_x000d_ · web view2021. 2. 9. · gh5: structural snapshots highlighting the involvement of two...

Acta Crystallographica Section D Research articles

GH5: Structural snapshots highlighting the involvement of two conserved residues in the catalysis

Authors

Laetitia Colletab*, Corinne Vander Wauvena, Yamina Oudjamaa, Moreno Gallenib and Raphael

Dutoita

a LABIRIS, 1 Avenue Emile Gryzon, Brussels, 1070, BelgiumbCenter for Protein Engineering (CIP), Biological Macromolecules, University of Liège, 13 Allée du 6

Août, Liège, 4000

Correspondence email: [email protected]

Synopsis Combined analysis of structural biology, enzymatic assays and mass spectrometry

highlighting the role of several conserved residues in the catalytic site of the GH5 transglycosylase

RBcel1

Abstract The ability of retaining glycoside hydrolases (GH) to transglycosylate is inherent to the

double displacement mechanism. Studying reaction intermediates, such as the glycosyl-enzyme

intermediate (GEI) and the Michaelis complex, could bring valuable information to better understand

the molecular factors governing the catalytic mechanism. We present the GEI structure of RBcel1, an

endo-1,4-β-glucanase of the GH5 family endowed with transglycosylase activity. It is the first

structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic

glutamate residues present. We also report the structure of the variant RBcel1_E135A in complex

with cellotriose, allowing the description of the entire binding cleft of RBcel1. Taken together, both

structures deliver different snapshots of the double displacement mechanism. The structural analysis

revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic

assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step

and contributes partly to deglycosylation. Moreover, a conserved tyrosine residue in the -1 subsite,

Tyr-201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI

was trapped in the RBcel1_Y201F variant. The approach to obtain the GEI presented herein could

easily be transposed to other retaining GH in the clan GH-A.

Keywords: glycosyl hydrolase family 5; cellulases; β-1,4-endoglucanases; TIM barrel; RBcel1; double displacement mechanism; transglycosylation; glycosyl-enzyme intermediate

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1. Introduction

In 2013, we reported the crystal structure of the psychrotolerant endo-1,4-β-glucanase RBcel1, cloned

by functional metagenomics from an Antarctic soil sample (Berlemont et al., 2009; Delsaute et al.,

2013). RBcel1 belongs to the glycoside hydrolase family 5, subfamily 5 (GH5_5) of the clan GH-A.

Its structure displays the typical TIM barrel and conserved residues of the GH5 family (Delsaute et

al., 2013). GH5 hydrolases are retaining enzymes proceeding through the double displacement

mechanism as described by Koshland in 1953 (Koshland, Jr, 1953). The mechanism involves two

catalytic residues, generally glutamate or aspartate residues, one acting as an acid/base (R A/B) and the

other as a nucleophile (RNUC) (Figure 1). During the first displacement, or glycosylation step, the RA/B

acts as an acidic residue by transferring a proton to the glycosidic bond oxygen. Simultaneously, the

nucleophile residue attacks the anomeric carbon leading to the formation of a covalent glycosyl-

enzyme intermediate (GEI). During the second displacement, or deglycosylation step, the RA/B, acting

as a base catalyst, deprotonates a water molecule, which attacks the covalently bound anomeric

carbon, releasing the GEI.

RBcel1 had aroused interest because of its ability to polymerize cello-oligosaccharides in

vitro under near-physiological conditions (Berlemont et al., 2009). Transglycosylating glycoside

hydrolases are known as transglycosylases (Danby & Withers, 2016; Bissaro et al., 2015). Their

inherent ability for glycosynthesis is a direct consequence of the double displacement mechanism.

After the glycosylation step, the donor sugar, located inside the negative numbered subsites, is

covalently bound to the enzyme. Transglycosylation can occur when a sugar hydroxyl group is used

as an acceptor instead of a water molecule during the deglycosylation step. Reaction yields, however,

remain low due to the constant hydrolysis of the products formed. It has been proposed that the ratio

of hydrolysis to transglycosylation could be modulated by subtle molecular adjustments, such as the

modification of the donor/acceptor binding sites, the orientation of the catalytic residues, and the

exclusion of water molecules from the catalytic site (Bissaro et al., 2015; Abdul Manas et al., 2018).

Studying reaction intermediates, such as the GEI and the Michaelis complex, could bring valuable

information to better understand the molecular factors governing the reaction.

A stable blocked GEI has been obtained by using 2-deoxy-2-fluoro sugar derivatives and

substituting the RA/B (Withers & Aebersold, 1995; Varrot & Davies, 2003; Varrot et al., 2000; Davies

et al., 1998; Wicki et al., 2002). The 2-deoxy-2-fluoro sugar derivatives destabilize the oxocarbenium

ion-like transition state, slowing down both the glycosylation and deglycosylation steps (Ly &

Withers, 1999; Mosi & Withers, 2002). To allow the accumulation of the GEI, it is critical to

compensate the decreased rate of glycosylation while maintaining deglycosylation as low as possible.

The glycosylation step can be rescued via the use of good leaving group such as 2,4-dinitrophenyl (Ly

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& Withers, 1999; Rempel & Withers, 2008). Since the RA/B is directly involved in the GEI release, its

substitution further reduces the deglycosylation rate (Noguchi et al., 2008; Ly & Withers, 1999). For

instance, such a strategy has been shown to trap the GEI without using 2-deoxy-2-fluoro sugar

derivatives for the β-glucosidase KLrP GBA3 (Noguchi et al., 2008). The structure of the Michaelis

complex, another reaction intermediate, also provides insight in the catalytic mechanism. To trap the

Michaelis complex, the main strategy is to co-crystallize catalytic inactive variants with sugar ligands

(Speciale et al., 2014; Ducros et al., 2002). Substituting the RA/B or the RNUC, however, presents the

major drawback that they are absents in the Michaelis complex structure. Kim & Ishikawa (2011)

reported an alternative substitution of a conserved tyrosine residue located in the -1 subsite to solve

the Michaelis complex structure of a GH5 β-1, 4-glucosidase from Pyrococcus horikoshii with

cellotetraose.

In this work, we focus on obtaining the structure of either the GEI or the Michaelis complex

of RBcel1. According to previous studies (Noguchi et al., 2008; Kim & Ishikawa, 2011), the former

should be trapped by substituting the acid/base residue Glu-135 (GluA/B), while the latter should be

stabilized by substituting Tyr-201. Quite unexpectedly, the substitution of Glu-135 did not lead to the

formation of any observable reaction intermediate. The Tyr-201 substitution, however, resulted in the

accumulation of the GEI. The resolution of their structures revealed molecular determinants for

substrate binding and catalytic mechanism. The structure of the Glu-135-Ala variant in complex with

cellotriose allowed to define the catalytic cleft of RBcel1, spanning from the -4 to +2 subsites. The

structure of the Tyr-201 variant was obtained with a cellotriose molecule covalently bound to the

nucleophilic residue Glu-245 (GluNUC). To our knowledge, it is the first structure reported in the GH5

family of a GEI obtained with a natural oligosaccharide including both catalytic residues. Indeed, the

few structures of GEI reported for the GH5 family have been obtained by using 2-deoxy-2-

fluoroglycosides and substituting the GluA/B. Moreover, the GEI complex structure of RBcel1 revealed

a displacement of the GluNUC during the glycosylation step in RBcel1. In addition, both Glu-135 and

Tyr-201 appear to play a key role in the deglycosylation step.

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Figure 1 (A) Double displacement mechanism of retaining glycoside hydrolases. The catalytic

residues are numbered according to RBcel1 sequence. R: sugar unit. (B) Effect of Glu-135 (RA/B)

substitution on activated substrates. R’: good leaving group as 2,4-dinitrophenyl. Adapted from

Noguchi et al. (2008).

2. Materials and methods

2.1. Cloning and mutagenesis of RBcel1

The original RBcel1 expression vector, pET22b-RBcel1, was described previously (Berlemont et al.,

2009). The gene coding RBcel1 was also cloned in the pBAD-TOPO vector (Thermo Fisher

Scientific) giving the pBAD-RBcel1 vector. Mutagenesis was performed using the QuikChange Site-

Directed Mutagenesis Kit (Agilent). The pET22b-RBcel1 and pBAD-RBcel1 vectors were used to

produce the RBcel1_E135A and RBcel1_Y201F variants, respectively (Table 1). For

RBcel1_E135A_Y201F, a synthetic gene harboring the desired mutations (GeneArt, Thermo Fisher

Scientific) was cloned into the pET30b vector (Novagen) using NdeI and XhoI restriction sites (Table

1). All genetic constructs were verified by sequencing (Genetic Service Facility, University of

Antwerp). All constructions comprised the RBcel1 original signal sequence and allowed for the

export of the recombinant protein into the periplasm. E. coli MC1061 was used for cloning and E coli

BL21 (DE3) for heterologous expression.

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2.2. RBcel1 production and purification

RBcel1_WT, RBcel1_E135A, and RBcel1_E135A_Y201F were produced as described previously

(Berlemont et al., 2009). For RBcel1_Y201F, cells were grown in LB media with 100 µM of

ampicillin at 37°C. At an OD660nm of 0.6, 13 mM arabinose was added for induction and cells were

further incubated overnight at 18°C. RBcel1_WT and its variants were purified as follows. After an

overnight induction at 18°C, a periplasmic extract was prepared as previously described (Garsoux et

al., 2004) and loaded onto an ion exchange column (SOURCE 15Q, 12 mL, GE Healthcare)

equilibrated in 20 mM Tris-HCl pH 8.5. The proteins were eluted by a linear NaCl gradient from 0 to

500 mM. After analysis by SDS-PAGE, the RBcel1-containing fractions were pooled and loaded on a

size exclusion chromatography column (Superdex 75, 120 mL, GE Healthcare), equilibrated in 20

mM sodium phosphate buffer (pH 6.5). The relevant fractions were pooled and concentrated on an

Amicon ultrafiltration unit (Millipore) with a 10-kDa cut-off. The purity was checked by SDS-PAGE.

Protein concentrations were calculated using the theoretical extinction coefficients [Δε280 = 80455

M-1 cm-1 for RBcel1_WT and RBcel1_E135A, Δε280 = 78965 M-1 cm-1 for RBcel1_Y201F and

RBcel1_E135A_Y201F].

2.3. Mass determination by ESI-Q-TOF-MS

50 µM of enzyme was incubated with 250 µM of 2-chloro-4-nitrophenyl β-cellotrioside (ClPNP β-

G3) in 20 mM sodium phosphate buffer pH 6.5 at 25°C. Several samples were collected at 10 min, 60

min, and 24 hours of incubation. The reaction was stopped by adding of 0.5% (v/v) of formic acid.

Samples were analysed at the GIGA Proteomics Facility (University of Liege). The analyses were

performed on an ESI-Q-ToF (Synapt G2 HDMS, Waters), in positive ion mode. Prior to MS (mass

spectrometry) analysis, the sample were ultrafiltrated on an Amicon ultrafiltration unit (Millipore)

with a 3 kDa cut-off to remove salts, and then conditioned in 25 mM ammonium acetate. Protein

concentration was adjusted to 40 µM. Peak intensity is given as the mass to charge ratio (m/Z) value

on spectra. Calibration was performed using clusters of phosphoric acid in the m/z range of 50 to

3500, corresponding to the raw spectra m/Z acquisition range. The maximum entropy calculation

method was used to deconvoluate spectra using MaxEnt 1 process of the MassLynx software

(Waters).

2.4. Crystallization

When incubated with ligands, RBcel1_Y201F crystallizes in another space group than the apo form.

Consequently, co-crystallization of RBcel1_Y201F with ClPNP β–G3 was done in two steps. The

enzyme, stored in 20 mM sodium phosphate pH 6.5 at a concentration of 400 µM, was incubated with

1 mM of ClPNP β–G3 for 1 hour at 4°C. The reaction mix was then crystallized in 100 mM Tris, 20.5

% PEG600 pH 7.0 (well buffer) using the hanging drop vapour diffusion method. Drops containing 2

µL of RBcel1_Y201F mixed with ClPNP β–G3 and 2 µL of well buffer were set up in easyXtal Tool 5


plates (Qiagen). Micro seeding was necessary to improve crystal shape and size. Monocrystal

appeared after a few hours and grew to maximum dimensions within 2 days at 292 °K. To optimize

the ratio of covalently linked enzymes, 0.2 µL of 20 mM ClPNP β–G3 was added to the drop with a

further incubation of 1 hour before crystal picking and cryogenization. RBcel1_E135A was directly

co-crystallized with cellotriose using the hanging drop method by mixing 2 µL of 385 µM protein in

20mM sodium phosphate buffer pH 6.5 and 5 mM cellotriose with 2 µL of 100 mM Tris HCl 17.5 %

PEG 600 pH 7.4. Drops were equilibrated for cryoprotection during 2 hours against a 500 µL

reservoir containing 0.1M Tris pH 7.4, 30% PEG 600 . The crystallization conditions are summarized

in Table 2.

2.5. Data collection and processing

Diffraction data were collected at Proxima 2 (RBcel1_E135A in complex with cellotriose) and

Proxima 1 (and RBcel1_Y201F in complex with ClPNP β–G3) beamlines at SOLEIL (Saint-Aubin,

France). Diffraction data were indexed using the XDS program package (Kabsch, 2010). The statistics

of data collection and indexation are summarized in Table 3. Both structures were determined by

molecular replacement with Phaser-MR in Phenix software (Adams et al., 2010) using the coordinates

of RBcel1 (PDB ID 4EE9) (Delsaute et al., 2013) as search model. The models were built using

phenix.autobuild (Adams et al., 2010) and Crystallographic Object-Oriented Toolkit (Coot) (Emsley

et al., 2010). The multiple rounds of refinement were performed using phenix.refine (Adams et al.,

2010). The final model of RBcel1_E135A consists of 638 amino-acid residues, 2 cellotriose

molecules, and 821 water molecules in the asymmetric unit. The final model of RBcel1_Y201F

consists of 1278 amino-acid residues, 4 cellotriose molecules, 1 Tris molecule, and 467 water

molecules in the asymmetric unit. The stereochemical quality of the model was assessed using

MolProbity (Chen et al., 2010). The structure solutions and refinement statistics of both structures are

presented in Table 4. Protein-ligand interactions were analysed using Ligplot 1 (Wallace et al., 1995).

Structures were illustrated using PyMOL Molecular Graphics System version 0.9 (Schrödinger, LLC).

2.6. Enzyme activity assays

2.6.1. Determination of kinetic parameters

The enzyme (0.2 µM) was incubated at 25°C with ClPNP β–G3 (Megazyme) in a range of

concentrations from 0.3 mM to 6 mM, in 20 mM sodium phosphate buffer pH 6.5. The release of 2-

chloro-4-nitrophenol (ClPNP) was followed through the increase of absorbance at 400 nm during 2

min. The kinetic parameters were determined under the initial rate conditions by a nonlinear

regression of the Michaëlis-Menten equation.

1

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2.6.2. Time-course ratio of ClPNP/G3

To evaluate if there was a delay between the release of cellotriose (G3) and 2-chloro-4-nitrophenol

(ClPNP), 2.5 µM of enzyme was incubated with 150 µM of ClPNP β-G3 at 25°C in 20 mM sodium

phosphate buffer pH 6.5. The absorbance was monitored at 400 nm and 50 µL samples of the reaction

mix were taken at different time-intervals to be analysed by PolyAcrylamide Carbohydrate

Electrophoresis (PACE). The results were expressed as percentages of product released. When the

hydrolysis of ClPNP β-G3 was complete, with a 100%-release of ClPNP, the OD 400nm reached 2.0. On

PACE analysis, 100% corresponds to the peak intensity of 150 µM of G3.

2.6.3. PACE analysis

PolyAcrylamide Carbohydrate Electrophoresis (PACE) was used to analyse the oligosaccharides

produced by hydrolysis and transglycosylation from cellohexaose (G6) and ClPNP β–G3. All

oligosaccharides or derivatives were purchased from Megazyme. Unless stated otherwise, the reaction

mixes contained 1mM of substrate in 20 mM sodium-phosphate buffer pH 6.5. The reaction was

started by adding 10µM of enzyme to the reaction mix kept at 4°C, and immediately incubated at

25°C. The incubation was finally stopped by adding formic acid (0.5 % final).

2.6.4. Derivatization with 8-Aminonaphthalene-1,3,6-trisulfonic acid (ANTS)

Before separation on gel, sugars were derivatized with 8-amino-naphthalene-1,3,6-trisulfonic acid

(ANTS) (Jackson, 1990; Goubet et al., 2002). 10 µL of reaction mixture were first air-dried, then

solubilized in 5 µL of 0.2 M ANTS (in water: acetic acid, 17:3 v/v) and 5 µL of 1 M NaCNBH 3 (in

DMSO). The derivatization was performed at 37°C for 18 h in dark. Samples were then briefly

vortexed and ventilated in a fume hood before being air dried 2 h at 45°C. The dried derivatized

sugars were solubilized in 50 µl of 3 M urea and 4 µL of each sample was loaded on the gel.

2.6.5. Polyacrylamide gel preparation and run

Polyacrylamide gel were prepared by mixing 6 mL of 40% acrylamide (37.5:1) (BioRad, 161-0148)

with 6 mL of TBE buffer containing 10 % glycerol, and adding 0.04 % ammonium persulfate and 5

µL of tetramethylethylenediamine for polymerization. The solution was quickly vortexed before

casting in a SureCast Gel Handcast Station (Thermo Fisher Scientific). After loading the samples (4

µL), the gel were run in 0.1 M Tris-borate buffer with a constant current of 18 mA for 60 min at 4°C,

in dark.

2.6.6. Gel analysis and quantification

Gels were visualized on a UV-transilluminator fitted with 365 nm UV light tubes and a CCD camera

system equipped with a short-pass (500–600 nm) filter. Gels were analysed using the Vision-Capt

software (Vilber Lourmat). The reaction products were identified by comparison with a size reference 7


ladder. Oligosaccharides from glucose to cellohexaose were used as standard sugars at different

concentration (from 0.025 mM to 0.9 mM) in order to quantify the reaction products.

3. Results and discussion

3.1. The role of Glu-135 and Tyr-201 in the activity of RBcel1

On the basis of the sequence alignment and the analysis of the RBcel1 binding cleft, Glu-135 and Tyr-

201 were designated as the acid/base catalyst and the conserved tyrosine residue, respectively

(Delsaute et al., 2013). With the expectation to trap the GEI, Glu-135 was substituted with an alanine

residue, while Tyr-201 was substituted with a phenylalanine residue to get the Michaelis complex.

The variants, hereafter named RBcel1_E135A and RBcel1_Y201F, were recombinantly produced in

E. coli and purified to homogeneity. To assess the impact of substitutions, the activity of RBcel1_WT,

RBcel1_E135A, and RBcel1_Y201F was assayed on cellohexaose (G6) and ClPNP β–G3. The

reaction products were analysed by polyacrylamide carbohydrates electrophoresis (PACE).

RBcel1_WT degraded G6 into smaller oligosaccharides ranging from cellopentaose (G5) to cellobiose

(G2) (Figure 2). On ClPNP -G3, it generated oligosaccharides of various sizes, including

transglycosylation products (G4, G5 and G6).

As expected, the Glu-135-Ala substitution resulted in a total loss of activity on G6 (Figure 2)

since natural sugars need the protonic assistance of RA/B for the glycosylation step (McCarter &

Withers, 1994). On the other hand, RBcel1_E135A still liberated ClPNP from ClPNP β–G3. Indeed,

activated substrates, like ClPNP β–G3, allow the glycosylation step to proceed in the absence of the

RA/B. The reaction, however, should stall at the deglycosylation step, with an oligosaccharide

remaining covalently bound to the GluNUC (Ly & Withers, 1999; Noguchi et al., 2008). Consequently,

the hydrolysis of ClPNP β–G3 should stop when all enzyme molecules are engaged in the GEI. Yet,

PACE results showed that RBcel1_E135A continuously released cellotriose (G3) from ClPNP β–G3,

indicating that deglycosylation could still proceed. Contrary to the wild type enzyme, RBcel1_E135A

could not transglycosylate, highlighting the role of Glu-135 in transglycosylation. Unlike

RBcel1_E135A, RBcel1_Y201F maintained a slight activity on G6, generating smaller

oligosaccharides (Figure 2). Although being active on G6, RBcel1_Y201F was less active than the

wild type since it required 1 h to achieve the same degradation pattern as of RBcel1_WT in 1 min.

Yet, PACE analysis did not show any product from ClPNP β–G3 hydrolysis, even though the reaction

mixture turned faintly yellowish. The release of ClPNP, however, was too slow to determine the

kinetic parameters of RBcel1_Y201F.

Further experiments were conducted to confirm the anomalous activity of RBcel1_E135A on

ClPNP β–G3. The rate of ClPNP release by RBcel1_E135A was compared to that of RBcel1_WT

(Table 5). The kinetics parameters strongly suggested that the Glu-135-Ala substitution affects the

deglycosylation step. Indeed, the KM of RBcel1_E135A was much lower than that of RBcel1_WT but 8


their kcat/KM remain constant. Consequently, the KM reduction correlates with a slowdown of the

deglycosylation step rate (Bissaro et al., 2014; Shallom et al., 2002). Despite being impaired,

deglycosylation could still proceed efficiently as G3 was continuously released as shown by PACE

analysis. To investigate if there was a delay between the release of ClPNP and G3, several samples

were collected at different time-intervals during incubation. The release of ClPNP, reflecting

glycosylation, was measured spectrophotometrically while the release of G3, reflecting

deglycosylation, was monitored by PACE (Figure 3). The data strongly support that the GEI was

unstable and quickly released since (i) 2.5 µM of RBcel1_E135A completely hydrolysed 150 µM of

substrate and (ii) no delay was observed between the release of ClPNP and G3.

The absence of hydrolysis on cellohexaose confirms that Glu-135 is the acid catalyst in the

glycosylation step. According to the double displacement mechanism, this residue would also act as a

general base in the deglycosylation step (Koshland, Jr, 1953). However, in RBcel1, the

deglycosylation step does not completely rely on Glu-135 and other residues seem involved. Tyr-201

is a possible candidate since its substitution with a phenylalanine residue dramatically impaired

ClPNP β-G3 hydrolysis.

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Figure 2 PACE analysis of reaction products from the activity of RBcel1_WT, RBcel1_E135A, and

RBcel1_Y201F on G6 and ClPNP β-G3. (1) size reference ladder containing 0.9 mM glucose (G1),

0.5 mM cellobiose (G2), 0.25 mM cellotriose (G3), 0.1 mM cellotetraose (G4), 0.05 mM

cellopentaose (G5), and 0.025 mM cellohexaose (G6); (2) G6 incubated without enzyme (Blk); (3) G6

incubated with RBcel1_WT (WT); (4) G6 incubated with RBcel1_E135A (E135A); (5) G6 incubated

with RBcel1_Y201F (Y201F); (6) ClPNP β-G3 incubated without enzyme; (7) ClPNP β-G3 incubated

with RBcel1_WT; (8) ClPNP β-G3 incubated with RBcel1_E135A; (9) ClPNP β-G3 incubated with

RBcel1_Y201F. 1 mM of substrate was incubated with 10 µM enzyme during 1 min. for RBcel1_WT

or 60 min. for RBcel1_E135A and RBcel1_Y201F. Reactions were stopped by adding 0.5 % (final)

formic acid and products derivatized with ANTS prior to PACE, as described in material and

methods.

0 100 200 300 400 500 600 7000

10

20

30

40

50

60

70

80

90

100

ClPNPG3

time (min.)

%

Figure 3 Release of ClPNP and G3 from ClPNP β-G3 hydrolysis by RBcel1_E135A. 2.5µM

RBcel1_E135A was incubated with 150 µM ClPNP β-G3. The release of ClPNP (▲) and G3 (×) is

expressed as a percentage of the quantity of product released after complete hydrolysis of 150 µM

ClPNP β-G3. A complete hydrolysis corresponds to an increase in OD400nm of 2, taken as 100% for the

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monitoring of ClPNP. The release of G3 was followed by PACE analysis. 100 % corresponds to the

band intensity of G3 after complete hydrolysis.

3.2. The role of Tyr-201 in deglycosylation

To clarify the results of the activity assays, ESI-Q-tof analysis was undertaken to investigate if the

GEI was effectively trapped in the RBcel1 variants. RBcel1_E135A and RBcel1_Y201F were

incubated with ClPNP β–G3 and several samples were analysed at different incubation times (from 10

min to 24 hour). Their respective apo form were also analysed for comparison. MS analysis of

RBcel1_E135A revealed a major peak of 36,272 ±3 Da (Figure 4a), corresponding to the relative

mass of the apo form. No peak corresponding to the trapped GEI was observed during the time-lapse

of the experiment (Figure 4a). On the other hand, an additional peak at 36,801 ±3 Da appeared during

incubation of RBcel1_Y201F with ClPNP β–G3 (Figure 4b). This mass correlates with a shift of 487

Da from the apo-enzyme relative mass of 36,314 Da. Such a difference corresponds to the mass

expected for the enzyme covalently bound to G3. The formation of GEI was already observed after 10

min of incubation. The peak height increased over time and, after 1 hour, about 90% of the enzyme

was engaged in a covalent bound with G3. Nevertheless, over longer incubation times, a decrease in

GEI was observed, indicating that deglycosylation still occurred. Our results showed that

deglycosylation in RBcel1 is more dependent on Tyr-201 than Glu-135. Subsequently, we decided to

use RBcel1_Y201F to get the structure of the GEI and RBcel1_E135A for the Michaelis complex.

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Figure 4 Analysis by ESI-Q-ToF-MS of the formation of a GEI: Deconvoluted spectra of: (a)

RBcel1_E135A (36,272 ±3 Da) and (b) RBcel1_Y201F (36,314 ±3 Da), for the apo form and for the

enzyme incubated with ClPNP β-G3 during 10 min and 1 hour (RBcel1_E135A and RBcel_Y201F)

or 24 hours (RBcel_Y201F). A G3 molecule covalently bound to the enzyme would represent a mass

shift of 487 Da, corresponding to a mass of 36,758 Da for RBcel_E135A and 36,800 Da for

RBcel1_Y201F. Samples were prepared as described in material and methods. Intensity peak heights

are given as a percentage of the peak height of the main species. The mass (Da) are determined with

an error of ± 3 Da.

3.3. Structural analysis

3.3.1. Structure of RBcel1_E135A in complex with cellotriose

RBcel1_E135A was co-crystallized with different oligosaccharides to obtain the structure of the

Michaelis complex. A data set was obtained for RBcel1_E135A co-crystallized with cellotriose and

the structure of the complex was solved at 1.7 Å. The asymmetric unit contains two monomers, each

in complex with a cellotriose differently positioned. In monomer A, the cellotriose molecule occupies

the -1 to +2 subsites while in monomer B, it occupies the -4 to -2 subsites. The cellotriose straddling

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the catalytic site of monomer A is not distorted, indicating that the Michaelis complex could not be

observed in the structure. The structural alignment of the RBcel1_E135A structure with the Michaelis

complex of the GH5 β-1,4 glucosidase from Pyrococcus horikoshii with cellotetraose strongly

supports the conclusion that the Michaelis complex is not being observed in the RBcel1_E135A

structure (Figure 5).

Figure 5 Comparison of the substrate distortion in the -1 subsite through structural alignment of the

RBcel1_E135A monomer A (cyan) and B (yellow) of RBcel1_E135A in complex with cellotriose

(PDB ID:5LJF) and the GH5 β-1,4 glucosidase from Pyrococcus horikoshii in Michaelis complex

with cellotetraose (pink) (PDB ID:3QHN). The subsites are identified from -4 to +2. The residue

annotations refer to the numbering in RBcel1_E135A.

Nevertheless, the structure of RBcel1_E135A allowed outlining the entire binding cleft from the -4 to

+2 subsites (Figure 5). In monomer A, the cellotriose molecule found in the -1 to +2 subsites

highlighted the interactions in the aglycone part (Figure 6a and b).

Determining such interactions is particularly relevant since the affinity of the positive numbered

subsites for the acceptor is critical for transglycosylation (Dilokpimol et al., 2011; Rosengren et al.,

2012; Dutoit et al., 2019; Bissaro et al., 2015). Trp-171 is part of both positive numbered subsites, as

previously suggested for the apo-enzyme structure (Delsaute et al., 2013). In monomer A of

RBcel1_E135A, the indole ring of Trp-171 adopts the same m-90° rotamer as in the RBcel1_WT

structure (Figure 7). Intriguingly, in monomer B, the indole ring of Trp-171 is flipped to a m-95°

rotamer (Figure 7). Since the two positions of the dual conformation of Trp-171 are observed in the

13


same structure, the flip is probably linked to the position of the cellotriose molecule in the catalytic

cleft (which is differently placed in each monomer) and not to the Glu-135 substitution. In the +2

subsite, the NH1 of Arg-176 interacts with the O2 of the sugar. The presence of a polar group in the

+2 subsite has been suggested to be a characteristic of transglycosylating GH as it could increase the

affinity of the aglycone subsites for the acceptor (Rosengren et al., 2012; Dutoit et al., 2019; Bissaro

et al., 2015). The backbone of Asp-205, Ala-206, and Ser-207 also takes part in shaping the +1 and

+2 subsites.

The -1 subsite principally consists of several residues conserved in the GH5 family: His-90, Tyr-92,

Glu-135-Ala, Tyr-201, and Glu-245. In monomer B, the side chain of the nucleophile Glu-245 adopts

a double conformation (Figure 5) perhaps due to the Glu-135 substitution. The loss of a carboxylate

at position 135 could lower structural constraints on the Glu-245 side chain. Indeed, the two catalytic

residues have been reported to be interconnected as they mutually influence their pKa via a pKa

cycling during the reaction (McIntosh et al., 1996). However, the displacement of Glu-245 side chain

was also observed in the structure of the GEI described below, highlighting its catalytic significance.

Since the cellotriose is not distorted in monomer A, the occupation of the negatively numbered

subsites seems to be important for catalysis. Indeed, as seen in monomer B, the -2 subsite imposes an

important constraint on the bound cellotriose molecule through a strong interaction between the sugar

moiety O6 and Glu-13 (see Figure 6c and d). In the -2 subsite, several aromatic residues also interact

with the sugar ring: Tyr-285, Trp-276, Trp-282, and Phe-14. The latter two take part in shaping the -3

subsite along with Thr-15. Finally, the -4 subsite consists of Thr-15, Thr-25, Phe-28, and Ser-281.

14


Figure 6 3D close-up view of the substrate-binding cleft of RBcel1_E135A in complex with

cellotriose. (a) Description of the -1 to +2 subsites in monomer A. Residues interacting with

cellotriose (CTR) are shown in stick representation, the 2Fo - Fc map around CTR is shown as blue

mesh. Glu-245 is hidden behind the CTR molecule in the graphical representation of monomer A. (b)

2D representation of the residues in interaction with CTR in monomer A. (c) Description of the -4 to

-2 subsites in monomer B. Residues interacting with cellotriose (CTR) are shown in stick

representation, the 2Fo - Fc map around CTR is shown as blue mesh. (d) 2D representation of the 15


residues in interaction with CTR in monomer B. Panels (c) and (d) were generated using LigPlot +

software (Laskowski & Swindells, 2011).

Figure 7 The conformations of the Trp-171 indole ring in RBcel1_WT (grey), RBcel1_E135A

monomer A (cyan) and monomer B (yellow).

3.3.2. Structure of the covalent glycosyl-enzyme intermediate with both catalytic glutamate residues

According to the MS data, the GEI formation was maximal when RBcel1_Y201F was incubated for 1

hour with ClPNP β–G3. Consequently, crystals of RBcel1_Y201F were soaked one hour with ClPNP

β-G3 prior to data collection. The structure of the GEI was solved at 2.1 Å resolution (PDB ID:

6ZZ3). The asymmetric unit contains four monomers, all of them with a cellotriose molecule

spanning from the -3 to -1 subsites. The distance between the nucleophilic oxygen and the anomeric

carbon of the glucose unit is 1.3 Å long (Figure 8), confirming the covalent link. The main structural

difference between the GEI and the apo form of RBcel1_WT is the displacement of the Glu-245 side

chain. Indeed, the carboxylate is rotated by 50° along the Cβ axis leading to a rotamer change, from

mt-10° to tt0° (Figure 8). This displacement is identical to that previously observed in the monomer

B of RBcel1_E135A.

16


Figure 8 Structural alignment of RBcel1_Y201F covalently bound with CTR (yellow) (PDB ID:

6ZZ3) and RBcel1_WT in the Apo form (green) (PDB ID: 4EE9). The side chains of residues

interacting with CTR are shown in stick representation. The 2Fo - Fc map (blue mesh) is shown

around CTR.

The mt-10° rotamer corresponds to the side chain conformation of Glu-245 in the apo wild-type

enzyme. The tt0° rotamer corresponds to Glu-245 covalently linked to a carbohydrate. This genuine

movement of the GluNUC is not a particularity of RBcel1. Indeed, a similar displacement of the Glu NUC

is also observed in four structures of GH5 enzymes forming a GEI (see Figure S1) (Varrot et al.,

2000; Varrot & Davies, 2003). In those cases, the displacement of the GluNUC has been suggested to be

a side effect of the use of 2-deoxy-2-fluoro sugar derivatives for the trapping of the GEI, as stated for

the GEI structure of a GH1 enzyme (Gloster et al., 2004). However, in RBcel1, the double

conformation of Glu-245 cannot be interpreted as an artefact since (i) the GEI was obtained with a

natural sugar and (ii) neither of the two catalytic residues were substituted.

Depending on its rotamer conformation, important changes are observed in the electronic environment

of Glu-245 (Figure 9). When Glu-245 adopts the mt-10° rotamer, the OE1 forms an H-bond with

Tyr-201, as in RBcel1_WT, while the OE2 forms a salt bridge with the NH1 of Arg-47. When Glu-

245 adopts the tt0° rotamer, the OE1 interacts with both His-199 and Glu-135 making an electron

relay network. The OE2 makes either a strong H-bond (2.1 Å) with Tyr-201, as in the RBcel1_E135A

structure, or a covalent bond to cellotriose. All residues interacting with Glu-245 in either of the

conformations are strictly conserved and their role in catalysis has already been discussed. Arg-47 and

His-199 are thought to be important for both enzyme activity and stability. The His-199 position has 17


been shown especially intolerant to substitution (Bortoli-German et al., 1995). The substitution of

His-199 with an alanine residue in RBcel1 dramatically impaired both activity and stability, while its

substitution with a lysine residue partly maintained the activity (data not shown). Arg-47 and His-199

are interconnected via their interactions with Asn-134.

Figure 9 Close-up view centered on the Glu-245 position of the structural alignment of

RBcel1_Y201F with a covalently bound cellotriose molecule (in yellow) and the monomer B of

RBcel1_E135A in complex with a cellotriose molecule (in cyan). OE1 and OE2 of Glu-245 are

marked by an asterisk (*) and an octothorpe (#), respectively.

3.3.3. Tyr-201 and Glu-135 are both involved in catalysis

Tyr-201 is highly conserved among GH of the clan GH-A and its role is still debated (Ducros et al.,

1995; Sakon et al., 1996; Petegem et al., 2002; Zheng et al., 2012; Kim & Ishikawa, 2011; Gonçalves

et al., 2012). Kim & Ishikawa (2011) described it as being important for the glycosylation step.

Indeed, they observed the Michaelis complex when Tyr-299 was substituted with a phenylalanine

residue in the GH5 endocellulase from Pyrococcus horikoshii. A link between the tyrosine residue

and the catalytic GluNUC has been proposed, either to stabilize the charge of the carboxylate (Ducros et

al., 1995) or to orientate the GluNUC (Gonçalves et al., 2012). These hypotheses, however, considered

only the mt-10° rotamer of the GluNUC.

For RBcel_Y201F, the activity assays with G6 and ClPNP-β-G3 confirmed the implication of Tyr-

201 in both the glycosylation and the deglycosylation steps. The structures of the GEI and

RBcel1_E135A showed that Tyr-201 makes a short H-bond with the OE2 of Glu-245 when the

nucleophile is positioned to form the GEI. This position of Tyr-201suggests that it could intervene to

maintain the tt0° during the glycosylation step.

Since the GEI was trapped in RBcel1_Y201F, Tyr-201 must be important for the deglycosylation

step. MS analysis, however, showed that the GEI was unstable over incubation times longer than one

hour. Thus, Tyr-201 is not the sole residue involved in deglycosylation. The role of the conserved

tyrosine residue in the deglycosylation step has already been discussed for other GH of Clan GH-A

18


like a GH2 β-galactosidase (Penner et al., 1999; Roth et al., 2003) and a GH1 β-glucosidase from

Agrobacterium faecalis (Gebler et al., 1995). For the latter, the authors reported that the substitution

Tyr-298-Phe resulted in trapping the GEI. They proposed that Tyr-298, equivalent to RBcel1 Tyr-201,

plays a crucial role in catalysis. It could also act as a catalytic nucleophile in a rescue system or attack

the glycosyl-enzyme link instead of a water molecule. Wang et al. (1995) proposed that Tyr-298

could play an independent role in the deglycosylation step since the effects of substituting Tyr-298

and the RA/B were cumulative on enzyme activity. Previously, Notenboom et al. (1998) reported

trapping a very stable GEI with Cex, a GH10 β-1, 4-glucanase from Cellulomonas fimi, by

substituting His-205 and Glu-127, equivalent to, respectively, Tyr-201 and Glu-135 in RBcel1. It is

worth noticing that Cex is an exception in the GH10 family as it displays a histidine residue instead of

the conserved tyrosine residue. Therefore, to confirm their implication in deglycosylation, a variant of

RBcel1 with the double substitution, Glu-135-Ala and Tyr-201-Phe, was generated, hereafter named

RBcel1_E135A_Y201F. The GEI formation was confirmed with this variant by MS. Indeed, a species

with a relative mass of 36,741 ± 3 Da was detected when RBcel1_E135A_Y201F was incubated with

ClPNP β-G3, (Figure 10). This represents a mass shift of 487 Da compared to the apo-enzyme. Such

a difference corresponds to a bound cellotriose molecule, proving the formation of the GEI. Contrary

to RBcel1_Y201F, the amount of GEI accumulated overtime remained stable, even after 24 hours of

incubation. Our data confirm that both Tyr-201 and Glu-135 contribute to deglycosylation, like His-

205 and Glu-127 in Cex.

19


Figure 10 Analysis by ESI-Q-ToF-MS of the formation of a GEI: Deconvoluted spectra of

RBcel1_E135A_Y201F (36,255 ± 3 Da), for the apo form and for the enzyme incubated with ClPNP

β-G3 during 10 min, 1 hour and 24 hours. A G3 molecule covalently bound to the enzyme would

represent a mass shift of 487 Da, corresponding to a mass of 36,742 Da for RBcel_E135A_Y201F.

Samples were prepared as described in material and methods. Intensity peak heights are given as a

percentage of the peak height of the main species. The mass (Da) are determined with an error of ±

3 Da.

4. Conclusion:

In this work, we combined structural biology with enzymatic assays and MS analysis to understand

the role of several conserved residues at the catalytic site of the GH5 transglycosylase RBcel1. The

Glu-135-Ala substitution confirmed the role of Glu-135 as the acidic catalyst during the glycosylation

step and as a base catalyst during transglycosylation. Yet, the substitution did not totally prevent

deglycosylation since the GEI could not be trapped with RBcel1_E135A. In addition, the absence of

transglycosylation products in this variant suggested that deglycosylation only occurs via hydrolysis.

Thus, Glu-135 is not indispensable in deglycosylation but is clearly involved in tranglycosylation as a

base catalyst.

The conserved tyrosine residue in the -1 subsite, Tyr-201, was found to be extremely determinant for

glycosylation and deglycosylation. The Tyr-201-Phe substitution clearly impaired the glycosylation

step, despite the presence of Glu- 135, since RBcel_Y201F was weakly active on G6 and ClPNP β-

G3. Unexpectedly, the GEI was trapped in that variant, allowing the resolution of the first structure of

a GH5 enzyme with both catalytic residues unmodified and a covalently bound natural sugar.

Our structural data, gathered from the structures of RBcel1_E135A and of the GEI in RBcel1 Y201F,

revealed successive snapshots of the retention mechanism in RBcel1. To our knowledge, those

structures showed for the first time that the side chain of the nucleophilic glutamate residue oscillates

between two alternate rotamers. Several conserved residues in its vicinity could play an important role

in positioning the Glu-245 side chain. Depending on its position, its electronic environment is

probably changing to modulate its reactivity during the GEI formation and its release. Finally, we

demonstrated that Glu-135 is required for deglycosylation. Indeed, the GEI in RBcel1_Y201F

appeared to be unstable over time, indicating that deglycosylation could still occurred. Yet, in the

variant RBcel1_Y201F_E135A, a stable GEI was formed, confirming the cumulative effects of both

substitutions. This approach seems promising to obtain GEI complexes for other retaining GH.

Source

organism

Uncultured bacterium Uncultured bacterium Uncultured bacterium

Expression pET22b pBAD-TOPO pET30b

20


vector

Expression

host Escherichia coli Escherichia coli Escherichia coli

Complete

amino acid

sequence of

the construct

produced

SVDLIGINVAGAEFTGGKLPGKHGTHYFFPPEGYFEYWSEQGIHTVRFPLKWERLQPSLNAELDDVYASLVDDMLDQAKENDIKVILDVHNYARYRKKVIGTEDVPVSAYQDLMERIAKRWQGHDALFAYDIMNAPYGSADKLWPAAAQAGIDGVRKYDKKRPLLIEGASWSSAARWPRYADELLKLKDPADNMVFSAHVYIDEDASGSYKKGPGKDFEPMIGVKRVEPFVNWLKEHGKKGHIGEFGIPNDDERWLDAMDKLLAYLNENCIPINYWAAGPSWGNYKLSIEPKDGEKRPQVALLKKYAAKDNCSDFGPAKAE

SVDLIGINVAGAEFTGGKLPGKHGTHYFFPPEGYFEYWSEQGIHTVRFPLKWERLQPSLNAELDDVYASLVDDMLDQAKENDIKVILDVHNYARYRKKVIGTEDVPVSAYQDLMERIAKRWQGHDALFAYDIMNEPYGSADKLWPAAAQAGIDGVRKYDKKRPLLIEGASWSSAARWPRYADELLKLKDPADNMVFSAHVFIDEDASGSYKKGPGKDFEPMIGVKRVEPFVNWLKEHGKKGHIGEFGIPNDDERWLDAMDKLLAYLNENCIPINYWAAGPSWGNYKLSIEPKDGEKRPQVALLKKYAAKDNCSDFGPAKAE

SVDLIGINVAGAEFTGGKLPGKHGTHYFFPPEGYFEYWSEQGIHTVRFPLKWERLQPSLNAELDDVYASLVDDMLDQAKENDIKVILDVHNYARYRKKVIGTEDVPVSAYQDLMERIAKRWQGHDALFAYDIMNAPYGSADKLWPAAAQAGIDGVRKYDKKRPLLIEGASWSSAARWPRYADELLKLKDPADNMVFSAHVFIDEDASGSYKKGPGKDFEPMIGVKRVEPFVNWLKEHGKKGHIGEFGIPNDDERWLDAMDKLLAYLNENCIPINYWAAGPSWGNYKLSIEPKDGEKRPQVALLKKYAAKDNCSDFGPAKAE

Table 1 Macromolecule production

Table 2 Crystallization

PDB ID 5LJF 6ZZ3

Method Vapor diffusion, hanging drop Vapor diffusion, hanging drop

Plate type EasyXtal (15-well) EasyXtal (15-well)

Temperature (K) 292 292

Protein concentration 192 µM 200 µM

Buffer composition of

protein solution 20 mM sodium phosphate pH 6.51 20 mM sodium phosphate pH 6.5

Composition of reservoir

solution

100 mM Tris HCl 17.5 % PEG600 pH

7.4

100 mM Tris, 20.5 % PEG600 pH

7.0

Volume and ratio of drop 1:1 1:1

Volume of reservoir 500 µL 500 µL

1 Crystals obtained in this crystallization condition were soaked 1 h in 100 mM Tris, 30 % PEG600

Table 3 Data collection and processing

Values for the outer shell are given in parentheses.

21


PDB ID 5LJF 6ZZ3

Diffraction sourceSOLEIL BEAMLINE

PROXIMA 2

SOLEIL BEAMLINE

PROXIMA 1

Wavelength (Å) 0.9801 0.9786

Temperature (K) 100 100

Detector ADSC quantum 315r CCD DECTRIS EIGER X

Crystal-detector distance (mm) 219.42 296.87

Rotation range per image (°) 0.5 0.1

Total rotation range (°) 220 360

Exposure time per image (s) 0.5 0.01

Space group P 21 21 21 P 1 21 1

a, b, c (Å) 45.69, 99.31, 148.58 57.00, 61.89, 177.26

α, β, γ (°) 90.00, 90.00, 90.00 90.00, 97.11, 90.00

Mosaicity (°) 0.137 0.129

Resolution range (Å) 38.9185–1.7343 (1.84–1.73) 45.10–2.09 (2.22-2.09)

Total No. of reflections 608773 499255

No. of unique reflections 70572 72135

Completeness (%)# 99.0 (96.4) 99.5 (97.1)

Redundancy 8.6 (8.00) 6.921(6.68)

⟨ I/σ(I)⟩ 15.81(3.01) 11.91 (1.39)

Rr.i.m.‡ 0.099 0.094

Overall B factor from Wilson plot

(Å2)

20.3 44.9

# relatively low overall completeness was related to ice-ring contamination.

‡ Estimated Rr.i.m. = Rmerge[N/(N − 1)]1/2, where N = data multiplicity.

Table 4 Structure solution and refinement

Values for the outer shell are given in parentheses.

PDB ID 5LJF 6ZZ3

Resolution range (Å) 38.92–1.73 (1.76-1.73) 45.10-2.10 (2.12-2.09)

Completeness (%) 99.3 99.5

22


σ cutoff F > 1.36 (F) F > 1.35(F)

No. of reflections, working set 70563 (2314) 72092 (2338)

No. of reflections, test set 3529 (122) 3607 (123)

Final Rcryst 0.176 (0.324) 0.195 (0.309)

Final Rfree 0.212 (0.330) 0.229 (0.353)

Cruickshank DPI 0.109 0.243

No. of non-H atoms

Protein 5065 10185

Ligand 68 140

Water 821 467

Total 5970 10792

R.m.s. deviations

Bonds (Å) 0.36 0.37

Angles (°) 0.57 0.56

Average B factors (Å2)

Protein 22.8 48.82

Ligand 23.3 44.33

Water 34.7 49.52

Ramachandran plot

Most favoured (%) 98 98.03

Allowed (%) 2 1.97

Table 5 Kinetic parameters of RBcel1_WT and RBcel1_E135A determined on ClPNP β–G3.

kcat (s-1) KM (mM) kcat/KM (s-1 mM-1)

RBcel1_WT 0.25 ± 0.012 2.35 ± 0.23 0.11

RBcel1_E135A 0.014 ± 0.0003 0.19 ± 0.02 0.07

23


Acknowledgements We thank the Giga proteomic facility (University of Liege) for the technical

support in MS analysis.

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26


Supporting information

Figure S1 (A) Cel5A from Bacillus agaradhaerens, close-up view of the GluNUC in the unliked form

(pink) (PDB ID: 1QI0) and covalently linked with 2’,4’-dinitrophenyl 2-deoxy-2-fluoro-β-D-

cellotrioside (yellow) (PDB ID: 1QI2). (B) Cel5A from Bacillus agaradhaerens, close-up view of the

GluNUC in the unliked form (green) (PDB ID:1A3H) and covalently linked with 2-deoxy-2-fluoro-β-D-

cellotriosyl (violet) (PDB ID: 1H11). (C) Endo-glyceramidase II from Rhodococcus sp close-up view

of the GluNUC in the unliked form (green) (PDB ID:2OSW) and in the Lactosyl-Enzyme Intermediate

(blue) (PDB ID:2OSY). (D) endo-xyloglucanase from Cellvibrio japonicus close-up view of the

GluNUC in the unlinked form (dark blue) (PDB ID:5OYC) and covalently linked with 2-deoxy-2-

fluoro-alpha-D-glucopyranose (grey) (PDB ID:6HAA).

(a) (b)

(c) (d)

27

_x000d_ · web view2021. 2. 9. · gh5: structural snapshots highlighting the involvement of two...

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