natural or de novo proteins or by
repurposing natural non-heme metalloenzymes, oen aided bycomputational design.11–13 The construction of endogenous
amino acid-ligated metal active centers within suitable protein
cavities to achieve simultaneous selective substrate binding andcatalytic activity is signicantly more challenging.
Recent crystal structures of mononuclear non-heme metalloenzymes have revealed the existence of a common metalbinding motif, which consists of two histidines and an aminoacid with a carboxylic acid side chain, that is, glutamate or
aspartate (His-His-Glu [HHE] or His-His-Asp [HHD]).2,14 This
motif has been designated as a 2-His-1-carboxylate facial triad,
where two nitrogen atoms and one oxygen atom coordinate to
the central metal in fac fashion (Fig. 1).2 The fac-type coordination of three amino acid residues at the octahedral coordination center oﬀers three vacant coordination sites on the
opposite side, which provide important space for the substrates
and coenzymes to bind.
Several ArMs containing His-His-Asp [HHD] fac-triad active
sites have been reported based on this triad.15,16 An articial CuDiels-Alderase was constructed by engineering an HHD fac-triad
within the non-metalated TIM barrel protein, imidazole glycerol
phosphate synthase (tHisF).17 The ArM possesses an engineered
metal binding site consisting of Leu50 and Ile52, which was
mutated to histidines, plus the native aspartic acid at position
11 and exhibited 73% conversion of 2-azachalcone compound 1
with good enantioselectivity (enantiomeric excess [ee] = 46%;
endo : exo = 93 : 7) in the reaction with cyclopentadiene.17
Another articial Cu-Diels-Alderase was generated by metal
swapping the natural Fe2+ of HHD fac-triad active site in 1aminocyclopropane carboxylic acid oxidase with the Cu2+ ion.
In same reaction, it gave same product with high quantitative© 2023 The Author(s). Published by the Royal Society of ChemistryView Article Online

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Chemical ScienceFig. 1 The extended metal ligand library with 2-His-1-carboxylate triad containing TM1459 cupin proteins for Michael addition reaction of
methyl acetoacetate. (A) Subunit structure of Cu-bound H52A variant (subunit A, PDB code 6L2E) and model structures of 3-His triad motifs
produced in silico. (B) Model structures of 2-His-1-Glu triad motifs manually produced in silico. (C) Subunit structure of Cu-bound H52A/H58E
variant (subunit A, PDB code 8HJX, see Tables S1–S4 and Fig. S1, S2†). Protein main chain is displayed as a ribbon, key amino acid residues as
sticks, Cu ions as green spheres, and water molecules as red spheres.

yield associated and excellent stereoselectivity (ee = 95%;
endo : exo = 99 : 1).18 Articial Fe proteins that simulate the
HHD fac-triad active site in non-heme monooxygenases havebeen developed by combining metal complexes and streptavidin.19 On the other hand, articial b-lactamases were built via
the self-assembly of a monomeric redox protein that has HisHis-Glu [HHE] triad zinc sites in its interfaces.20,21
We constructed ArMs using the homodimeric protein from
Thermotoga maritima, TM1459,22,23 which belongs to the cupin
superfamily and has a small molecular mass of 12 977 Da(residues 1–114). The protein possesses a metal-binding site
consisting of four histidines, which is reminiscent of trispyridylamine (TPA) ligand, that can also be used as a macromolecular metal ligand.24 We repurposed Os-TM1459 into articialperoxygenase to catalyze stereoselective cis-1,2-dihydroxylation
reaction.24 Moreover, we reported articial metallolyase basedon the Cu-TM1459 cupin to accelerate stereoselective Michael
addition reactions using nitroalkane and 2-azachalcone 1 asmodel substrates to simultaneously screen the primary andsecondary coordination sphere with mini TM1459 variant
library.25 Several TM1459 variants containing three histidinebearing metal-binding site (3-His triad, Fig. 1A) and two
histidine-bearing site (2-His dyad) are employed in the reaction
screening.25 Michael addition reactions are very useful in theconstruction of carbon–carbon bonds that can be used in
combination with various electrophiles and nucleophiles.
Consequently, excellent enantioselectivities have been reported
for Michael addition reactions of dimethylmalonate to unsaturated 2-acyl imidazoles or 2-azachalcone 1 with the catalytic
system of ArMs based on DNA26–28 and small proteins.29 In
particular, compounds obtained with chalcone derivatives as
acceptors and malonate derivatives as donors can yield transcyclopropanes and oxetane derivatives, which are used as
structural motifs for some pharmaceuticals and bioactive
substances.30,31 Herein, we report our recent eﬀorts to expand
our approach by creating a 2-His-1-carboxylate triad (HHE,
Fig. 1B and C, see Tables S1–S4 and Fig. S1, S2†), which can© 2023 The Author(s). Published by the Royal Society of Chemistry

catalyze the Michael addition reaction of a,b-unsaturated
ketone, 2-azachalcone 1, with representative active methylene
compounds, such as dimethylmalonate and methyl
acetoacetate.

Results and discussion
We rst screened TM1459 variants containing 3-His triads(H52A, H54A, H58A, and H92A, Fig. 1A) and 4-His tetrad (wildtype) as metal-binding motifs. When employing dimethylmalonate as a Michael donor (Tables S5–S7 and Fig. S1†), the wildtype TM1459 did not show enantioselectivity. On the other
hand, H52A mutant could produce the (−)-isomer of compound
2 with excellent selectivity (enantiomeric excess (ee) = 86%;
Table S7,† entry 3), whereas the H92A mutant generated theinverted stereoisomer (+)-compound 2 with moderate selectivity(ee = 58%; Table S7,† entry 6), demonstrating that the nucleophilic attack of active methylene species can be stericallycontrolled by the protein matrix of these Cu-TM1459 variants(Fig. S3†). The absolute conguration of (−)-compound 2 wasdetermined to be (R)-compound 2 by comparison with the corresponding pyridine N-oxide adduct (N-oxygenated, following
the treatment of (−)-compound 2 with H2O2, see ESI† Experimental section).
The substrate scope of the reaction was then examined using
methyl acetoacetate as the donor. In the case of methyl acetoacetate, the active methylene species attack a,b-unsaturated
ketones, producing diastereomers because of the diﬀerent
substituents and acetyl and methyl ester groups. First, thecatalytic reactions were conducted using the same series of
TM1459 variants bearing the 3-His triad and wild-type containing one 4-His tetrad. Under the optimized conditions (Table
S8†), the diastereomeric ratio (d.r.; Table 1, entry 1) was notobserved without TM1459 variants. In contrast, TM1459 variants exhibited various d.r. and ee values (Table 1, entry 2–6).
H52A and H92A, in particular, preferred the anti-form andshowed very good enantioselectivity and very good yield, partly

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Table 1

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Addition of methyl acetoacetates to azachalcones 1 catalyzed by Cu-TM1459 mutantsa,b

Entry

TM1459 variant

Yieldb,c (%)

d.r. (anti : syn)eed (%) (anti)

Selectivitye

TON

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

—
Wild type
H52A
H54A
H58A
H92A
H52A/H54E
H52A/H58E
H52E/H54A
H54A/H58E
H52E/H58A
H54E/H58A
H52E/H92A
H52A/H58E/V19L
H52A/H58E/F41W
H52A/H58E/I49Q
H52A/H58E/F104W
H52A/H58Q

36
35
82
4
31
72
33
64
37
42
70
76
37
93
97
85
76
10

51 : 49
57 : 43
55 : 45n.d.
46 : 54
70 : 30
61 : 39
52 : 48
60 : 40
56 : 4456 : 44
59 : 41
55 : 45
57 : 43
62 : 38
52 : 48
71 : 29
n.d.

n.d.
38(+)
80(−)
n.d.
16(+)
86(+)
24(−)
96(−)
36(−)
4(+)
12(−)
4(−)
20(−)
88(−)
94(−)
94(−)
90(−)
n.d.

n.d.
39%
50%
n.d.
27%
65%
38%
52%
41%
29%
31%
31%
33%
54%
60%
50%
68%
n.d.

12
12
27
1
10
24
11
21
12
14
23
25
12
31
32
28
25
3

a
Reaction conditions: TM1459 (0.3 mM), CuSO4 (0.3 mM), 1 (10 mM), methyl acetoacetate (60 mM), potassium phosphate buﬀer (pH 6.5)/CH3CN(9 : 1), 20 °C, 16 h. b Yields, diastereomeric ratio (d.r.) and enantiomeric excesses (ee) were determined by chiral HPLC analysis. c Yields were
calculated based on the total amount of stereoisomers. d (+) or (−) was determined by polarimeter on HPLC. e Single isomer/total.

reecting that the pKa value of a-proton of methyl acetoacetate(y11) is lower than that of dimethylmalonate (y13).32 Then, we
chose the variant with 3-His triad as a scaﬀold, which was
repurposed into 2-His-1-carboxylate triads, introduced bymutating one histidine to metal-coordinating glutamate. Thismutation could slightly perturb the substrate-binding cavity
and Cu-coordinating water molecule because of its smaller size
than the imidazole and bidentate ability of the carboxyl group,
while maintaining a coordination geometry similar to that of
the 3-His triad. (Fig. 1B and C).
While the 3-His triad of H54A and H92A mutants exhibited
meridional coordination geometry, those of H52A and H58A
mutants presented facial coordination geometry in silico,although the 3-His facial triad structure was conrmed in the
crystal structure of H52A (Fig. 1A). As a result of the mutation ofa His residue to Glu, the corresponding mutants to H54A and
H92A, and H52A and H58A displayed meridional and facial
triads, respectively (Fig. 1B). The 7 corresponding mutants of 12(Fig. S1 and S2†) were screened for the reaction with methyl
acetoacetate (Table 1, entry 7–13). We found a drastic increase inenantioselectivity in the case of the H52A/H58E mutant, withgood yield (Table 1, entry 8 and Fig. S4†). Typically, the 2-His-1carboxylate facial triads in natural enzymes are consideredbenecial structural motifs. In this experiment, we used mutants
with meridional and facial triads. Screening revealed that the
H52A/H58E mutant was obtained as a variant that exhibited highenantioselectivity and was postulated to have a facial triad, whichis required for selective and eﬃcient catalysis.

3934 | Chem. Sci., 2023, 14, 3932–3937

To clarify the stereochemical mechanism that can accountfor the improvement in enantioselectivity, the crystal structureof the Cu-bound H52A/H58E mutant was determined with

Fig. 2 (A) Crystal structure of Cu-binding site of H52A/H58E (chain B,
PDB code 8HJX). (B) Superimposed structures of the Cu-binding sitesof H52A/H58E (pink) and H52A (blue, chain B, PDB code 6L2E) (C)Surface models and close-up views of Cu site of H52A/H58E of insilico-obtained azachalcone-TM1459 complex. 2Fo − Fc and anomalous maps contoured at 1.5 and 5.0s are shown in gray and magenta
mesh, respectively.

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highly reliable crystallographic statistics at a resolution of 1.15
Å (Fig. 1C, 2A, S5A, S5B, and Tables S1–S3†). The coordination
structure of the Cu center was shown to be a 2-His-1-carboxylate
facial triad, completing the square pyramidal geometry of the
N2O3 donor set, with two nitrogen atoms of histidines (His54
and His92), two water molecules in the equatorial position (O1and O2), and an oxygen atom of glutamate (Glu58) in the axial
position (Fig. 2A). In the reported crystal structure of H52A (PDB
code 6L2E), Cu ions are supported by a 3-His triad with twowater molecules. His54, His58, and two water molecules arelocated in the equatorial position and His92 sits in the axial
position in chain A, whereas His58, His92, and two watermolecules are located in the equatorial position and His54 islocated in the axial position of chain B (O2′ and O3′, Fig. 2B).
This change in the coordination structure implies that there areat least three types of binding modes between the Cu center andcompound 1 as bidentate ligands, causing low stereoselectivity.
In the case of H52A/H58E, the glutamate placed at 58 position
supported the Cu ion by one carboxyl oxygen atom in a monodentate fashion, and the other carboxyl oxygen connected with
Cu-coordinating water via hydrogen bonds (O1, Fig. 2A), likely
preventing the ligand exchange of this water molecule with
substrate compound 1. Therefore, compound 1 could access theCu center only from the distal side of Glu58, xing the binding
mode to determine its chiral plane. In stark contrast to the Cu
center of H52A, electron density due to minor Cu species wasobserved (Cu(B), Fig. 2A). Because of the rearrangement of
coordination sphere, the ligand geometry of minor species isslightly diﬀerent from major ones, where coordinate bond
between Glu58 and Cu is slightly elongated from 2.20 ± 0.03 Å
to 2.73 ± 0.04 Å, generating a sterically less hindered Cu center(Table S3†). This possibly makes it easy for the ketone oxygen
and pyridine nitrogen of 1 to coordinate to the Cu ion from thepositions trans to His92 and Glu58 (O2 and O3 positions,Fig. 2B), respectively, forming an octahedral intermediate containing the Jahn–Teller axis.
To scrutinize the binding mode of 1, a docking simulation
with azachalcone was performed using this structural coordinate (PDB code 8HJX) and AutoDock Vina soware. The lowest
energy conformation of 1 is very similar to that of the H52A
mutant in our previous study,15 and the ketone and pyridine
moieties pointed to the Cu center (calculated free energy ofbinding [DGb]: −5.1 (kcal mol−1), Fig. 2C). Together with the
crystal structure, it appears likely that the ketone oxygen andpyridine nitrogen bind to the Cu center from the positions transto Glu58 and His92, respectively, to form the intermediate(Fig. 2C). In the azachalcone-docked structure, b-Si-face attackby methyl acetoacetate probably occurs at the position indicatedby the dotted circle (Fig. 2C). This space is tightly packed by vehydrophobic amino acid residues, Val19, Phe41, Ile49, Phe94,
and Phe104, while the opposite side is solvent-accessible(Fig. 2C).
To substantiate this assumption, we selected four aminoacid residues (Val19, Phe41, Ile49, and Phe104) in the secondary
coordination sphere as mutation targets for H52A/H58E basedon the results of the docking simulation. To inuence theinteraction of this cavity with methyl acetoacetate, we replaced© 2023 The Author(s). Published by the Royal Society of Chemistry

Chemical Science
them with larger amino acid residues. The additional mutationof V19L and I49Q in H52A/H58E did not aﬀect the diastereoselectivity, although the yields were enhanced to an excellent
extent (Table 1; entries 14 and 16). On the other hand, H52A/
H58E/F41W and H52A/H58E/F104W were found to give antipreference up to 62 : 38 and 71 : 29 (d.r.), respectively, whichwere higher than those of H52A and H52A/H58E, retaining
excellent enantioselectivity (ee = 94 and 90%, respectively;
Table 1, entries 15, 17, and Fig. S4†). These results also partly
support the hypothesis that methyl acetoacetate can access 1
from the Si-face of the double bond b-carbon of azachalcone in
the docked structure.
To elucidate the substrate-binding mode in the H52A/H58E/
F104W mutant, the crystal structure was determined at 1.18 Å
resolution (Tables S1–S3†). The overall structure and coordination geometry of the Cu center were almost identical to thoseof the H52A/H58E mutant (Fig. 3A, B and S5A, S5C†). In thiscrystal structure, the major and minor copper species (Cu(A)and Cu(B), respectively, Fig. 3A) were observed, although Cu–Cu
distance (0.83 ± 0.02 Å) was slightly larger than that in the
H52A/H58E mutant (0.54 ± 0.02 Å, Fig. 3B and C; Tables S3†).
Interestingly, the electron density of another water molecule
was observed (O4, Fig. 3A), which was not observed in the H52A/
H58E mutant because of the introduction of tryptophan at
position 104 (Fig. 3B). This water molecule bridges the nitrogen
atom of Trp104 and the carboxylic oxygen of Glu58 via hydrogenbonds, inducing the conguration of the hydrogen-bonding
network starting from the Cu-coordinated water molecule totryptophan. This water molecule sits near the aforementioned(A) Crystal structure of the Cu-binding site of H52A/H58E/
F104W (chain B, PDB code 8HJY). (B) Superimposed structures of the
Cu-binding sites of H52A/H58E/F104W(green) and H52A/H58E (pink).
(C) Schematic representation of copper transition. (D) Plausible transition state of the quaternary complex of enzyme, Cu, azachalcone,and methyl acetoacetate. (E) Crystal structure of Cu-binding site of
H52A/H58Q (chain B, PDB code: 8HJZ). 2Fo − Fc and anomalous maps
contoured at 1.5 and 5.0s are shown in gray and magenta mesh,respectively.
Fig. 3Chem. Sci., 2023, 14, 3932–3937 | 3935

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Chemical Sciencesmall cavity, which is surrounded by ve amino acid residues,with methyl acetoacetate approaching. An additional F104W
mutation improved the diastereoselectivity of the Michael
addition reaction, perhaps by spatially xing another watermolecule between Glu58 and Trp104. This result suggests thatthe water molecule may have facilitated the acetyl group to
intrude by the interaction with the ketone of the acetyl group via
hydrogen bonding, and the bulky Trp104 residue prevented theslightly larger methoxycarbonyl side of the methyl acetoacetate
molecule from intruding into this cavity rather than its acetylgroup side (Fig. 3D). Reecting such substrate binding mode,
this mutant showed Michaelis–Menten saturation curve in the
kinetic assay (Fig. S6†).
To further investigate the pliable copper center, we also
determined the crystal structure of Cu-bound H52A/H58Q
mutants (1.22 Å resolution; Fig. S5A and S5D; Tables S1–S3†),
the Cu center of which was supported by the fac-triad of His-HisGln (HHQ). As in the H52A/H58E mutant, the nitrogen atom of
the amide of the glutamine residue is hydrogen-bonded to thewater molecule in the equatorial position. However, the dative
bond length of Cu-amide oxygen of Gln58(A) (2.49 ± 0.11 Å) is
longer than that of Cu-carboxyl oxygen of Glu58 in the H52A/
H58E mutant (2.20 ± 0.03 Å), suggesting that the strength ofthe coordinate bond is weakened (Fig. 3E and Table S3†). In
addition, we found the existence of not only minor Cu species
but also another conformation of Gln58, indicating that Cu ionsspatially uctuate in the active center to a greater extent. In
support of this notion, the minor Cu species was more signicantly distant from the major Cu species (1.23 ± 0.02 Å)
compared to that in the H52A/H58E mutant (0.54 ± 0.02 Å),
breaking the coordination bond between Cu(B) and Gln58(B)residues and the hydrogen bond between Cu-coordinatingwater and Gln58(B) residue. This suggests that the binding
aﬃnity to Cu2+ ions for the H52A/H58Q mutant is relatively low.
Actually, the H52A/H58Q mutant was found to have higher
dissociation constant (Kd = 0.53 mM) than that of the H52A/
H58E mutant (Kd = 0.11 mM, Fig. S7†). During the catalytic
cycle, Cu ions can strip from the active center resulting ina decrease in reactivity, which leads to a drastic decrease inyields (Table 1; entry 18).
Cu active centers of three TM1459 variants (H52A, H52A/
H58E, and H52A/H58Q) exhibited diﬀerent degrees of exibility, reecting the pKa values of the amino acid residues at the
58 position (His, Glu, and Gln, respectively). In the H52A/H58E
mutant, the appropriate exibility of the Cu center appears toplay a signicant role in the binding of azachalcone 1, where thecarboxyl group of Glu58 loosely restrains the location of the Cu
ion via its moderate donating ability and somewhat immobilizes the Cu-coordinating water molecule by bridging the Cu ion
and water molecule in a bidentate manner (Fig. 3C and D).

Conclusions
To the best of our knowledge, this study is the rst to construct
His-His-Glu [HHE] facial-triad-coordinating copper-containing
ArMs that can catalyze the abiological reactions, although
there are several literature that reported ArMs containing His-

3936 | Chem. Sci., 2023, 14, 3932–3937

Edge Article
His-Asp [HHD] facial-triad copper active sites.15–18 The 2-His-1carboxylate facial triad motif is one of the most pivotal
common structural motifs frequently observed in the active
centers of natural metalloenzymes. In this system, three vacantsites located on the distal side of the facial triad play a signicant role in the catalytic mechanisms, such as complex formation between cofactors and substrates. Small variations in thetriad have also been discovered, such as the bidentate coordination of the carboxylato group in Rieske oxygenases, where thecoordination geometry of the metal center by the three residues
cannot be strictly referred to as the facial triad. In this context,
the Cu active site near the heme moiety doesn't have the HHE
triad motif in the articial nitric oxide reductase.33 ACC oxidasemutant with the repurposed Cu center supported by the HHE
triad did not show any catalytic activity, and concrete evidence
is required for the clarication of its coordination geometry.34
In our study, the HHE motif, which was constructed to reduce
the steric hindrance, allowed us to achieve high stereoselectivity
in the Michael addition reaction, conrming the versatility ofthis fac-triad motif. Additionally, natural mononuclear copper
metalloenzymes with the 2-His-1-carboxylate facial triad motif
have not yet been reported.35 One of the variations, 3-His-1-Glu
tetrad with Cu ion, was reported in quercetin 2,4-dioxygenase.
This copper binding site forms two geometries in the restingstate: a tetrahedral site of the 3-His motif plus water with Glu73
in an oﬀ-copper conformation (major form) and a distorted vecoordinate site in which Glu73 is an additional monodentate
ligand (minor form).36,37 Gratifyingly, a similar equilibrium
between the two geometries was observed in our ArMs, HHE factriad-coordinating Cu-TM1459 mutants. We envision that theuse of the extended library of mutants of TM1459 for ArMs canhelp to expand the repertoire of articial non-heme metalloenzymes. Moreover, investigating the detailed catalytic
mechanisms of ArMs thus obtained in future studies will
provide profound insights into natural enzymes and theircatalytic activities.

Data availability
All the data supporting this article have been included in themain text and the ESI.†

Author contributions
Ryusei Matsumoto: investigation, methodology, data curation,
visualization, writing – original dra. Saho Yoshioka: investigation, data curation. Miho Yuasa: investigation. Yoshitsugu
Morita: methodology, investigation. Genji Kurisu: methodology, writing – review and editing. Nobutaka Fujieda: conceptualization, methodology, visualization, writing – review andediting, supervision, funding acquisition.

Conﬂicts of interest
There are no conicts to declare.

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Acknowledgements
N. F. received nancial support/funding from JSPS, MEXT,
Japan, (JSPS KAKENHI 18H04270 and 21H01954), the NOVARTIS Foundation (Japan) for the Promotion of Science, and theSumitomo Foundation. This work was performed using
a synchrotron beamline BL44XU at SPring-8 (Harima, Japan)
under the Cooperative Research Program of the Institute forProtein Research, Osaka University (proposal numbers
2021A6500, 2021B6500, 2022B6500, and 2022B6500). We would
also like to thank Dr E. Yamashita, Dr K. Sakurai, and Dr A.
Nakagawa of SPring-8 BL44XU for their assistance with crystallographic data collection.

Notes and references
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