Functional and structural characterization of an α-?-arabinofuranosidase from Thermothielavioides terrestris and its exquisite domain-swapped β-propeller fold crystal packing

Article abstract of DOI:10.1016/j.bbapap.2020.140533

The fungus Thermothielavioides terrestris plays an important role in the global carbon cycle with enzymes capable of degrading polysaccharides from biomass, therefore an attractive source of proteins to be investigated and understood. From cloning to a three-dimensional structure, we foster a deeper characterization of an α-?-arabinofuranosidase, a glycoside hydrolase from the family 62 (TtAbf62), responsible to release arabinofuranose from non-reducing ends of polysaccharides. TtAbf62 was tested with synthetic (pNP-Araf) and polymeric substrates (arabinan and arabinoxylan), showing optimal temperature and pH (for pNP-Araf) of 30 °C and 4.5–5.0, respectively. Kinetic parameters revealed different specific activity for the three substrates, with a higher affinity for pNP-Araf (K_M: 4 ± 1 mM). The hydrolyzing activity of TtAbf62 on sugarcane bagasse suggests high efficiency in the decomposition of arabinoxylan, abundant hemicellulose presented in the sugarcane cell wall. The crystal packing of TtAbf62 reveals an exquisite domain swapping, located at the supramolecular arrangement through a disulfide bond. All crystallographic behaviors go against its monomeric state in solution, indicating a crystal-induced artifact. Structural information will form the basis for further studies aiming the development of optimized enzymatic properties to be used in biotechnological applications.

Full text of DOI:10.1016/j.bbapap.2020.140533

BBA-ProteinsandProteomics1868(2020)140533
Contents lists available at ScienceDirect
BBA - Proteins and Proteomics
journal homepage: www.elsevier.com/locate/bbapap
Functional and structural characterization of an α-ʟ-arabinofuranosidase
from Thermothielavioides terrestris and its exquisite domain-swapped β-
propeller fold crystal packing
Suelen Camargoa,1, Evandro J. Mulinaria,1, Leonardo R. de Almeidaa,1, Amanda Bernardesa,
⁎
Rolf A. Pradeb, Wanius Garciac, Fernando Segatod, João R.C. Muniza,
b Department of Microbiology and Molecular Genetics, Oklahoma State University (OSU), Stillwater, OK, USA
c Centro de Ciências Naturais e Humanas, Universidade Federal do ABC (UFABC), Santo André, SP, Brazil
d Department of Biotechnology, Lorena School of Engineering, University of São Paulo (USP), Lorena, SP, Brazil
a São Carlos Institute of Physics (IFSC), University of São Paulo (USP), São Carlos, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
The fungus Thermothielavioides terrestris plays an important role in the global carbon cycle with enzymes capable
of degrading polysaccharides from biomass, therefore an attractive source of proteins to be investigated and
understood. From cloning to a three-dimensional structure, we foster a deeper characterization of an α-ʟ-ara-
binofuranosidase, a glycoside hydrolase from the family 62 (TtAbf62), responsible to release arabinofuranose
from non-reducing ends of polysaccharides. TtAbf62 was tested with synthetic (pNP-Araf) and polymeric sub-
strates (arabinan and arabinoxylan), showing optimal temperature and pH (for pNP-Araf) of 30 °C and 4.5–5.0,
respectively. Kinetic parameters revealed different specific activity for the three substrates, with a higher affinity
Thermothielavioides terrestris
α-ʟ-arabinofuranosidase
Domain-swapping
GH62
TtAbf62
for pNP-Araf (KM: 4
1 mM). The hydrolyzing activity of TtAbf62 on sugarcane bagasse suggests high effi-
ciency in the decomposition of arabinoxylan, abundant hemicellulose presented in the sugarcane cell wall. The
crystal packing of TtAbf62 reveals an exquisite domain swapping, located at the supramolecular arrangement
through a disulfide bond. All crystallographic behaviors go against its monomeric state in solution, indicating a
crystal-induced artifact. Structural information will form the basis for further studies aiming the development of
optimized enzymatic properties to be used in biotechnological applications.
1. Introduction
These enzymes can release arabinose from many substrates and they
are part of the xylanolytic system, being essential to disrupt arabinox-
The α-ʟ-arabinofuranosidases (Abfs; EC 3.2.1.55) play a key role in
the releasing process of ʟ-arabinose from polysaccharides or arabino-
oligosaccharides acting into α-(1 → 2)-, α-(1 → 3)- or α-(1 → 5)- linked
ʟ-arabinofuranosyl residues from the non-reducing end of poly- and
oligosaccharides [1], increasing the hydrolysis of glycosidic bonds of
polysaccharides by other enzymes [2]. According to the Carbohydrate-
Active Enzymes database (CAZy; www.cazy.org), Abfs are grouped into
six distinct families of glycoside hydrolases: GH2, GH3, GH5, GH39,
GH43, GH51, GH54, and GH62 [3–5]. Based on their amino acid se-
quences, members of GH62 family has been divided into GH62_1 and
GH62_2 subfamilies [6]. Additionally, the Abf families are further
classified into three types based on divergence among themselves
concerning substrates specificity [7,8].
ylan bonds [1,9]. Therefore, Abfs are of remarkably biotechnological
importance [1], which extends from the use in industries oriented to
agriculture [10], food [11], and also in cocktails for digestion of animal
feed [10], enhancement of wines flavor [10,12], improvement in the
clarification of fruit juices [10,13], and for the delignification of cel-
lulose on biofuels production [14,15].
The filamentous fungus Thermothielavioides terrestris (previously
Thielavia terrestris), belongs to the Ascomycota phylum and is among a
limited number of eukaryotic species classified as thermophiles
showing optimal growth above 45 °C [16]. Many enzymes character-
ized from T. terrestris, such as cellulases and hemicellulases, have been
shown attractive for biotechnological applications involving biomass
hydrolysis due to its favorable catalytic and stability profiles [16–19].
⁎ Corresponding author at: n 1100, Jardim Santa Angelina - CEP 13563-120, São Carlos, São Paulo, Brazil.
E-mail address: jrcmuniz@ifsc.usp.br (J.R.C. Muniz).
1 Authors contributed equally to this manuscript.
https://doi.org/10.1016/j.bbapap.2020.140533
Received 19 April 2020; Received in revised form 25 July 2020; Accepted 12 August 2020
Availableonline29August2020
1570-9639/©2020ElsevierB.V.Allrightsreserved.

S. Camargo, et al.
BBA-ProteinsandProteomics1868(2020)140533
Aiming to contribute towards a better comprehension of these
with 2 mL Ni Sepharose 6 Fast Flow (GE Healthcare Life Sciences) was
performed as the first purification step and after washing the column
with buffer A, the recombinant protein was eluted with the same buffer
with 150 mM imidazole added on it. Eluted solution was dialyzed
overnight at 4 °C with buffer A. TEV protease (1:50 mg) was used for
cleavage of the N-terminal His6-Trx-tag. The second purification step
repeats the IMAC purification step but elutes the protein with 20 mL of
buffer A without imidazole. A third purification step was performed
classes of enzymes, in this study we describe an α-ʟ-arabinofur-
anosidase from the thermophilic fungus T. terrestris (subfamily GH62_2;
named here TtAbf62). The biochemical characteristics of purified
TtAbf62 were determined using sugar beet arabinan (SBA), wheat
arabinoxylan (WAX), and pNP-Araf as substrates. Moreover, the results
presented here allowed inferences to be drawn on the hydrolysis of
SBA, WAX, and pNP-Araf by T. terrestris. The biophysical and bio-
chemical outcomes aim to promote a better understanding of its cata-
lytic mechanism when degrading renewable biomass, making them
potential candidates for biotechnological applications in the biofuel and
food industries. Furthermore, structural analyzes revealed the presence
of a domain-swapping, probably a crystallization artifact. However, this
new and beautiful shape elegantly preserves the active site of the en-
zyme where the catalytic pocket was structurally conserved between
the 5-bladed β-propeller fold and confers protein stability.
using size exclusion chromatography (SEC) on
a HiLoad 16/60
Superdex 200 column (GE Healthcare Life Sciences) with buffer A. The
purity of the recombinant protein was monitored by SDS-PAGE.
2.3. Biochemical characterization
Optimal pH for TtAbf62 was determined by measuring the release of
4-nitrophenol from p-nitrophenyl-α-ʟ-arabinofuranoside (pNP-Araf;
2 mM; Sigma-Aldrich) as the substrate in 100 μL reactions containing
10 μg of the purified enzyme, and 50 mM McIlvaine buffer [26] with
the pH ranging from 2.0 to 8.0 at 50 °C for 10 min. The optimal tem-
perature was evaluated as described before, using the optimal pH
varying the temperature from 10.0 to 60.0 °C, incubated for 15 min.
After incubation, all reactions were quenched by the addition of 100 μL
of a stop reagent (saturated sodium tetraborate solution) and the ab-
sorbances of the released p-nitrophenol was measured at 410 nm. For
kinetic parameters determination, the end-point velocities of the en-
zymes were measured in 50 mM McIlvaine buffer at optimal pH and
temperature, using pNP-Araf in concentrations ranging from 0.1 to
50.0 mM, sugar beet arabinan (SBA) and wheat arabinoxylan (WAX)
(Megazyme) in concentrations ranging from 1 to 27 mg.mL−1 for
25 min for polysaccharides and 7 min for pNP-Araf. All reactions were
carried out in 100 μL and using 2 μg of the enzyme. The reducing sugars
released in enzymatic reactions with polysaccharides was measured by
the 3,5-dinitrosalicylic acid method (DNS) by measuring the absor-
bances at 540 nm [27]. The kinetics parameters (KM and kcat) were
estimated by using direct weighting nonlinear least-squares regression
analysis with Origin software (version 2018). The adjusted R-Square
(Adj. R-Square) value was determined as a measure of the goodness of
fit of experimental data. A calibration curve for the DNS method was
plotted and used to calculate the amount of non-reducing end released.
The 4-nitrophenol (pNP) was used to calculate the amount of arabinose
released in pNP-Araf reactions. All assays were carried out in triplicate
and the averaged values reported.
2. Methods
2.1. Fungus cultivation
The T. terrestris strain UAMH 3264 was purchased at Microfungus
Collection of University of Alberta, Devonian Botanic Garden
Edmonton. The fungus was maintained in Potato Dextrose Agar
medium (Sigma-Aldrich), at 45 °C for 5 days and the resulting myce-
lium was used for further cultivation that was performed in 1 L of the
minimal medium [20] containing: 50 mL of 20× Clutterbuck solution
[21], 1 mL of a trace elements solution (100 mL MilliQ® water: 2.2 g of
ZnSO4, 1 g of H3BO3, 0.5 g of MnCl2.4H2O, 0.5 g of FeSO4.7H2O, 0.16 g
of CoCl2.5H2O, 0.16 g of CuSO4.5H2O, 0.11 g of Na2MoO4.4H2O and
5 g of Na2EDTA at pH 6.5) and supplemented with 5 mM glutamine and
2.4 μM thiamine at pH 5.0 [22] using a rotary shaker (150 rpm) at 45 °C
for 48 h.
2.2. Cloning, heterologous expression, and purification of TtAbf62
A total of 10 mg of the mycelium of T. terrestris was frozen in liquid
nitrogen and then triturated using a pistil, placed in a 1.5 mL tube
where 600 μL and the protocol for genomic DNA isolation was followed
according to Damásio and collaborators [23]. To confirm the quality of
the extracted DNA, the sample was analyzed on agarose gel (0.8%) by
using the technique of electrophoresis.
The gene encoding the TtAbf62 protein (GenBank: AEO64662.1)
was amplified from T. terrestris genomic DNA by Polymerase Chain
Reaction (PCR) and cloned into pETTrx-1-a/LIC expression vector
(Novagen) allowing the expression of recombinant protein with a His6-
Thioredoxin-tag (His6-Trx-tag) fused at its N-terminal [24,25] using
specific oligonucleotides (Supplemental SM1). The vector containing
the gene fragment of interest was transformed into E. coli Rosetta-
gami™ 2 (DE3) (Novagen) expression system and the positive colonies
were selected based on its resistance of growing in the presence of
kanamycin and chloramphenicol. The recombinant TtAbf62 was over-
expressed in a 2 L Erlenmeyer containing 1 L of auto-induction medium
(Supplemental SM2) added with kanamycin (0.05 mg.mL−1) and
chloramphenicol (0.036 mg.mL−1), incubated in an orbital shaker with
150 rpm at 37 °C until an optical density of 0.6 at 600 nm; then the
temperature was lowered and stabilized at 17 °C to induce protein ex-
pression for 17 h. The expression was monitored by SDS-PAGE. Cells
were separated by centrifugation at 4500 xg for 20 min at 4 °C, and the
pellet stored at −80 °C.
The activity of TtAbf62 in the presence of sugarcane bagasse in
natura was evaluated by incubating the reaction in the agitation of
150 rpm in a rotatory shaker at 50 °C for 20 h. The duplicate reaction
with 1.25 mL contained 20 mg of sugarcane bagasse in natura in
1.25 mL reaction with 40 mM McIlvaine buffer pH 4.5, 30 μg TtAbf62
and 0.02% sodium azide. In the enzyme-free reaction (control), the
volume corresponding to the enzyme was replaced by 50 mM Tris-HCl
at pH 8.0 (stock buffer). The reaction was centrifuged at 13,000 xg,
aliquoted in 100 μL, and the amount of reducing sugars was measured
by DNS [27]. The thermal stability of TtAbf62 was evaluated by mea-
suring the residual activity at optimal pH and incubating the enzyme in
temperatures of 30 and 50 °C. Amounts corresponding to 10 μg of the
enzyme were aliquoted at time intervals and assays carried out using
pNP-Araf (0.5 mM final concentration) as the substrate in 100 μL re-
actions up to the total time of 36 h of incubation. All measurements
were performed in triplicates and conducted in a 96-well microplate
(Greiner) by using a Spectramax Plus 384 spectrophotometer (Mole-
cular Device).
The pellet was resuspended in buffer A (50 mM Tris-HCl at pH 7.5,
150 mM NaCl and 10% (w/v) of glycerol) for cell disruption. The
treated pellet was sonicated on ice in 3 cycles (30 s sonication, 30 s
rest), in a sonicator (Fisher Scientific™ Model 505 Sonic Dismembrator)
set with 45% amplitude. The lysate was then centrifuged at 14,000 xg at
4 °C for 40 min. Immobilized metal affinity chromatography (IMAC)
2.4. Thermal denaturation analysis
2.4.1. Differential scanning fluorimetry (DSF)
The DSF method was used to evaluate the structural integrity of
TtAbf62 in different conditions including a variety of buffer salts and
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BBA-ProteinsandProteomics1868(2020)140533
pH values. A CFX96 Real-Time System® (Bio-Rad) was used to detect
the dye fluorescence allowing the determination of TtAbf62 melting
temperature Tm [28]. The assay was carried out applying 30 μg of
purified TtAbf62 to a 96 well plate containing different buffer solutions
(Supplementary Table A.1), in a final concentration of 50 mM and
1:2000 of SYPRO Orange (Molecular Probes) and analysis were per-
formed within the temperature range from 25 to 90 °C (with 1 °C in-
crement of each 30 s). The fluorescence variation of SYPRO Orange was
measured using excitation at 490 nm and emission at 530 nm. The
construction and analysis of the curves, including the determination of
the Tm of each sample, were carried out using Origin, version 2018
(OriginLab Corporation).
2.7. Size exclusion chromatography with multiangle light scattering (SEC-
MALS)
Using Waters 600 HPLC coupled to a Wyatt DAWN EOS light scat-
tering instrument, Wyatt Optilab refractometer and Wyatt dynamic
light scattering module (Wyatt Technology Corp., Santa Barbara, CA),
SEC-MALS experiments were employed for chromatographic separa-
tions to verify the oligomeric state of TtAbf62 using a Superdex 75 10/
300 GL (GE Healthcare Life Sciences) equilibrated with 20 mM Tris-HCl
at pH 8.0 and 300 mM NaCl buffer, with an eluent flow rate of
0.5 mL.min−1. Molecular mass calculations were performed by ASTRA
software.
2.8. Glutaraldehyde cross-linking
2.4.2. Circular dichroism (CD) spectroscopy
CD transition curve of TtAbf62 was obtained using a JASCO J-815
CD spectropolarimeter in 50 mM McIlvaine, pH 4.5, and 50 mM Tris-
HCl, 0.3 M NaCl, pH 7.5 buffers, using a 0.1 cm quartz cuvette, mon-
itoring the ellipticity at 220 nm from 20 °C to 80 °C at a heating rate of
1 °C/min.
Glutaraldehyde treatment with cross-linkers was performed in
20 mM HEPES at pH 8.5. Reaction mix containing 1 μg of protein
(control and TtAbf62) in a total volume of 100 μL were treated with 5
and 10 μL of 2.3% freshly prepared solution of glutaraldehyde for 5 min
at 37 °C. The reaction was stopped by the addition of 10 μL of 1 M Tris-
HCl at pH 8.5. Cross-linked proteins were solubilized by the addition of
an equal volume of Laemmli buffer [40], and verified by SDS-PAGE
[41].
2.5. Protein crystallization, data collection, and processing
Crystals of TtAbf62 were obtained using the purified protein in a
concentration of 20 mg.mL−1 in previously described buffer A. The
protein concentration was determined by NanoDrop® 2000 spectro-
photometer (Thermo Scientific). The initial screening of crystallization
conditions was performed using a Crystal Gryphon LCP® crystallization
robot (Art Robbins Instruments). Crystals were obtained after 30 days
during exposure to 0.1 M sodium acetate (pH 4.6) and 2 M ammonium
sulfate as reservoir solution at 18 °C.
3. Results and discussion
3.1. Expression and purification of TtAbf62
The TtAbf62 gene was localized in T. terrestris chromosome I be-
tween positions 9,454,228 and 9,455,223 at the complement strand.
The gene sequence did not present introns and showed a region of 78 bp
that correspond to the signal peptide (SP) in the TtAbf62 protein. To
oligonucleotides synthesis, the SP region was not considered and the
gene showing 903 bp was amplified by PCR, cloned and expressed. The
recombinant TtAbf62 was expressed in E. coli system containing a tag of
6 histidine residues (His6) and a thioredoxin (Trx) fused-protein. The
total protein yield was approximately 2 mg.L−1 of culture medium.
Before TEV protease treatment, the SDS-PAGE analysis revealed a mo-
lecular mass bellow 45 kDa, which was consistent with the product of
cloning on pETTrx-1-a/LIC (Fig. 1). After TEV cleavage the final mo-
lecular mass was around 31 kDa, which contains the residual site of
TEV, plus one methionine, resulting in a molecular mass calculated
from the mature amino acid sequence of 32.8 kDa (Fig. 1).
The crystal diffraction and data collection were performed at the
synchrotron radiation source at the Brazilian National Laboratory of
Synchrotron Light from National Center for Energy and Materials
(LNLS-CNPEM, Brazil). Diffraction datasets from the TtAbf62 crystal
were collected on beamline W01-MX2 using a PILATUS2M detector
(Dectris). The data-collection strategy was determined using the
iMosflm Strategy function [29], targeting > 95% completeness for data
set collected to the given resolutions. The data were indexed and in-
tegrated using the programs XIA2 [30] and XDS [31] and scaled using
Aimless [32,33] from the CCP4 suite [34]. The data completeness and
CC1/2 were used to determine the resolution limit.
2.6. Structure determination and refinement
Structure of TtAbf62 was initially solved using molecular replace-
ment with Phaser program [35]. The structure of a thermostable family
GH62 α-ʟ-arabinofuranosidase from Streptomyces thermoviolaceus (PDB
id; 4O8N) [2] was used as a search model (79% of sequence identity).
Crystallographic refinement was carried out using REFMAC5 [36] and
manual model building was performed with Coot program [37] using
weighted 2Fobs – Fcalc and Fobs – Fcalc electron-density maps. Water
molecules and additional fortuitous ligands such as sulfate ions and
ethylene glycol molecules were manually placed appropriately by using
Coot. The behavior of Rwork and Rfree was used as the main criteria to
validate the refinement protocol and its convergence. The stereo-
chemical quality of the model was evaluated with the MolProbity
program [38]. The PISA server [39] was applied to verify oligomeric
states by evaluating interfaces and their strength of the interaction
between neighboring monomers generated by applying symmetry to
the input file coordinates on the macromolecular assembly of TtAbf62,
consequently, predict the most likely biological form of the structure
established through domain swapping.
Fig. 1. Purification of TtAbf62. Mr – molecular mass; 1 –TtAbf62 after an IMAC
step of purification (43 kDa); 2 – The protein with approximately 33 kDa after
cleavage with TEV protease; 3 – Purified protein after size exclusion chroma-
tography.
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S. Camargo, et al.
BBA-ProteinsandProteomics1868(2020)140533
3.2. Thermal denaturation analysis
and 50 °C (Supplementary Fig. A.3). At 50 °C, the enzyme maintained a
residual activity of 30.6% after 36 h, and in the same period, the re-
sidual activity at 30 °C was 43.5%. Even at the higher temperature
tested (50 °C), the enzymatic activity demonstrated that the TtAbf62
tolerates this condition for extended periods, whereas the complete
inactivation was not observed, also the complete inactivation at 30 °C
was not observed as well.
To investigate the thermostability under miscellaneous buffer con-
ditions, the DSF was performed for TtAbf62 and its melting point
temperatures (Tm), at different buffering conditions, were determined,
as shown in the Supplementary Table A.1. An overview of the analyzed
conditions indicates that the higher Tm was 64 °C in 50 mM Bis-Tris
buffer at pH 6.7. These results also demonstrated a reasonable thermal
tolerance at temperatures near to 60 °C, decreasing the values of Tm
only when the buffer pH was extremely acidic or alkaline regardless of
the composition (more details in the Supplementary Table A.1).
The DSF analyses established that Bis-Tris buffer at pH 6.7 was the
best condition for protein stability with a Tm of 64 °C, followed by the
same buffer at pH 6.0 and Tris-HCl buffer at pH 7.5, both with a Tm of
63 °C. The addition of NaCl did not affect the Tm. Thermal denaturation
was also examined by CD as a complementary technique providing
additional information on Tm. Both CD conditions resulted in Tm values
3.3.2. Kinetic parameters
The kinetic parameters were determined on substrates with all Adj.
R-square greater than 0.95 where TtAbf62 had the uppermost activity
based on the substrate panel: pNP-Araf, WAX and SBA (Table 1). As
described in Table 1 equivalent values of kcat was achieved for the
substrates SBA (with a KM = 9
3 mg.mL−1) and pNP-Araf
(KM = 4
1 mM−1), wherein the TtAbf62 shows higher affinity by
pNP-Araf, as well as other enzymes previously characterized. Com-
paring kcat towards arabinan 5.8
0.8 s−1
, and pNP-Araf
6.8
0.3 s−1, the turnover number is in the same order of magnitude,
of 59.7
0.4 and 62.6
0.2 °C for McIlvaine and Tris buffers, re-
spectively (Supplementary Fig. A.1). These data suggest that the en-
zyme has great structural stability over a wide spectrum of pH values
and buffers conditions.
suggesting the similar conversions rate independently of the polymeric
or synthetic substrate. Nevertheless, the enzymatic preference (kcat/KM)
is higher for pNP-Araf when compared to SBA, whereas SthAbf62A,
AnAbf62A and ScAbf62A (Table 1) prefer WAX. TtAbf62 also presented
the highest catalytic efficiency and enzymatic activity (U mg−1) in pNP-
Araf and SBA when compared to other enzymes as described in Table 1.
The activity towards pNP-Araf can be explained by the insights from
the structural analysis of the TtAbf62 (section 3.4), which has the cat-
alytic triad composed of two aspartates (Asp58 and Asp165) and one
glutamate (Glu217) coordinating the substrate. Also, the residues
Asp58 and Asp165 are acting as catalytic residues, interacting with a p-
nitrophenolate ring that stabilizes the intermolecular interactions.
Considering that increased thermal stability of the enzyme is cor-
related with enhanced structural arrangement often followed by con-
formational integrity of protein [42–44], the crystallization tests of
TtAbf62 were performed in Tris-HCl buffer at pH 7.5, one of the best
conditions pointed out by the DSF experiment.
3.3. Biochemical characterization
3.3.1. Optimum pH and temperature for TtAbf62 activity
Some enzymes of family GH62 can hydrolyze the substrate pNP-
Araf, and also display arabinofuranosidase activity when assayed with
polymeric substrates [2,45,46]. The activity of TtAbf62 was verified on
polymeric substrates (Supplementary Fig. A.2) and did not show ac-
tivity in the synthetic substrates as p-nitrophenyl-β-ᴅ-mannopyrano-
side, p-nitrophenyl-β-ᴅ-glucopyranoside, p-nitrophenyl-β-ᴅ-galactopyr-
anoside, p-nitrophenyl-β-ᴅ-xylopyranoside (data not shown) although
only against pNP-Araf the activity was detected and applied to further
analysis. The activity on arabinoxylan and arabinan reveals its capacity
to release arabinose from the branches of water-soluble α-ʟ-Araf-(1 →
3) and α-ʟ-Araf-(1 → 2) bonds [47]. Beyond arabinoxylan, it is im-
portant to note that the enzymatic activity in branched arabinan, which
contains a backbone of (1 → 5)-α-ʟ-Araf linkage, shows that TtAbf62
may be acting in both polysaccharides from hemicellulose and pectin of
sugarcane cell wall sources. The preference of the enzyme to hydrolyze
substrates decorated with Araf-α-(1 → 3) and Araf-α-(1 → 2) bonds is
clear since the significant activity is observed for WAX, indicating that
the arabinosyl side chain is critical for the substrate recognition [48],
making it plausible to classify the enzyme as arabinoxylan arabinofur-
anohydrolases [49].
3.3.3. Activity of TtAbf62 in sugarcane bagasse
The sugarcane cell wall contains 40% arabinoxylan (AX), where
arabinoside side chain may cause a steric hindrance for xylanases [55].
The hydrolysis with TtAbf62 was qualitatively tested against natural
biomass to estimate the potential of this enzyme to act on complex
substrates. Evaluated by DNS method [27], the addition of TtAbf62
resulted in a higher concentration of reducing terminals (4.5 μmol
glucose equivalent), compared with the enzymatic cocktail itself
(0.2 μmol glucose equivalent). This might be related to its ability to
remove arabinose side chains from the AX, as well as arabinan, de-
creasing steric restraint in branched polysaccharides, making it more
susceptible to hydrolysis by exo and endoxylanases.
3.4. Structural characterization
3.4.1. Overall structure
After the crystallization process, orthorhombic crystals of TtAbf62
were obtained (I222; a = 75.267 Å, b = 79.747 Å, c = 82.922 Å) and
diffracted up to 1.87 Å resolution. The structure was determined by
molecular replacement with one molecule in the asymmetric unit. The
protein chain was comprised of 303 amino acid residues (from Gly28 to
Arg331), two ions: calcium (Ca2+), sulfate (SO42−), and one molecule
of ethylene glycol (EDO). All amino acids and ligands built in the
TtAbf62 exhibits high activity for pNP-Araf over the pH range from
4.5 to 5.0 (Fig. 2a), with an abrupt decrease in its catalytic strength for
pH values lower than 4.0 and over 5.0, suggesting an activity sensitive
to pH. Since the DSF results pointed high structural stability for TtAbf62
towards pH changes, the decrease in the enzyme activity should not be
related with the loss in structural stability, but probably due to an
impact on the overall charge of the amino acid residues involved in its
activity.
structure exhibited
a well-defined electron density. Final crystal-
lographic Rwork and Rfree values were 14.7% and 19.5%, respectively,
with more than 96% of the residues located on the sterically allowed
regions of the Ramachandran plot. Further refinement statistics are
described in Table 2.
When analyzed the optimal temperature of activity the highest va-
lues are in a range between 25 and 35 °C (Fig. 2b). Even being a
thermophilic fungus, these values are compatible with the optimum pH
and temperature previously observed for GH62 family towards pNP-
Araf as a substrate, e.g.: Podospora anserina pH 5.0 and 37 °C [6]; Us-
tilago maydis pH 5.0 and 37 °C [6] (Table 2).
The TtAbf62 asymmetric unit is shown in Fig. 3a. The N-terminus
began in a loop from Gly28 to Thr54 residue, followed by five β-sheets
(called blades I to V), usually composed of four antiparallel β-strands.
Blades I, II, and III are composed of four antiparallel β-strands; blade IV
by five mixed β-strands plus a η4 helix; and blade V by three anti-
parallel β-strands, respectively, forming a set of 20 β-strand, from β1 to
β20, linked by loops with variant lengths. Also, five helices (called η1 to
Furthermore, the enzyme stability was assessed by measuring the
residual activity after prolonged periods (up to 36 h) of incubation at 30
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S. Camargo, et al.
BBA-ProteinsandProteomics1868(2020)140533
Fig. 2. Optimal pH and temperature of TtAbf62 for pNP-Araf. In both cases (a) and (b), the solid line is just a guide for the eye.
Table 1
Kinetic parameters of TtAbf62. Substrate specificity and kinetic data of GH62 enzymes with known structure deposited on PDB. pNP: para-nitrophenol, pNP-Araf:
para-nitrophenyl α-ʟ-arabinofuranoside, pNP-X: para-nitrophenyl β-D-xylopyranoside, pNP-Arap: pNP-α-ʟ-arabinopyranoside, SBA: sugar beet arabinan, WAX: wheat
arabinoxylan, HV: high viscosity, LV: low viscosity. Sp. act.: Specific activity, U: unit defined as the release of 1 μmol of product per min per mg of the substrate under
the specific assay conditions. *values are normalized according to the unit.
Source,
Assay conditions,
Activity
KM
kcat
kcat/KM
Reference
(protein ID),
accession no.
substrate
Thermothielavioides terrestris (TtAbf62),
pH 4.5, 30 °C
Sp. Act
(U mg−1
0.78
mM
4
s−1
s−1.mM−1
This study
AEO64662.1
)
pNP-Araf
1
6.8
0.3
0.8
2
mg.mL−1
s−1
s−1.mg−1.mL
WAX
0.12
0.21
SBA
9
3
5.8
0.6
Streptomyces
coelicolor A3
(ScAbf62A),
CAA16189.1
Podospora anserina
S mat+
pH 5.5, 30 °C
pNP-Araf
mM
1.9
s−1
*
s−1.mM−1
0.01
*
[46]
0.02
s−1
mg.mL−1
7.3
s−1.mg−1.mL
0.04
WAX
0.30
a
pH 5.0, 37 °C
Sp. Act
(U mg−1
0.61a
mM
s-1⁎
s−1.mM-1⁎
[6];b [50];
)
(PaAbf62A),
CAP62336.1
pNP-Araf
6.1a,b
0.38a,b
0.06a,b
0.38b
WAX-LV
SBA
1b
0 .11b
Sp. Act
Streptomyces
pH 7.0, 55 °C
mM
3
s−1
1.3
s−1 mM
0.5
[2,51]
thermoviolaceus
OPC-520
(U mg−1)*
0.003
pNP-Araf
pNP-X
(SthAbf62A-m2,3),
BAB84113.1
7.8·10−6
5.2·10−7
pNP-Arap
8.2
0.01
s−1
0.001
mg.mL−1
12
s−1.mg−1.mL
15
WAX-HV
WAX-LV
0.04
180
0.02
SBA
0.001
Sp. Act
32
6
0.2
a
b
Ustilago maydis
pH 5.0, 37 °Ca
pH 4.5, 40 °Cb
pNP-Araf
WAX-LV
mM
s-1⁎
s−1.mM-1⁎
[6]; [52]
521 (UmAbf62Am2,3),
EAK85571.1
(U mg−1
)
0.06a; 0.012b
30b
7.5a
0.04a
0.005a
SBA
0 .003b
Sp. Act
Aspergillus nidulans
FGSC A4
pH 5.5, 37 °C
s−1.mM−1
[53]
[47]
[54]
(U mg−1
1.6
)
)
(AnAbf62A-m2,3),
EAA59562.1
pNP-Araf
0.26
mg.mL−1
4.9
s−1
17
8
s−1.mg−1.mL
36
WAX-LV
67
Scytalidium
pH 7.0, 50 °C
pNP-Araf
Sp. Act
(U mg−1
0.02
thermophilum CBS
625.91 (StAbf62Cm2,3),
AHZ56660.1
mg.mL−1
3.66
s−1
s−1.mg−1.mL
0.07
WAX-HV
SBA
0.28
1.5
0.4
Coprinopsis cinerea
(CcAbf62A),
pH 7.0, 40 °C
Sp. Act
(U mg−1)*
0.15
BAK14423.1
WAX
5

S. Camargo, et al.
BBA-ProteinsandProteomics1868(2020)140533
Table 2
enzyme TtAbf62 adopts a homodimeric blade-swapped β-propeller fold
(see scheme in Supplementary Fig. A.5), each part containing five
blades (I to V) radially arranged in two pseudo-5-fold axes wherein the
beta-sheets (β1 to β20) are repeated, as shown in Fig. 4a and Supple-
mentary Fig. A.5, maintaining the catalytic residues on its appropriated
position (Fig. 4b).
Data collection and refinement statistics. Values in parentheses are for the
highest resolution shell.
Name
TtAbf62 (PDB ID: 6CC7)
Data collection
Beamline
W01A–MX2, LNLS
1.4586
The domain swapping occurs when two or more molecules make a
partial exchange of domains among their structures, usually to form
oligomers, from their monomeric subunits [59–63]. It represents not
only an isolated event of structural innovation but differently, these
swaps enable the creation of new interfaces of interactions previously
absent in the monomer or a closed arrangement [59]. Considered an
important innovative aspect in the structure and function of a protein,
even been commonly found, such a mechanism is still poorly under-
stood structurally.
Wavelength (Å)
Space group
I222
a, b, c (Å)
75.27, 79.75, 82.92
1.79
VM (Å3.Da−1
)
a
Solvent content (%)
Resolution (Å)
Unique reflections
Completeness (%)
Multiplicity
31.3
33.05–1.87 (1.91–1.87)
20,946 (1318)
99.8 (97.4)
5.5 (2.8)
CC1/2 (%)
99.8 (83.8)
7.1 (38.6)
Rmerge (%)
A search for similar structures of TtAbf62 was performed using the
Dali server [64] (Supplementary Table A.2) and the results are dis-
played as aligned sequences with graphical enhancements performed
by ESPript 3.0 [65] (Supplementary Fig. A.6). Comparisons to similar
structures from Streptomyces coelicolor ScAbf62A (PDB ID 3WMY;
3WN0; 3WN1; 3WN2) [46], S. thermoviolaceus SthAbf62A (PDB ID
4O8N; 4O8O and 4O8P) [2], Podospora anserina PaAbf62A (PDB ID
4N4B and 4N2Z), Ustilago maydis UmAbf62A (PDB ID 4N1I and 4N2R)
[6], Aspergillus nidulans AnAbf62A (PDB ID 5UBJ) [9], Scytalidium
thermophilum SthAbf62C (PDB ID 4PVA and 4PVI) [47], Coprinopsis ci-
nerea CcAbf62A (PDB ID 5B6S) [66] and Talaromyces pinophilus
TpAbf62 (PDB ID 6F1J) [67].
Rmeas (%)
8.4 (52.2)
Rp.i.m. (%)
4.5 (34.9)
Mean I/σ(I)
11.9 (2.2)
Refinement
Resolution (Å)
No. of reflections
Completeness (%)
Rwork (%)
57.48–1.87
19,767
99.65
14.74
19.53
23.69
23.78
35.26
39.66
39.33
29.19
2334
b
Rfree (%)
Average B factor (Å2)
Protein
Waters
Metal
Inorganic
Organic
No. of protein atoms
No. of solvent atoms
No. of molecules in the asymmetric unit
R.m.s.d.b, bonds (Å)
R.m.s.d., angles (°)
Ramachandran plot
Favored (%)
3.4.2. Catalytic pocket description
298
In-depth analysis side-by-side of structurally characterized GH62
enzymes revealed that the catalytic triad [68]: an aspartate (acting as a
general acid); glutamate (acting as a general base); and an aspartic acid
supposedly modulating the pKa of the catalytic general acid, as ob-
served in many crystallographic structures [2,6,46] and supported by
site-directed mutagenesis and kinetic data [2,46], were also conserved
in TtAbf62, named Asp58 (from β1), Asp165 (from β9), and Glu217
(from β13) (Fig. 4b, Supplementary Table A.3, and Fig. A.4). These
residues interact with the ligands similarly as described in ScAbf62A
(3WN0) [46], SthAbf62A (4O8O) [2] and UmAbf62A (4N2R) [6] for an
arabinose residue; in SthAbf62A (4O8P) [2] for a xylotetraose residue;
in ScAbf62A (3WN1) [46] and SthAbf62C (4PVI) [47] for xylotriose
residues; in ScAbf62A (3WN2) [46] for a xylohexaose residue; and in
PaAbf62A (4N2Z) [6] for a cellotriose residue. They are also conserved
independently of their subclassification of the family, GH43, and GH62.
For TtAbf62 (6CC7), a molecule of ethylene glycol (EDO, from the
crystallization condition) is observed at the catalytic site, interacting
via hydrogen bonds directly with Lys57 (dNZ-O1 = 2.84 Å) and Asp58
(dOD2-O1 = 2.72 Å).
1
0.014
1.628
96.36
3.31
0.33
0.44
0.90
Allowed (%)
Outliers (%)
Clashscore
MolProbity score [38]
a
b
Matthews coefficient.
The test set uses ~5% of the data.
η5) are distributed along the chain (Fig. 3b).
Analysis using PISA server [39] showed that the opened interface
adopted by the structure of TtAbf62 allows contacts between chains
related by a symmetry operation (−x + 1, y, −z) throughout 125
residues (41.3% of the whole structure). These interactions embrace a
variety of hydrogen bonds, a salt bridge between His283A and Asp58B
(dND1-OD1 = 3.10 Å), and also a disulfide bond between Cys32A (from
chain A) and Cys300B (from chain B) with a distance dS-S of 2.03 Å.
This interchain disulfide is connecting a loop (formed by residues 28 to
54) located at the N-terminus of chain A (Cys32A) and a η4 located at
the C-terminus of chain B (Cys300) (Fig. 4).
Alignments of the three-dimensional structures (Fig. 5) and amino
acid sequences (Supplementary Fig. A.6) revealed that in the catalytic
pocket, the residues at the position equivalent to 57 and 283 in TtAbf62
(6CC7) are conserved in all structures, except in TpAbf62 (6F1J), since
the Lys57 and His283 were mutated by an arginine (Arg) and serine
(Ser), respectively. The key residues at position 58 (acting as a general
acid), position 125 and 165 (acting as a pKa modulator), position 217
(acting as a general base), and positions 307 and 317 are fully con-
served in all available structures.
The disulfide bond parameters are calculated according to the em-
pirical formula described by Katz et al. [56] and Schmidt et al. [57]
(https://services.mbi.ucla.edu/disulfide/). Interestingly, the presence
of a disulfide bond summed with the opened interface adopted by the
structure provided the possibility of an exquisite phenomenon of do-
main swapping, first observed in the GH62 family. This domain swap-
ping generates an interface of contacts among neighbor protein chains
involving blade I from chain B (blade IB) fitted onto chain A and blade I
from chain A (blade IA) shuffled to chain B. Additionally, this phe-
nomenon also relies on the so-called “molecular velcro”, joining the N-
terminal β1 in one of the modular β-sheets (β20) via hydrogen bonds
[58]. However, in this particular case, the molecular velcro is arranged
by hydrogen bonds connecting the loop (28–54) from N-terminal upon
β20 from C-terminal (details in Supplementary Fig. A.4). As a result, the
The calcium ions (Ca2+) listed in Table A.3 are usually hydrogen-
bonded to
a histidine residue (Ca-His), equivalent to His283
(Supplementary Fig. A.6), with a few exceptions for TtAbf62 (6CC7)
and SthAbf62C (4PVA and 4PVI). The presence of this histidine residue
in the catalytic pocket has been proposed to be an asset in combination
with the catalytic triad residues [47].
Some particular water molecules (H2O) also appear to play a sig-
nificant role in the catalytic site. In TtAbf62, the H2O 533, 542, 574,
and 594 are present, and their equivalent positions in other structures
6

S. Camargo, et al.
BBA-ProteinsandProteomics1868(2020)140533
Fig. 3. Topology and top view of the asymmetric unit. a) The overall structure of TtAbf62 revealing a five-bladed β-propeller fold, top view showing the numbering of
5 blades (blades I to V), a disulfide bond between Cys32A and Cys300B, an ethylene glycol (EDO) molecule located at partial catalytic cleft correspondent to channel
of the fold, two sulfate ions (SO42−) close to Blade V, and a calcium ion (Ca2+) coordinated close to Blade IV. b) The overall topology of TtAbf62 revealing a five-
bladed β-propeller fold. Amino acid residue numbers are given at each start and end of the respective secondary structure elements, β-strands are indicated as arrows
β1–β20 (shown in blue) and helices are indicated by cylinders η1–η5 (shown in red), respectively.
are often connected to the His283 (His-H2O) as shown in 3WN1, 3WN2,
4PVI and 5B6S. The H2O542 intermediates an interaction between re-
sidues Gln307 and Tyr317 (Gln-H2O-Tyr), however, it was not observed
in 3WN0 and 4N2R, where the water molecule is replaced by β-ʟ-ara-
binofuranose, and for 5UBJ and 4PVA was replaced by a calcium and
phosphate ion, respectively. The list of structural elements in the cat-
alytic pocket for the Abfs GH62 family is presented in Supplementary
Table A.3.
share a high degree of similarity, adopting the β-propeller fold with five
blades (I-V), and their active site is composed of previously highlighted
catalytic triad [68], that often interacts with the substrate.
The structure of TtAbf62 was compared to other Abfs belonging to
the family GH62 in complex with xylooligosaccharides and revealed
that their orientations were similar (Supplementary Fig. A.8).
According to Maehara et al. [46], the residues Trp270 and Tyr461 in
ScAbf62A are interacting with xylotriose (PDB ID 3WN1) and xylo-
hexaose (PDB ID 3WN2) ring at subsites +3NR and + 1, respectively.
The same positions were found in TtAbf62, corresponding to Trp126
and Tyr317, respectively. The residue Asn318 in TtAbf62 (PDB ID
6CC7), also has the same orientation as described in the structures
complexed with xylooligosaccharides: ScAbf62A (PDB IDs 3WN1 and
3WN2) [46] with an equivalent position of Asn462; Asn339 in
SthAbf62C (PDB ID 4PVI) [47]; Asn321 in PaAbf62A (PDB ID 4N2Z)
[6]; and Asp350 in SthAbf62A (PDB ID 4O8P) [2]. Amino acids at this
position are usually involved in the hydrogen bond to subsite +2NR
and reported as responsible for affecting the activity specifically in
polysaccharides [46].
The calcium ion (Ca2+) presented in the structure of TtAbf62 is
located in a completely different position when compared to other
solved structures for the same family. In TtAbf62 the Ca2+ is located at
the interface of contacts between two dimeric arrangements, specifi-
cally by the residue Thr212 and (H2O)730 both within 3.9 Å of distance
as detailed in Supplementary Fig. A.7a. Furthermore, the chelation of
Ca2+ by EDTA had no apparent effect on the enzymatic activity (data
not shown). Fortuitous ligands from crystallization condition, the sul-
fate ions (SO42−) 402 and 403 are coordinated on the surface of the
structure by residues Trp91 and Asn312, respectively, with the addition
of some water molecules (Supplementary Fig. A.7b-c).
To demonstrate the most likely bio-assembly of TtAbf62 in solution,
SEC-MALS and cross-linking experiments were carried out. Differently
of the crystal structure, assays with TtAbf62 in solution demonstrate
3.4.3. Structural insights
According to CAZy, all the structures of characterized GH62 family
7

S. Camargo, et al.
BBA-ProteinsandProteomics1868(2020)140533
Fig. 4. Detailed view of the swapped domain from TtAbf62, its catalytic pocket, and disulfide bond. a) TtAbf62 represented as a homodimer through a domain swap.
In detail, the N- (dark blue) and C- (dark red) terminus from their monomer chains respectively, which are connected via a disulfide bond. Five blades of β-strands
can be observed assembling the dimeric system required for this structure. Calcium ions (Ca2+) are represented as spheres colored in green; sulfate ions (SO42−)
and ethylene glycol (EDO) ligands can also be observed. b) Detailed view of the catalytic pocket with hydrogen bonds between water molecules mediating inter-
actions to EDO and also a salt bridge evolving H283 and *D58 residues. Isomesh level 1.0. c) Detailed view of the disulfide bond between A:C32 (belonging to the N
terminus) and B:C300 (belonging to the C terminus) with its respective parameters.
evidence of a monomeric arrangement, suggesting that a closed inter-
face regarding all blades in a classical β-propeller fold is most reliable.
In general, the domain swapping phenomena has no clear ex-
planations, and one possible reason could be due to specific conditions
that the protein had been exposed [60]. These conditions can be jus-
tified through some processes such as pH and temperature changes,
mutation, and denaturing agents [63,69]. Also, the physiological con-
ditions of T. terrestris are at acidic pH and temperature of 45 °C [16],
providing the basis to believe that the new molecular arrangement
(dimeric) of the enzyme with domain swapping and the complement of
catalytic pocket by surrounding molecule to be an artifact arising from
molecular alterations caused by the environmental and physiological
conditions during crystallization.
or even a homotetramer (if considering the dimer-dimer interface for
Ca2+). According to Yang et al. [70], domain swapping happens at high
protein concentration where a monomer becomes less favorable en-
tropically and proteins tend to form homodimers. The exchanged blade
generates an oligomerization in such matter to afford to rise in protein
stability by providing additional contacts between chains [71]. There-
fore, measurements of the molecular mass for a sample with higher
protein concentration, close to the condition of the drop in the condi-
tion of crystallization were carried out. Thus, the graphs of Fig. 6b-c
show two more concentrations:
2
mg.mL−1 in buffer
A and
54 mg.mL−1 in 1:1 buffer A and crystallization solution, respectively,
with both confirming the protein as a monomer in solution. Although
there are small peaks with a mass of approximately (51
1) and
(52
2) kDa, they do not correspond to another oligomeric state of
TtAbf62, but rather a contaminant not eliminated during the purifica-
3.5. Size exclusion chromatography with multiangle light scattering (SEC-
MALS) and glutaraldehyde cross-linking
tion processes, as can be seen in the SDS-PAGE gel of Fig. 6a in two
concentrations: 2 and 54 mg.mL−1
.
Moreover, TtAbf62 was also analyzed by SDS-PAGE after glutar-
aldehyde cross-linking reaction, and again a single band corresponding
a monomer predominates, indicating that TtAbf62 does not form an-
other oligomeric state in solution (Fig. 7). To conclude, TtAbf62 is a
monomer in solution, adopting a more compact structure like all other
proteins from the GH62 family.
When analyzed by SEC-MALS, TtAbf62 exhibited a symmetrical
peak with an average molecular mass of (32.1
0.2) kDa (Fig. 6b).
The expected theoretical molecular mass under denaturing conditions
for TtAbf62 is 32.8 kDa, which represents only 2% of error for the es-
timative mass. This result indicates that TtAbf62 is likely a monomer in
solution; despite the crystal structure has shown a possible homodimer
8

S. Camargo, et al.
BBA-ProteinsandProteomics1868(2020)140533
Fig. 5. Structural superposition of TtAbf62 (6CC7) with representative Abf from the GH62 family with similar three-dimensional structures. a) Superimposition of
representative Abf structures; b) Color scheme adopted for identification of the structures; c) Residues in the active site of similar Abf with their equivalent in
TtAbf62, represented by one letter code (colored as described in b). the catalytic residues *D165 and *E217 belong to chain A of TtAbf62 and residue *D58 belongs to
chain B of TtAbf62, forming its catalytic site. Water molecules with structurally conserved positions are represented as small spheres (colored by chains and structure
as described in b). Calcium ions (Ca2+) are represented as spheres with arbitrary radii colored by chains. Residues are labeled by one-letter code. Additional
structural comparisons of TtAbf62A are summarized in Supplemental Table A.3.
4. Conclusion
I) are necessary for complementarity, and it is only possible through
this molecular architecture. Therefore, the domain swapping reveals a
new structure within the GH62 family proteins and suggests a novel
structural arrangement. The nature of this behavior may be related to
the favorable entropic as a form of storage or compacting of the enzyme
in high concentrations.
The enzyme TtAbf62 catalyzes the hydrolysis of arabinose residues
of the arabinan branches, arabinoxylan as well as acts in sugarcane
bagasse in natura, whereas, suggesting a great versatility of the fungal
enzyme against the natural substrates, emerging its biotechnological
potential. The structural differences assisted in the clarification of the
reasons for these observed functional differences. Sequence alignments
of glycoside hydrolase GH62 show conserved blocks. The catalytic
pocket was structurally conserved between the 5-bladed-propeller fold.
The β-propeller architecture followed a pattern, with the junction of C
and N-terminus blades forming a pseudo-5-fold symmetry, which con-
fers protein stability. This stability is also strengthened by disulfide
bonds and the “non-classical molecular velcro”. The structural outcome
for TtAbf62 is unexpected, considering the predominant monomeric
nature of the homologous proteins adopting classical β-propeller folds.
Therefore, the possibility of evolution or mere crystallization artifact on
protein oligomerization for this family of enzymes was shown. Such
things may lead to its thermostability to be related to the folding. All
analyses presented conclude that the quaternary structure of the en-
zyme TtAbf62, in crystal, is in a dimeric arrangement, once the inter-
dependence between the catalytic residues and domain-swapped (blade
Author contributions
S.C., E.J.M., L.R.A., and J.R.C.M. designed the research. S.C. and
E.J.M. purified TtAbf62. L.R.A. and J.R.C.M. prepared cryo-crystals
samples and collected datasets. S.C., E.J.M., A.B., and F.S. characterized
TtAbf62 enzymatically. W.G. characterized TtAbf62 biophysically.
L.R.A. and J.R.C.M. processed X-ray diffraction images and refined the
atomic coordinates. S.C., E.J.M., L.R.A., R.P., and J.R.C.M. wrote the
paper with support from all other authors.
Footnote
This article contains supplementary data: SM1, SM2, Table A.1,
Table A.2, Table A.3, Fig. A.1, Fig. A.2, Fig. A.3, Fig. A.4, Fig. A.5, Fig.
A.6, Fig. A.7, and Fig. A.8.
9

S. Camargo, et al.
BBA-ProteinsandProteomics1868(2020)140533
Fig. 6. Multiangle Light Scattering (SEC-MALS) analysis for different concentrations of TtAbf62 in solution. a) 12% SDS-PAGE after SEC. Mr – molecular mass; lanes 2
and 3 show TtAbf62 in two different concentrations: 2 and 54 mg.mL−1, respectively. SEC-MALS of purified TtAbf62 at 2 mg.mL−1 (b) and 54 mg.mL−1 (c). For all
samples, the small peak represents a possible contaminant confirmed by SDS-PAGE (a) with the highest peak representing TtAbf62 in the monomeric state in solution
in agreement with the theoretical molar mass of 32.8 kDa. MALS calculations are dependent on a good estimate of the refractive index increment (dn/dc) of the
sample, near to 0.185 for proteins [72]. The peaks in (b) correspond to (51
(32.8 0.7) kDa, respectively.
1) and (32.1
0.2) kDa, respectively; peaks in (c) correspond to (52
2) and
The atomic coordinates and structure factors (code 6CC7) have been
deposited in the Protein Data Bank, Research Collaboratory for
Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
Declaration of Competing Interest
All authors of this work declare that have no potential conflict of
interest and that there is no financial, consultant, institutional or other
relationships that might lead to bias or conflicts of interest in this re-
search. Financial grants, infrastructure and fellowships supporting this
Fig. 7. Glutaraldehyde Cross-linking reaction. a)
10% SDS-PAGE of the reaction (positive control)
using a known sample of a homodimer in solution;
Mr – molecular mass reference; 1 – protein control of
84.9 kDa (monomeric, red box); 2 – after crosslink
control reaction with 5 μL of glutaraldehyde mixture
(dimers - yellow box and monomers - red box). b)
15% SDS-PAGE after reaction with TtAbf62. Mr
molecular mass; 1 – only the purified protein. The
TtAbf62 after crosslink reaction with 5 μL and 10 μL
of glutaraldehyde mixture: lanes 2 and 3, respec-
tively.
10

S. Camargo, et al.
BBA-ProteinsandProteomics1868(2020)140533
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