annotation_IOB/PMC4817029.tsv

Molecular	O
characterization	O
of	O
a	O
family	B-protein_type
5	I-protein_type
glycoside	I-protein_type
hydrolase	I-protein_type
suggests	O
an	O
induced	O
-	O
fit	O
enzymatic	O
mechanism	O

Glycoside	B-protein_type
hydrolases	I-protein_type
(	O
GHs	B-protein_type
)	O
play	O
fundamental	O
roles	O
in	O
the	O
decomposition	O
of	O
lignocellulosic	O
biomaterials	O
.	O

Here	O
,	O
we	O
report	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
structure	B-evidence
of	O
a	O
cellulase	B-protein_type
from	O
Bacillus	B-species
licheniformis	I-species
(	O
BlCel5B	B-protein
),	O
a	O
member	O
of	O
the	O
GH5	B-protein_type
subfamily	I-protein_type
4	I-protein_type
that	O
is	O
entirely	O
dependent	O
on	O
its	O
two	O
ancillary	B-structure_element
modules	I-structure_element
(	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
and	O
CBM46	B-structure_element
)	O
for	O
catalytic	O
activity	O
.	O

Using	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
,	O
small	B-experimental_method
-	I-experimental_method
angle	I-experimental_method
X	I-experimental_method
-	I-experimental_method
ray	I-experimental_method
scattering	I-experimental_method
and	O
molecular	B-experimental_method
dynamics	I-experimental_method
simulations	I-experimental_method
,	O
we	O
propose	O
that	O
the	O
C	O
-	O
terminal	O
CBM46	B-structure_element
caps	O
the	O
distal	O
N	O
-	O
terminal	O
catalytic	B-structure_element
domain	I-structure_element
(	O
CD	B-structure_element
)	O
to	O
establish	O
a	O
fully	B-protein_state
functional	I-protein_state
active	B-site
site	I-site
via	O
a	O
combination	O
of	O
large	O
-	O
scale	O
multidomain	O
conformational	O
selection	O
and	O
induced	O
-	O
fit	O
mechanisms	O
.	O

The	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
is	O
pivoting	O
the	O
packing	O
and	O
unpacking	O
motions	O
of	O
CBM46	B-structure_element
relative	O
to	O
CD	B-structure_element
in	O
the	O
assembly	O
of	O
the	O
binding	B-site
subsite	I-site
.	O

This	O
is	O
the	O
first	O
example	O
of	O
a	O
multidomain	O
GH	B-protein_type
relying	O
on	O
large	O
amplitude	O
motions	O
of	O
the	O
CBM46	B-structure_element
for	O
assembly	O
of	O
the	O
catalytically	B-protein_state
competent	I-protein_state
form	O
of	O
the	O
enzyme	O
.	O

Plant	B-taxonomy_domain
biomass	O
-	O
the	O
most	O
abundant	O
source	O
of	O
carbohydrates	B-chemical
on	O
Earth	O
-	O
is	O
primarily	O
composed	O
of	O
cellulose	B-chemical
microfibrils	O
surrounded	O
by	O
a	O
hydrated	O
heteropolymeric	O
matrix	O
of	O
hemicellulose	B-chemical
and	O
lignin	B-chemical
.	O

Plant	B-taxonomy_domain
biomass	O
may	O
be	O
subjected	O
to	O
thermo	O
-	O
chemical	O
pretreatments	O
and	O
enzymatic	O
reactions	O
to	O
produce	O
soluble	O
fermentable	O
sugars	B-chemical
.	O

The	O
canonical	O
model	O
of	O
hydrolytic	O
degradation	O
of	O
cellulose	B-chemical
requires	O
at	O
least	O
three	O
classes	O
of	O
enzymes	O
.	O

Cellobiohydrolases	B-protein_type
(	O
CBHs	B-protein_type
)	O
processively	O
cleave	O
the	O
glycosidic	O
bonds	O
at	O
the	O
reducing	O
and	O
non	O
-	O
reducing	O
ends	O
of	O
cellulose	B-chemical
chains	O
in	O
crystalline	O
regions	O
to	O
produce	O
cellobiose	B-chemical
.	O

Endoglucanases	B-protein_type
(	O
EGs	B-protein_type
)	O
introduce	O
random	O
cuts	O
in	O
the	O
amorphous	O
regions	O
of	O
cellulose	B-chemical
and	O
create	O
new	O
chain	O
extremities	O
for	O
CBH	B-protein_type
attack	O
;	O
thus	O
,	O
these	O
enzymes	O
act	O
synergistically	O
.	O

The	O
released	O
cellobiose	B-chemical
molecules	O
are	O
then	O
enzymatically	O
converted	O
into	O
glucose	B-chemical
by	O
β	B-protein_type
-	I-protein_type
glucosidases	I-protein_type
.	O

The	O
molecular	O
architecture	O
of	O
glycoside	B-protein_type
hydrolases	I-protein_type
(	O
GHs	B-protein_type
)	O
frequently	O
consists	O
of	O
a	O
catalytic	B-structure_element
domain	I-structure_element
(	O
CD	B-structure_element
),	O
where	O
hydrolysis	O
occurs	O
,	O
and	O
one	O
or	O
more	O
ancillary	B-structure_element
modules	I-structure_element
(	O
AMs	B-structure_element
),	O
which	O
are	O
usually	O
connected	O
by	O
less	B-protein_state
structured	I-protein_state
linkers	B-structure_element
.	O

The	O
most	O
common	O
type	O
of	O
AMs	B-structure_element
are	O
carbohydrate	B-structure_element
-	I-structure_element
binding	I-structure_element
modules	I-structure_element
(	O
CBMs	B-structure_element
),	O
which	O
are	O
able	O
to	O
recognize	O
and	O
bind	O
specific	O
carbohydrate	B-chemical
chains	O
.	O

Generally	O
distinct	O
and	O
independent	O
structural	O
domains	O
,	O
the	O
CBMs	B-structure_element
facilitate	O
carbohydrate	B-chemical
hydrolysis	O
by	O
increasing	O
the	O
local	O
concentration	O
of	O
enzymes	O
at	O
the	O
surface	O
of	O
insoluble	O
substrates	O
,	O
thereby	O
targeting	O
the	O
CD	B-structure_element
component	O
to	O
its	O
cognate	O
ligands	O
.	O

CBMs	B-structure_element
might	O
also	O
disrupt	O
the	O
crystalline	O
structure	O
of	O
cellulose	B-chemical
microfibrils	O
,	O
although	O
the	O
underlying	O
mechanism	O
remains	O
poorly	O
understood	O
.	O

Thus	O
,	O
CBMs	B-structure_element
enhance	O
the	O
accessibility	O
of	O
CDs	B-structure_element
to	O
carbohydrate	B-chemical
chains	O
to	O
improve	O
enzymatic	O
activity	O
,	O
making	O
them	O
important	O
candidates	O
for	O
the	O
development	O
of	O
effective	O
biomass	O
-	O
degrading	O
enzymes	O
in	O
industrial	O
settings	O
.	O

Although	O
there	O
are	O
examples	O
of	O
active	B-protein_state
GHs	B-protein_type
that	O
lack	B-protein_state
AMs	B-structure_element
,	O
the	O
majority	O
of	O
the	O
enzymes	O
depend	O
on	O
AMs	B-structure_element
for	O
activity	O
.	O

In	O
several	O
cases	O
,	O
CBMs	B-structure_element
were	O
shown	O
to	O
extend	O
and	O
complement	O
the	O
CD	B-structure_element
substrate	B-site
-	I-site
binding	I-site
site	I-site
in	O
multimodular	O
carbohydrate	B-protein_type
-	I-protein_type
active	I-protein_type
enzymes	I-protein_type
,	O
such	O
as	O
endo	B-protein_type
/	I-protein_type
exocellulase	I-protein_type
E4	B-protein
from	O
Thermobifida	B-species
fusca	I-species
,	O
chitinase	B-protein
B	I-protein
from	O
Serratia	B-species
marcescens	I-species
,	O
a	O
starch	B-protein_type
phosphatase	I-protein_type
from	O
Arabidopsis	B-species
thaliana	I-species
and	O
a	O
GH5	B-protein_type
subfamily	I-protein_type
4	I-protein_type
(	O
GH5_4	B-protein_type
)	O
endoglucanase	B-protein_type
from	O
Bacillus	B-species
halodurans	I-species
(	O
BhCel5B	B-protein
).	O

A	O
pioneer	O
work	O
of	O
Sakon	O
et	O
al	O
.	O
revealed	O
that	O
rigid	O
structural	O
extension	O
of	O
the	O
GH9	B-protein_type
CD	B-structure_element
by	O
a	O
type	B-structure_element
C	I-structure_element
CBM3	I-structure_element
imprints	O
a	O
processive	O
mode	O
of	O
action	O
to	O
this	O
endoglucanase	B-protein_type
.	O

Further	O
publications	O
showed	O
that	O
CBM	B-structure_element
-	O
based	O
structural	O
extensions	O
of	O
the	O
active	B-site
site	I-site
are	O
important	O
for	O
substrate	O
engagement	O
and	O
recognition	O
.	O

Recently	O
,	O
Venditto	O
et	O
al	O
.	O
reported	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
structure	I-evidence
of	O
the	O
tri	B-structure_element
-	I-structure_element
modular	I-structure_element
GH5_4	B-protein_type
endoglucanase	B-protein_type
from	O
Bacillus	B-species
halodurans	I-species
(	O
31	O
%	O
sequence	O
identity	O
to	O
BlCel5B	B-protein
),	O
with	O
the	O
CBM46	B-structure_element
extension	O
of	O
the	O
active	B-site
site	I-site
appended	O
to	O
the	O
CD	B-structure_element
via	O
an	O
immunoglobulin	B-structure_element
(	I-structure_element
Ig	I-structure_element
)-	I-structure_element
like	I-structure_element
module	I-structure_element
.	O

Removal	B-experimental_method
of	I-experimental_method
the	O
CBM46	B-structure_element
caused	O
a	O
~	O
60	O
-	O
fold	O
reduction	O
of	O
the	O
activity	O
of	O
the	O
enzyme	O
against	O
β	B-chemical
-	I-chemical
glucans	I-chemical
,	O
but	O
showed	O
little	O
or	O
no	O
effect	O
against	O
xyloglucan	B-chemical
hydrolysis	O
.	O

Moreover	O
,	O
the	O
CBM46	B-structure_element
mediated	O
a	O
significant	O
increase	O
in	O
the	O
BhCel5B	B-protein
activity	O
in	O
plant	B-taxonomy_domain
cell	O
wall	O
settings	O
.	O

Modeling	B-experimental_method
of	O
cellotriose	B-chemical
in	O
the	O
negative	B-site
subsites	I-site
of	O
the	O
active	B-site
site	I-site
of	O
BhCel5B	B-protein
demonstrated	O
the	O
structural	B-protein_state
conservation	I-protein_state
of	O
the	O
-	B-residue_number
1	I-residue_number
position	O
,	O
but	O
provided	O
little	O
information	O
about	O
direct	O
interactions	O
between	O
CBM46	B-structure_element
and	O
the	O
substrate	O
.	O

It	O
was	O
speculated	O
that	O
β	O
-	O
1	O
,	O
3	O
kink	O
of	O
the	O
β	B-chemical
-	I-chemical
glucan	I-chemical
might	O
allow	O
the	O
ligand	O
to	O
reach	O
for	O
the	O
CBM46	B-structure_element
,	O
whereas	O
pure	O
β	O
-	O
1	O
,	O
4	O
linkages	O
in	O
the	O
backbone	O
of	O
xyloglucan	B-chemical
chains	O
would	O
restrict	O
binding	O
to	O
the	O
CD	B-structure_element
,	O
thus	O
explaining	O
the	O
lack	O
of	O
influence	O
of	O
the	O
CBM46	B-structure_element
on	O
the	O
enzymatic	O
activity	O
of	O
BhCel5B	B-protein
against	O
xyloglucans	B-chemical
in	O
solution	O
.	O

It	O
was	O
also	O
argued	O
that	O
the	O
CBM46	B-structure_element
could	O
potentialize	O
the	O
activity	O
by	O
driving	O
BhCel5B	B-protein
towards	O
xyloglucan	B-structure_element
-	I-structure_element
rich	I-structure_element
regions	I-structure_element
in	O
the	O
context	O
of	O
the	O
plant	B-taxonomy_domain
cell	O
walls	O
,	O
but	O
no	O
large	O
-	O
scale	O
conformational	O
adjustments	O
of	O
the	O
AMs	B-structure_element
have	O
been	O
shown	O
to	O
occur	O
or	O
suggested	O
to	O
take	O
part	O
in	O
the	O
enzymatic	O
activity	O
.	O

Although	O
initially	O
introduced	O
as	O
contradictory	O
theories	O
,	O
these	O
two	O
limiting	O
cases	O
can	O
be	O
unified	O
considering	O
the	O
flux	O
description	O
concept	O
or	O
the	O
extended	B-protein_state
conformational	O
selection	O
model	O
.	O

While	O
local	O
ligand	O
-	O
induced	O
conformational	O
adjustments	O
have	O
been	O
reported	O
for	O
carbohydrate	B-protein_type
-	I-protein_type
active	I-protein_type
enzymes	I-protein_type
,	O
cognate	O
ligands	O
recognition	O
and	O
hydrolysis	O
mediated	O
by	O
a	O
large	O
-	O
scale	O
conformational	O
mobility	O
of	O
distinct	O
domains	O
in	O
multidomain	O
settings	O
is	O
uncommon	O
for	O
endoglucanases	B-protein_type
.	O

Here	O
,	O
we	O
report	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
a	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
GH5_4	B-protein_type
enzyme	O
from	O
Bacillus	B-species
licheniformis	I-species
(	O
BlCel5B	B-protein
)	O
that	O
exhibits	O
two	O
AMs	B-structure_element
(	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
and	O
CBM46	B-structure_element
)	O
appended	O
to	O
the	O
CD	B-structure_element
.	O

We	O
structurally	B-experimental_method
and	I-experimental_method
functionally	I-experimental_method
characterize	I-experimental_method
the	O
enzyme	O
using	O
a	O
combination	O
of	O
protein	B-experimental_method
crystallography	I-experimental_method
,	O
small	B-experimental_method
-	I-experimental_method
angle	I-experimental_method
X	I-experimental_method
-	I-experimental_method
ray	I-experimental_method
scattering	I-experimental_method
(	O
SAXS	B-experimental_method
),	O
molecular	B-experimental_method
dynamics	I-experimental_method
computer	I-experimental_method
simulations	I-experimental_method
and	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
,	O
and	O
show	O
that	O
the	O
AMs	B-structure_element
and	O
their	O
conformational	O
mobility	O
are	O
essential	O
for	O
the	O
enzymatic	O
activity	O
of	O
BlCel5B	B-protein
.	O

We	O
find	O
that	O
the	O
large	O
-	O
scale	O
conformational	O
adjustments	O
of	O
the	O
distal	O
CBM46	B-structure_element
mediated	O
by	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
hinge	I-structure_element
domain	I-structure_element
are	O
crucial	O
in	O
active	B-site
-	I-site
site	I-site
assembly	O
for	O
optimal	O
substrate	O
binding	O
and	O
hydrolysis	O
.	O

We	O
propose	O
that	O
the	O
BlCel5B	B-protein
conformational	O
selection	O
/	O
induced	O
-	O
fit	O
mechanism	O
of	O
hydrolysis	O
represents	O
a	O
novel	O
paradigm	O
that	O
applies	O
to	O
several	O
GH5_4	B-protein_type
members	O
and	O
,	O
possibly	O
,	O
to	O
a	O
number	O
of	O
other	O
multidomain	O
GHs	B-protein_type
.	O

BlCel5B	B-protein
Crystal	B-evidence
Structure	I-evidence

BlCel5B	B-protein
crystals	B-evidence
in	O
the	O
substrate	B-protein_state
-	I-protein_state
free	I-protein_state
form	O
and	O
complexed	B-protein_state
with	I-protein_state
cellopentaose	B-chemical
(	O
C5	B-chemical
)	O
were	O
obtained	O
and	O
diffracted	O
to	O
1	O
.	O
7	O
Å	O
and	O
1	O
.	O
75	O
Å	O
resolutions	O
,	O
respectively	O
(	O
Supplementary	O
Table	O
1	O
).	O

The	O
substrate	B-protein_state
-	I-protein_state
free	I-protein_state
and	O
complexed	B-protein_state
structures	B-evidence
exhibited	O
no	O
substantial	O
conformational	O
differences	O
(	O
with	O
the	O
exception	O
of	O
the	O
substrate	O
).	O

Because	O
of	O
minor	O
variations	O
in	O
the	O
loops	B-structure_element
located	O
distal	O
to	O
the	O
substrate	B-site
-	I-site
binding	I-site
site	I-site
,	O
a	O
root	B-evidence
mean	I-evidence
squared	I-evidence
deviation	I-evidence
(	O
rmsd	B-evidence
)	O
of	O
0	O
.	O
33	O
Å	O
between	O
the	O
complexed	B-protein_state
and	O
substrate	B-protein_state
-	I-protein_state
free	I-protein_state
structures	B-evidence
was	O
observed	O
.	O

A	O
single	O
protein	O
chain	O
occupies	O
the	O
asymmetric	O
unit	O
,	O
and	O
most	O
of	O
the	O
residues	O
were	O
built	O
,	O
with	O
the	O
exception	O
of	O
the	O
first	B-residue_range
17	I-residue_range
residues	I-residue_range
and	O
those	O
in	O
the	O
loop	B-structure_element
between	O
L398	B-residue_name_number
and	O
P405	B-residue_name_number
due	O
to	O
weak	O
electron	B-evidence
density	I-evidence
.	O

The	O
BlCel5B	B-protein
structure	B-evidence
comprises	O
three	O
distinct	O
domains	O
:	O
an	O
N	O
-	O
terminal	O
CD	B-structure_element
(	O
residues	O
18	B-residue_range
to	I-residue_range
330	I-residue_range
),	O
an	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
(	O
residues	O
335	B-residue_range
to	I-residue_range
428	I-residue_range
)	O
and	O
a	O
family	B-structure_element
46	I-structure_element
CBM	I-structure_element
(	O
residues	O
432	B-residue_range
to	I-residue_range
533	I-residue_range
)	O
(	O
Fig	O
.	O
1A	O
,	O
B	O
).	O

Similarly	O
to	O
other	O
members	O
of	O
the	O
GH5	B-protein_type
family	O
,	O
the	O
CD	B-structure_element
of	O
BlCel5B	B-protein
has	O
a	O
typical	O
TIM	B-structure_element
barrel	I-structure_element
fold	I-structure_element
with	O
eight	O
inner	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
and	O
eight	O
outer	O
α	B-structure_element
helices	I-structure_element
that	O
are	O
interconnected	O
by	O
loops	B-structure_element
and	O
three	O
short	O
α	B-structure_element
helices	I-structure_element
.	O

Very	O
short	O
linkers	B-structure_element
,	O
D429	B-structure_element
-	I-structure_element
D430	I-structure_element
-	I-structure_element
P431	I-structure_element
and	O
V331	B-structure_element
-	I-structure_element
P332	I-structure_element
-	I-structure_element
N333	I-structure_element
-	I-structure_element
A334	I-structure_element
,	O
connect	O
the	O
CBM46	B-structure_element
to	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
and	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
to	O
the	O
CD	B-structure_element
,	O
respectively	O
.	O

Both	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
and	O
CBM46	B-structure_element
have	O
a	O
β	B-structure_element
-	I-structure_element
sandwich	I-structure_element
fold	I-structure_element
composed	O
of	O
two	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
of	O
four	O
and	O
three	O
antiparallel	B-structure_element
β	I-structure_element
-	I-structure_element
strands	I-structure_element
interconnected	O
by	O
loops	B-structure_element
and	O
a	O
short	O
α	B-structure_element
helix	I-structure_element
between	O
strands	B-structure_element
β3	B-structure_element
and	O
β4	B-structure_element
(	O
Fig	O
.	O
1C	O
).	O

A	O
structural	B-experimental_method
comparison	I-experimental_method
between	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
and	O
the	O
CBM46	B-structure_element
using	O
the	O
Dali	B-experimental_method
server	I-experimental_method
yielded	O
an	O
rmsd	B-evidence
of	O
2	O
.	O
3	O
Å	O
and	O
a	O
Z	B-evidence
-	I-evidence
score	I-evidence
of	O
10	O
.	O
2	O
.	O

A	O
structure	B-experimental_method
-	I-experimental_method
based	I-experimental_method
search	I-experimental_method
performed	O
using	O
the	O
same	O
server	O
showed	O
that	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
is	O
similar	O
to	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
from	O
a	O
recently	O
solved	B-experimental_method
crystal	B-evidence
structure	I-evidence
of	O
a	O
tri	B-structure_element
-	I-structure_element
modular	I-structure_element
GH5_4	B-protein_type
enzyme	O
from	O
Bacillus	B-species
halodurans	I-species
,	O
BhCel5B	B-protein
,	O
with	O
rmsd	B-evidence
=	O
1	O
.	O
3	O
Å	O
and	O
Z	B-evidence
-	I-evidence
score	I-evidence
=	O
15	O
.	O
3	O
.	O

The	O
CBM46	B-structure_element
from	O
BhCel5B	B-protein
is	O
the	O
most	O
structurally	O
similar	O
to	O
BlCel5B	B-protein
CBM46	B-structure_element
,	O
with	O
rmsd	B-evidence
=	O
1	O
.	O
6	O
Å	O
and	O
Z	B-evidence
-	I-evidence
score	I-evidence
=	O
12	O
.	O
4	O
.	O

The	O
sequence	O
identity	O
relative	O
to	O
BhCel5B	B-protein
,	O
however	O
,	O
is	O
low	O
(	O
28	O
%	O
for	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
and	O
25	O
%	O
for	O
CBM46	B-structure_element
).	O

The	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
,	O
adjacent	O
to	O
the	O
CD	B-structure_element
,	O
contains	O
only	O
one	O
tyrosine	B-residue_name
(	O
Y367	B-residue_name_number
)	O
exposed	O
to	O
solvent	O
and	O
no	O
tryptophan	B-residue_name
residues	O
.	O

Because	O
aromatic	O
residues	O
play	O
a	O
major	O
role	O
in	O
glucose	B-chemical
recognition	O
,	O
this	O
observation	O
suggests	O
that	O
substrate	O
binding	O
may	O
not	O
be	O
the	O
primary	O
function	O
of	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
.	O

In	O
contrast	O
,	O
the	O
CBM46	B-structure_element
has	O
three	O
tryptophan	B-residue_name
residues	O
,	O
two	O
of	O
which	O
face	O
the	O
CD	B-structure_element
substrate	B-site
binding	I-site
site	I-site
(	O
Fig	O
.	O
1A	O
),	O
indicating	O
that	O
it	O
may	O
be	O
actively	O
engaged	O
in	O
the	O
carbohydrate	B-chemical
binding	O
.	O

Electron	B-evidence
density	I-evidence
maps	I-evidence
clearly	O
reveal	O
the	O
presence	B-protein_state
of	I-protein_state
a	O
cellotetraose	B-chemical
(	O
C4	B-chemical
)	O
and	O
not	O
a	O
soaked	O
cellopentaose	B-chemical
(	O
C5	B-chemical
)	O
in	O
the	O
CD	B-structure_element
negative	B-site
substrate	I-site
-	I-site
binding	I-site
subsites	I-site
(	O
Fig	O
.	O
1D	O
),	O
indicating	O
that	O
BlCel5B	B-protein
is	O
catalytically	B-protein_state
active	I-protein_state
in	O
the	O
crystal	O
state	O
and	O
able	O
to	O
cleave	O
a	O
C5	B-chemical
molecule	O
.	O

The	O
lack	B-evidence
of	I-evidence
electron	I-evidence
density	I-evidence
verifies	O
the	O
absence	B-protein_state
of	I-protein_state
the	O
fifth	B-residue_number
glucose	B-chemical
moiety	O
from	O
the	O
soaked	O
C5	B-chemical
,	O
and	O
a	O
closer	O
inspection	O
of	O
the	O
structure	B-evidence
confirmed	O
that	O
the	O
presence	B-protein_state
of	I-protein_state
a	O
fifth	B-residue_number
glucose	B-chemical
unit	O
would	O
be	O
sterically	O
hindered	O
by	O
the	O
catalytic	B-site
residues	I-site
on	O
the	O
reducing	O
end	O
and	O
by	O
residue	O
R234	B-residue_name_number
of	O
a	O
symmetry	O
-	O
related	O
enzyme	O
molecule	O
on	O
the	O
non	O
-	O
reducing	O
end	O
.	O

The	O
ability	O
of	O
BlCel5B	B-protein
to	O
cleave	O
C5	B-chemical
into	O
glucose	B-chemical
and	O
C4	B-chemical
molecules	O
in	O
solution	O
was	O
demonstrated	O
by	O
enzymatic	B-experimental_method
product	I-experimental_method
profile	I-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
analysis	O
(	O
Fig	O
.	O
2A	O
).	O

The	O
C4	B-chemical
oligomer	O
in	O
the	O
BlCel5B	B-protein
binding	B-site
site	I-site
is	O
coordinated	B-bond_interaction
by	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
to	O
residues	O
N36	B-residue_name_number
,	O
H113	B-residue_name_number
,	O
H114	B-residue_name_number
,	O
N158	B-residue_name_number
,	O
W301	B-residue_name_number
,	O
and	O
N303	B-residue_name_number
and	O
by	O
a	O
CH	B-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
interaction	I-bond_interaction
with	O
residue	O
W47	B-residue_name_number
(	O
Fig	O
.	O
1D	O
).	O

These	O
residues	O
belong	O
to	O
the	O
CD	B-structure_element
and	O
are	O
conserved	B-protein_state
in	O
the	O
GH5	B-protein_type
family	O
.	O

BlCel5B	B-protein
enzymatic	O
activity	O

BlCel5B	B-protein
exhibits	O
optimum	O
activity	O
toward	O
carboxymethylcellulose	B-chemical
(	O
CMC	B-chemical
;	O
8	O
.	O
7	O
U	O
/	O
mg	O
)	O
at	O
a	O
pH	O
of	O
4	O
.	O
0	O
and	O
55	O
°	O
C	O
and	O
retains	O
approximately	O
half	O
of	O
its	O
maximum	O
activity	O
at	O
80	O
°	O
C	O
,	O
demonstrating	O
considerable	O
thermal	O
stability	O
(	O
Fig	O
.	O
2B	O
,	O
C	O
).	O

BlCel5B	B-protein
is	O
also	O
active	B-protein_state
on	O
β	B-chemical
-	I-chemical
glucan	I-chemical
(	O
34	O
U	O
/	O
mg	O
),	O
lichenan	B-chemical
(	O
17	O
.	O
8	O
U	O
/	O
mg	O
)	O
and	O
xyloglucan	B-chemical
(	O
15	O
.	O
7	O
U	O
/	O
mg	O
)	O
substrates	O
(	O
Table	O
1	O
),	O
whereas	O
no	O
activity	O
was	O
detected	O
on	O
galactomannan	B-chemical
,	O
rye	B-taxonomy_domain
arabinoxylan	B-chemical
,	O
1	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
β	I-chemical
-	I-chemical
mannan	I-chemical
or	O
the	O
insoluble	O
substrate	O
Azo	B-chemical
-	I-chemical
Avicel	I-chemical
.	O

Kinetic	O
parameters	O
were	O
calculated	O
assuming	O
Michaelis	B-experimental_method
-	I-experimental_method
Menten	I-experimental_method
behavior	I-experimental_method
with	O
CMC	B-chemical
as	O
substrate	O
:	O
KM	B-evidence
=	O
1	O
.	O
78	O
g	O
L	O
−	O
1	O
and	O
Vmax	B-evidence
=	O
1	O
.	O
41	O
×	O
10	O
−	O
4	O
g	O
s	O
−	O
1	O
mg	O
protein	O
−	O
1	O
(	O
Fig	O
.	O
2D	O
).	O

Although	O
BlCel5B	B-protein
is	O
not	O
a	O
highly	O
active	B-protein_state
enzyme	O
against	O
one	O
specific	O
substrate	O
as	O
compared	O
to	O
others	O
GH5_4	B-protein_type
,	O
it	O
has	O
the	O
advantage	O
of	O
being	O
active	B-protein_state
against	O
different	O
substrates	O
with	O
β	O
-	O
1	O
,	O
3	O
and	O
/	O
or	O
β	O
-	O
1	O
,	O
4	O
glycosidic	O
linkages	O
.	O

To	O
understand	O
the	O
importance	O
of	O
the	O
ancillary	B-structure_element
modules	I-structure_element
for	O
BlCel5B	B-protein
activity	O
,	O
enzymatic	B-experimental_method
assays	I-experimental_method
were	O
carried	O
out	O
using	O
four	O
enzyme	O
mutants	B-protein_state
:	O
a	O
CBM46	B-structure_element
deletion	B-experimental_method
(	O
ΔCBM46	B-mutant
)	O
and	O
an	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
+	O
CBM46	B-structure_element
deletion	B-experimental_method
(	O
ΔIg	B-mutant
-	I-mutant
CBM46	I-mutant
)	O
as	O
well	O
as	O
point	B-experimental_method
mutations	I-experimental_method
of	O
the	O
CBM46	B-structure_element
inner	O
surface	O
residues	O
W479A	B-mutant
and	O
W481A	B-mutant
.	O

These	O
mutants	B-protein_state
were	O
expressed	B-experimental_method
and	I-experimental_method
purified	I-experimental_method
as	O
described	O
for	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
enzyme	O
.	O

Strikingly	O
,	O
neither	O
of	O
the	O
deletion	B-protein_state
variants	I-protein_state
exhibited	O
detectable	O
activity	O
toward	O
any	O
of	O
the	O
substrates	O
tested	O
using	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
BlCel5B	B-protein
(	O
Table	O
1	O
),	O
demonstrating	O
that	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
and	O
the	O
CBM46	B-structure_element
are	O
essential	O
for	O
BlCel5B	B-protein
activity	O
.	O

Thermal	B-experimental_method
shift	I-experimental_method
assays	I-experimental_method
were	O
conducted	O
to	O
confirm	O
structural	O
stability	O
of	O
the	O
mutants	B-protein_state
(	O
Supplementary	O
Fig	O
.	O
1	O
).	O

All	O
of	O
the	O
constructs	O
showed	O
similar	O
melting	B-evidence
temperatures	I-evidence
:	O
62	O
°	O
C	O
for	O
BlCel5B	B-protein
,	O
58	O
°	O
C	O
for	O
BlCel5BΔCBM46	B-mutant
,	O
56	O
°	O
C	O
for	O
BlCel5BΔIg	B-mutant
-	I-mutant
CBM46	I-mutant
,	O
65	O
°	O
C	O
for	O
BlCel5BW479A	B-mutant
and	O
59	O
°	O
C	O
for	O
BlCel5BW479A	B-mutant
,	O
thus	O
confirming	O
their	O
proper	O
overall	O
fold	O
.	O

We	O
also	O
examined	O
the	O
function	O
of	O
the	O
CBM46	B-structure_element
inner	O
surface	B-site
residues	O
W479	B-residue_name_number
and	O
W481	B-residue_name_number
(	O
Fig	O
.	O
1A	O
)	O
in	O
BlCel5B	B-protein
activity	O
by	O
performing	O
enzymatic	B-experimental_method
assays	I-experimental_method
with	O
W479A	B-mutant
and	O
W481A	B-mutant
mutants	B-protein_state
.	O

Both	O
mutations	B-experimental_method
reduced	O
enzymatic	O
activity	O
toward	O
all	O
tested	O
substrates	O
(	O
Table	O
1	O
),	O
with	O
W481A	B-mutant
having	O
a	O
stronger	O
effect	O
than	O
W479A	B-mutant
(~	O
64	O
%	O
vs	O
.	O
79	O
%	O
activity	O
relative	O
to	O
wt	B-protein_state
BlCel5B	B-protein
using	O
β	B-chemical
-	I-chemical
glucan	I-chemical
and	O
~	O
10	O
%	O
vs	O
.	O
50	O
%	O
using	O
CMC	B-chemical
).	O

This	O
indicates	O
that	O
CBM46	B-structure_element
must	O
interact	O
with	O
the	O
substrate	O
via	O
residues	O
W479	B-residue_name_number
and	O
W481	B-residue_name_number
.	O

However	O
,	O
since	O
the	O
BlCel5B	B-protein
crystal	B-evidence
structure	I-evidence
exhibits	O
no	O
close	B-protein_state
contact	O
between	O
these	O
residues	O
and	O
the	O
substrate	O
,	O
these	O
results	O
suggest	O
the	O
existence	O
of	O
large	O
-	O
amplitude	O
interdomain	O
motions	O
that	O
may	O
enable	O
direct	O
interactions	O
between	O
CBM46	B-structure_element
and	O
the	O
carbohydrate	B-chemical
.	O

BlCelB5	B-protein
dynamics	O
and	O
binding	B-site
-	I-site
site	I-site
architecture	O

Molecular	B-experimental_method
dynamics	I-experimental_method
(	O
MD	B-experimental_method
)	O
simulations	B-experimental_method
were	O
performed	O
to	O
investigate	O
the	O
conformational	O
mobility	O
of	O
BlCel5B	B-protein
.	O

In	O
the	O
simulations	B-experimental_method
of	O
the	O
crystal	B-evidence
structure	I-evidence
for	O
BlCel5B	B-protein
bound	B-protein_state
to	I-protein_state
C4	B-chemical
,	O
the	O
substrate	O
dissociates	O
from	O
the	O
protein	O
within	O
the	O
first	O
100	O
ns	O
of	O
the	O
simulation	B-experimental_method
time	O
(	O
Supplementary	O
Fig	O
.	O
2A	O
).	O

This	O
observation	O
suggests	O
that	O
cellotetraose	B-chemical
does	O
not	O
exhibit	O
detectable	O
affinity	O
for	O
this	O
specific	O
BlCel5B	B-protein
conformation	O
in	O
solution	O
,	O
as	O
one	O
might	O
otherwise	O
expect	O
for	O
a	O
reaction	O
product	O
.	O

No	O
changes	O
beyond	O
local	O
fluctuations	O
were	O
observed	O
in	O
any	O
of	O
the	O
three	O
BlCel5B	B-protein
domains	O
within	O
the	O
time	O
scale	O
of	O
these	O
runs	O
(	O
400	O
ns	O
;	O
Supplementary	O
Fig	O
.	O
2B	O
).	O

However	O
,	O
the	O
CBM46	B-structure_element
and	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
domains	I-structure_element
did	O
exhibit	O
rigid	O
body	O
-	O
like	O
motions	O
relative	O
to	O
the	O
CD	B-structure_element
,	O
with	O
rmsd	B-evidence
values	O
around	O
2	O
.	O
3	O
Å	O
and	O
1	O
.	O
8	O
Å	O
,	O
respectively	O
,	O
suggesting	O
that	O
BlCel5B	B-protein
may	O
execute	O
large	O
-	O
amplitude	O
interdomain	O
motions	O
over	O
longer	O
time	O
scales	O
(	O
Supplementary	O
Fig	O
.	O
2B	O
,	O
C	O
).	O

Accordingly	O
,	O
simulations	B-experimental_method
were	O
then	O
performed	O
using	O
accelerated	B-experimental_method
molecular	I-experimental_method
dynamics	I-experimental_method
(	O
aMD	B-experimental_method
)	O
techniques	O
to	O
probe	O
BlCel5B	B-protein
interdomain	O
motions	O
.	O

aMD	B-experimental_method
enhances	O
conformational	O
sampling	O
by	O
raising	O
the	O
basins	O
of	O
the	O
dihedral	B-evidence
potential	I-evidence
energy	I-evidence
surface	I-evidence
without	O
affecting	O
the	O
general	O
form	O
of	O
the	O
atomistic	O
potential	O
,	O
thereby	O
increasing	O
transition	O
rates	O
between	O
different	O
local	O
minima	O
.	O

aMD	B-experimental_method
trajectories	B-evidence
corresponding	O
to	O
more	O
than	O
1	O
.	O
0	O
μs	O
of	O
conventional	O
MD	B-experimental_method
runs	O
were	O
generated	O
.	O

During	O
these	O
simulations	B-experimental_method
,	O
we	O
observed	O
occlusive	O
conformations	O
between	O
CBM46	B-structure_element
and	O
CD	B-structure_element
that	O
resulted	O
in	O
a	O
rearrangement	O
of	O
the	O
enzyme	O
’	O
s	O
architecture	O
around	O
the	O
active	B-site
site	I-site
(	O
Video	O
S1	O
).	O

Figure	O
3A	O
shows	O
BlCel5B	B-protein
in	O
the	O
crystallographic	B-experimental_method
conformation	O
(	O
red	O
)	O
and	O
in	O
a	O
selected	O
configuration	O
obtained	O
with	O
aMD	B-experimental_method
(	O
blue	O
)	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
the	O
substrate	O
.	O

Interdomain	O
motions	O
were	O
gauged	O
by	O
the	O
time	O
evolution	O
of	O
the	O
distance	B-evidence
between	O
the	O
α	O
carbons	O
of	O
residues	O
I120	B-residue_name_number
and	O
E477	B-residue_name_number
(	O
represented	O
as	O
spheres	O
in	O
Fig	O
.	O
3A	O
),	O
belonging	O
to	O
the	O
CD	B-structure_element
and	O
CBM46	B-structure_element
,	O
respectively	O
.	O

Figure	O
3C	O
shows	O
that	O
the	O
I120	B-residue_name_number
-	O
E477	B-residue_name_number
distance	B-evidence
(	O
red	O
curve	O
)	O
gradually	O
decreases	O
from	O
~	O
35	O
Å	O
to	O
~	O
7	O
Å	O
within	O
the	O
first	O
half	O
of	O
the	O
1	O
.	O
0	O
μs	O
aMD	B-experimental_method
trajectory	B-evidence
,	O
indicating	O
a	O
transition	O
between	O
the	O
semi	B-protein_state
-	I-protein_state
open	I-protein_state
(	O
crystallographic	B-experimental_method
)	O
and	O
occluded	B-protein_state
(	O
aMD	B-experimental_method
sampled	O
)	O
configurations	O
.	O

During	O
the	O
second	O
half	O
of	O
the	O
aMD	B-experimental_method
simulation	I-experimental_method
,	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
enzyme	O
remained	O
in	O
the	O
closed	B-protein_state
conformation	O
,	O
with	O
the	O
CBM46	B-structure_element
covering	O
the	O
carbohydrate	B-site
-	I-site
binding	I-site
site	I-site
.	O

These	O
results	O
suggest	O
that	O
BlCel5B	B-protein
undergoes	O
large	O
-	O
scale	O
interdomain	O
movements	O
that	O
enable	O
interactions	O
between	O
CBM46	B-structure_element
and	O
the	O
substrate	O
bound	B-protein_state
to	I-protein_state
the	O
CD	B-structure_element
.	O

To	O
study	O
the	O
interactions	O
of	O
BlCel5B	B-protein
with	O
a	O
non	O
-	O
hydrolyzed	O
glucan	B-chemical
chain	O
,	O
we	O
built	O
a	O
model	O
structure	B-evidence
with	O
a	O
cellooctaose	B-chemical
(	O
C8	B-chemical
)	O
chain	O
spanning	O
the	O
entire	O
positive	B-site
(+	I-site
1	I-site
to	I-site
+	I-site
4	I-site
)	I-site
and	O
negative	B-site
(−	I-site
4	I-site
to	I-site
−	I-site
1	I-site
)	I-site
subsites	B-site
of	O
the	O
enzyme	O
.	O

Starting	O
from	O
the	O
crystallographic	O
BlCel5B	B-protein
conformation	O
,	O
the	O
C8	B-chemical
molecule	O
deviated	O
significantly	O
from	O
the	O
active	B-site
site	I-site
and	O
assumed	O
a	O
non	O
-	O
productive	O
binding	O
mode	O
(	O
Supplementary	O
Fig	O
.	O
2D	O
).	O

This	O
observation	O
suggests	O
that	O
the	O
open	B-protein_state
conformation	O
of	O
BlCel5B	B-protein
is	O
not	O
able	O
to	O
hold	O
the	O
substrate	O
in	O
a	O
position	O
suitable	O
for	O
hydrolysis	O
(	O
Supplementary	O
Fig	O
.	O
2E	O
).	O

However	O
,	O
after	O
subjecting	O
the	O
BlCel5B	B-complex_assembly
-	I-complex_assembly
C8	I-complex_assembly
complex	O
to	O
a	O
0	O
.	O
5	O
μs	O
aMD	B-experimental_method
simulation	I-experimental_method
with	O
harmonic	O
restraints	O
on	O
the	O
C8	B-chemical
chain	O
to	O
prevent	O
it	O
from	O
deviating	O
from	O
the	O
productive	O
binding	O
mode	O
,	O
the	O
CBM46	B-structure_element
readily	O
closed	B-protein_state
over	O
the	O
CD	B-structure_element
and	O
trapped	O
the	O
C8	B-chemical
chain	O
in	O
position	O
for	O
hydrolysis	O
(	O
Fig	O
.	O
3B	O
).	O

In	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
substrate	O
,	O
CBM46	B-structure_element
adopts	O
a	O
final	O
conformation	O
intermediate	O
between	O
the	O
crystallographic	B-evidence
structure	I-evidence
and	O
that	O
observed	O
in	O
the	O
substrate	B-protein_state
-	I-protein_state
free	I-protein_state
BlCel5B	B-protein
aMD	B-experimental_method
simulations	I-experimental_method
;	O
this	O
is	O
illustrated	O
by	O
the	O
I120	B-residue_name_number
-	O
E477	B-residue_name_number
distance	B-evidence
,	O
which	O
stabilizes	O
near	O
20	O
Å	O
in	O
the	O
closed	B-protein_state
configuration	O
that	O
traps	O
the	O
C8	B-chemical
molecule	O
(	O
in	O
contrast	O
to	O
~	O
7	O
Å	O
for	O
substrate	B-protein_state
-	I-protein_state
free	I-protein_state
BlCel5B	B-protein
)	O
(	O
Fig	O
.	O
3C	O
).	O

This	O
BlCel5B	B-complex_assembly
-	I-complex_assembly
C8	I-complex_assembly
configuration	O
remains	O
stable	O
over	O
an	O
additional	O
500	O
ns	O
of	O
conventional	O
MD	B-experimental_method
simulation	I-experimental_method
with	O
no	O
restraints	O
(	O
Fig	O
.	O
3C	O
cyan	O
line	O
,	O
Supplementary	O
Fig	O
.	O
2E	O
,	O
F	O
).	O

A	O
closer	O
inspection	O
of	O
the	O
productive	O
binding	O
mode	O
obtained	O
from	O
these	O
extensive	O
simulations	B-experimental_method
reveals	O
that	O
the	O
CBM46	B-structure_element
tryptophan	B-residue_name
residues	O
W479	B-residue_name_number
and	O
W481	B-residue_name_number
(	O
along	O
with	O
CD	B-structure_element
tryptophan	B-residue_name
residues	O
)	O
play	O
important	O
roles	O
in	O
carbohydrate	B-chemical
recognition	O
and	O
orientation	O
by	O
creating	O
a	O
tunnel	B-site
-	O
like	O
topology	O
along	O
the	O
BlCel5B	B-protein
binding	B-site
cleft	I-site
,	O
as	O
depicted	O
in	O
Fig	O
.	O
3D	O
.	O

Together	O
,	O
these	O
results	O
indicate	O
that	O
CBM46	B-structure_element
is	O
a	O
key	O
component	O
of	O
the	O
catalytic	B-protein_state
active	I-protein_state
complex	O
,	O
providing	O
an	O
explanation	O
as	O
to	O
why	O
CBM46	B-structure_element
is	O
essential	O
for	O
the	O
enzymatic	O
activity	O
of	O
BlCel5B	B-protein
.	O

To	O
enable	O
substantially	O
longer	O
time	O
scales	O
compared	O
to	O
atomistic	B-experimental_method
simulations	I-experimental_method
,	O
we	O
further	O
explored	O
the	O
dynamics	O
of	O
BlCel5B	B-protein
using	O
coarse	B-experimental_method
-	I-experimental_method
grained	I-experimental_method
MD	I-experimental_method
(	O
CG	B-experimental_method
-	I-experimental_method
MD	I-experimental_method
)	O
simulations	B-experimental_method
.	O

We	O
performed	O
three	O
independent	O
~	O
120	O
μs	O
CG	B-experimental_method
-	I-experimental_method
MD	I-experimental_method
simulations	I-experimental_method
,	O
for	O
a	O
total	O
of	O
approximately	O
360	O
μs	O
of	O
sampling	O
.	O

The	O
distance	B-evidence
between	O
the	O
α	O
carbons	O
of	O
two	O
residues	O
centrally	O
positioned	O
in	O
the	O
CD	B-structure_element
and	O
CBM46	B-structure_element
(	O
Fig	O
.	O
4A	O
)	O
was	O
monitored	O
,	O
and	O
the	O
results	O
shown	O
in	O
Fig	O
.	O
4B	O
indicate	O
that	O
the	O
wide	O
-	O
amplitude	O
events	O
described	O
above	O
frequently	O
appear	O
in	O
this	O
time	O
scale	O
.	O

The	O
computed	B-evidence
distance	I-evidence
distribution	I-evidence
depicted	O
in	O
Fig	O
.	O
4C	O
indicates	O
three	O
main	O
conformational	O
states	O
ranging	O
from	O
(	O
I	O
)	O
closed	B-protein_state
conformations	O
similar	O
to	O
those	O
encountered	O
in	O
the	O
substrate	B-protein_state
-	I-protein_state
free	I-protein_state
aMD	B-experimental_method
simulations	I-experimental_method
,	O
in	O
which	O
CBM46	B-structure_element
interacts	O
with	O
the	O
CD	B-structure_element
to	O
shape	O
the	O
substrate	B-site
binding	I-site
site	I-site
,	O
to	O
(	O
II	O
)	O
semi	B-protein_state
-	I-protein_state
open	I-protein_state
conformations	O
similar	O
to	O
the	O
crystallographic	B-evidence
structure	I-evidence
,	O
and	O
(	O
III	O
)	O
extended	B-protein_state
BlCel5B	B-protein
conformations	O
in	O
which	O
the	O
CD	B-structure_element
and	O
CBM46	B-structure_element
are	O
even	O
further	O
apart	O
than	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
.	O

BlCel5B	B-protein
conformers	O
fit	O
the	O
SAXS	B-experimental_method
envelope	B-evidence

SAXS	B-experimental_method
experiments	O
were	O
conducted	O
to	O
assess	O
BlCel5B	B-protein
conformational	O
states	O
in	O
solution	O
,	O
and	O
the	O
results	O
revealed	O
the	O
enzyme	O
in	O
its	O
monomeric	B-oligomeric_state
form	O
,	O
with	O
average	O
values	O
of	O
Rg	B-evidence
=	O
27	O
.	O
17	O
Å	O
and	O
Dmax	B-evidence
=	O
87	O
.	O
59	O
Å	O
(	O
Supplementary	O
Table	O
2	O
).	O

The	O
ab	B-experimental_method
initio	I-experimental_method
dummy	I-experimental_method
atom	I-experimental_method
model	I-experimental_method
(	O
DAM	B-experimental_method
)	O
demonstrated	O
that	O
the	O
SAXS	B-experimental_method
-	O
derived	O
BlCel5B	B-protein
molecular	O
envelope	B-evidence
could	O
not	O
be	O
single	O
-	O
handedly	O
filled	O
by	O
any	O
of	O
the	O
main	O
conformational	O
states	O
encountered	O
in	O
the	O
simulations	B-experimental_method
(	O
Fig	O
.	O
4D	O
).	O

It	O
is	O
known	O
that	O
a	O
Kratky	B-evidence
plot	I-evidence
exhibits	O
a	O
peak	O
with	O
an	O
elevated	O
baseline	O
at	O
high	O
q	O
for	O
a	O
monodisperse	O
system	O
composed	O
of	O
multi	O
-	O
domain	O
particles	O
with	O
flexible	O
extensions	O
.	O

Indeed	O
,	O
an	O
elevation	O
of	O
the	O
baseline	O
toward	O
a	O
hyperbolic	O
-	O
like	O
curve	O
was	O
observed	O
for	O
BlCel5B	B-protein
,	O
indicating	O
a	O
considerable	O
degree	O
of	O
molecular	O
mobility	O
in	O
solution	O
(	O
Supplementary	O
Fig	O
.	O
3	O
).	O

Thus	O
,	O
the	O
conformational	O
heterogeneity	O
of	O
the	O
enzyme	O
can	O
be	O
decomposed	O
in	O
structural	O
terms	O
as	O
a	O
combination	O
of	O
conformational	O
states	O
identified	O
in	O
our	O
crystallographic	B-experimental_method
and	I-experimental_method
MD	I-experimental_method
studies	I-experimental_method
.	O

We	O
found	O
that	O
the	O
SAXS	B-experimental_method
envelope	B-evidence
can	O
be	O
well	O
represented	O
by	O
considering	O
the	O
superimposition	B-experimental_method
of	O
three	O
different	O
representative	O
molecular	O
conformations	O
of	O
BlCel5B	B-protein
(	O
Fig	O
.	O
4E	O
):	O
a	O
closed	B-protein_state
or	O
CBM46	B-structure_element
/	O
CD	B-structure_element
-	O
occluded	B-protein_state
conformation	O
extracted	O
from	O
the	O
simulations	B-experimental_method
with	O
a	O
relative	O
weight	O
of	O
26	O
%,	O
a	O
semi	B-protein_state
-	I-protein_state
open	I-protein_state
conformation	O
represented	O
by	O
the	O
crystal	B-evidence
structure	I-evidence
corresponding	O
to	O
40	O
%,	O
and	O
an	O
extended	B-protein_state
conformation	O
based	O
on	O
simulations	B-experimental_method
that	O
is	O
responsible	O
for	O
34	O
%	O
of	O
the	O
SAXS	B-experimental_method
envelope	B-evidence
.	O

The	O
resulting	O
average	B-evidence
scattering	I-evidence
curve	I-evidence
from	O
this	O
model	O
fits	O
the	O
experimental	O
protein	O
scattering	B-evidence
intensity	I-evidence
,	O
with	O
χ	B-evidence
=	O
1	O
.	O
89	O
(	O
Supplementary	O
Fig	O
.	O
3	O
).	O

GH5_4	B-protein_type
phylogenetic	B-experimental_method
analysis	I-experimental_method

After	O
the	O
exclusion	O
of	O
partial	O
sequences	O
and	O
the	O
suppression	O
of	O
highly	O
identical	O
members	O
(	O
higher	O
than	O
90	O
%	O
identity	O
),	O
144	O
sequences	O
containing	O
between	O
277	B-residue_range
and	I-residue_range
400	I-residue_range
residues	O
were	O
aligned	B-experimental_method
and	O
used	O
to	O
construct	O
a	O
phylogenetic	B-evidence
tree	I-evidence
(	O
Supplementary	O
Fig	O
.	O
4A	O
).	O

According	O
to	O
PFAM	O
database	O
conserved	O
domain	O
classification	O
,	O
128	O
GH5	B-protein_type
enzymes	O
have	O
an	O
architecture	O
consisting	O
of	O
an	O
N	O
-	O
terminal	O
catalytic	B-structure_element
module	I-structure_element
,	O
a	O
CBM_X2	B-structure_element
module	O
and	O
an	O
unknown	O
module	O
of	O
approximately	O
100	O
residues	O
at	O
the	O
C	O
-	O
terminus	O
(	O
Supplementary	O
Fig	O
.	O
4B	O
).	O

Of	O
these	O
,	O
12	O
enzymes	O
have	O
an	O
additional	O
CBM1	B-structure_element
,	O
and	O
5	O
have	O
a	O
CBM2	B-structure_element
at	O
the	O
N	O
-	O
terminal	O
region	O
.	O

Based	O
on	O
this	O
PFAM	O
architecture	O
and	O
CAZy	O
subfamily	O
classification	O
,	O
all	O
the	O
144	O
enzymes	O
(	O
including	O
BlCel5B	B-protein
)	O
belong	O
to	O
the	O
GH5_4	B-protein_type
subfamily	O
and	O
group	O
together	O
in	O
the	O
same	O
branch	O
of	O
the	O
phylogenetic	B-evidence
tree	I-evidence
,	O
evidencing	O
a	O
common	O
ancestor	O
.	O

These	O
results	O
support	O
the	O
hypothesis	O
that	O
the	O
enzymes	O
may	O
employ	O
the	O
same	O
mechanism	O
by	O
which	O
ligand	O
binding	O
is	O
mediated	O
by	O
an	O
extensive	O
conformational	O
breathing	O
of	O
the	O
enzyme	O
that	O
involves	O
the	O
large	O
-	O
scale	O
movement	O
of	O
CBM46	B-structure_element
around	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
(	O
CBM_X2	B-structure_element
)	O
as	O
a	O
structural	B-structure_element
hinge	I-structure_element
.	O

Here	O
,	O
we	O
elucidate	O
the	O
trimodular	B-protein_state
molecular	O
architecture	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
BlCel5B	B-protein
,	O
a	O
member	O
of	O
the	O
GH5_4	B-protein_type
subfamily	O
,	O
for	O
which	O
large	O
-	O
scale	O
conformational	O
dynamics	O
appears	O
to	O
play	O
a	O
central	O
role	O
in	O
its	O
enzymatic	O
activity	O
.	O

Full	B-protein_state
-	I-protein_state
length	I-protein_state
BlCel5B	B-protein
is	O
active	B-protein_state
on	O
both	O
cellulosic	B-chemical
and	O
hemicellulosic	B-chemical
substrates	O
and	O
auxiliary	O
modules	O
are	O
crucial	O
for	O
its	O
activity	O
.	O

Most	O
carbohydrate	B-protein_type
-	I-protein_type
active	I-protein_type
enzymes	I-protein_type
are	O
modular	O
and	O
consist	O
of	O
a	O
catalytic	B-structure_element
domain	I-structure_element
appended	O
to	O
one	O
or	O
more	O
separate	O
AMs	B-structure_element
.	O

AMs	B-structure_element
,	O
such	O
as	O
CBMs	B-structure_element
,	O
typically	O
recognize	O
carbohydrates	B-chemical
and	O
target	O
their	O
cognate	O
catalytic	B-structure_element
domains	I-structure_element
toward	O
the	O
substrate	O
.	O

Because	O
the	O
structural	B-experimental_method
analysis	I-experimental_method
of	O
the	O
protein	O
is	O
challenging	O
if	O
the	O
linkers	B-structure_element
connecting	O
the	O
structural	O
subunits	O
of	O
the	O
enzyme	O
are	O
long	O
and	O
flexible	O
,	O
the	O
standard	O
approach	O
is	O
to	O
study	O
the	O
domains	O
separately	O
.	O

In	O
this	O
work	O
,	O
a	O
combination	O
of	O
protein	B-experimental_method
crystallography	I-experimental_method
,	O
computational	B-experimental_method
molecular	I-experimental_method
dynamics	I-experimental_method
,	O
and	O
SAXS	B-experimental_method
analyses	O
enabled	O
the	O
identification	O
of	O
a	O
new	O
conformational	O
selection	O
-	O
based	O
molecular	O
mechanism	O
that	O
involves	O
GH5	B-protein_type
catalytic	B-structure_element
domain	I-structure_element
and	O
two	O
AMs	B-structure_element
in	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
BlCel5B	B-protein
.	O

We	O
observed	O
that	O
the	O
BlCel5B	B-protein
distal	O
CBM46	B-structure_element
is	O
directly	O
involved	O
in	O
shaping	O
the	O
local	O
architecture	O
of	O
the	O
substrate	B-site
-	I-site
binding	I-site
site	I-site
.	O

Although	O
the	O
CD	B-structure_element
alone	B-protein_state
appears	O
unable	O
to	O
bind	O
the	O
substrate	O
for	O
catalysis	O
,	O
the	O
AMs	B-structure_element
exhibit	O
open	B-protein_state
-	O
close	B-protein_state
motions	O
that	O
allow	O
the	O
substrate	O
to	O
be	O
captured	O
in	O
a	O
suitable	O
position	O
for	O
hydrolysis	O
.	O

Here	O
,	O
we	O
advocate	O
that	O
large	O
-	O
amplitude	O
motions	O
of	O
AMs	B-structure_element
are	O
crucial	O
for	O
assembling	O
the	O
enzyme	O
into	O
its	O
active	B-protein_state
conformation	O
,	O
highlighting	O
a	O
new	O
function	O
of	O
CBMs	B-structure_element
.	O

This	O
mechanism	O
of	O
substrate	O
binding	O
closely	O
resembles	O
the	O
extended	B-protein_state
conformational	O
selection	O
model	O
,	O
with	O
the	O
induced	O
-	O
fit	O
mechanism	O
of	O
reaction	O
as	O
its	O
limiting	O
case	O
.	O

To	O
the	O
best	O
of	O
our	O
knowledge	O
,	O
this	O
enzymatic	O
mechanism	O
has	O
not	O
been	O
proposed	O
previously	O
for	O
any	O
GH	B-protein_type
.	O

The	O
CD	B-site
binding	I-site
site	I-site
of	O
BlCel5B	B-protein
is	O
open	O
and	O
relatively	O
flat	O
and	O
is	O
thus	O
barely	O
able	O
to	O
properly	O
hold	O
the	O
substrate	O
in	O
position	O
for	O
catalysis	O
without	O
assistance	O
from	O
the	O
CBM46	B-structure_element
.	O

In	O
contrast	O
,	O
other	O
GH5s	B-protein_type
belonging	O
to	O
subfamily	O
4	O
listed	O
in	O
the	O
Protein	O
Data	O
Bank	O
exhibit	O
a	O
deep	O
binding	B-site
cleft	I-site
or	O
tunnel	B-site
that	O
can	O
effectively	O
entrap	O
the	O
substrate	O
for	O
catalysis	O
(	O
Fig	O
.	O
5	O
).	O

Due	O
to	O
the	O
marked	O
interdomain	O
conformational	O
rearrangement	O
observed	O
in	O
our	O
simulations	B-experimental_method
,	O
the	O
CBM46	B-structure_element
generates	O
a	O
confined	O
binding	B-site
site	I-site
in	O
BlCel5B	B-protein
that	O
resembles	O
the	O
binding	B-site
site	I-site
architecture	O
of	O
the	O
other	O
GH5	B-protein_type
enzymes	O
that	O
lack	B-protein_state
AMs	B-structure_element
.	O

Thus	O
,	O
BlCel5B	B-protein
appears	O
to	O
have	O
adopted	O
a	O
strategy	O
of	O
CBM46	B-structure_element
-	O
mediated	O
interactions	O
for	O
proper	O
functioning	O
.	O

Although	O
the	O
homologous	O
BhCel5B	B-protein
has	O
the	O
same	O
domain	O
architecture	O
of	O
BlCel5B	B-protein
and	O
belongs	O
to	O
the	O
same	O
subfamily	O
(	O
a	O
comparison	O
of	O
the	O
sequence	O
and	O
structure	B-evidence
of	O
BlCel5B	B-protein
and	O
BhCel5B	B-protein
is	O
presented	O
in	O
Supplementary	O
Fig	O
.	O
5	O
),	O
its	O
binding	B-site
site	I-site
exhibits	O
important	O
differences	O
that	O
may	O
impact	O
the	O
catalytic	O
mechanism	O
.	O

The	O
BhCel5B	B-protein
binding	B-site
site	I-site
is	O
V	B-protein_state
-	I-protein_state
shaped	I-protein_state
and	O
deeper	O
than	O
the	O
BlCel5B	B-protein
binding	B-site
site	I-site
(	O
Figs	O
5	O
and	O
6	O
).	O

This	O
is	O
due	O
to	O
the	O
loop	B-structure_element
between	O
residues	O
F177	B-residue_name_number
and	O
R185	B-residue_name_number
from	O
BhCel5B	B-protein
(	O
absent	B-protein_state
in	O
the	O
BlCel5B	B-protein
),	O
which	O
contains	O
residue	O
W181	B-residue_name_number
that	O
forms	O
part	O
of	O
the	O
binding	B-site
cleft	I-site
(	O
Fig	O
.	O
6	O
).	O

Consistently	O
,	O
although	O
BhCel5B	B-protein
CBM46	B-structure_element
is	O
important	O
for	O
β	B-chemical
-	I-chemical
1	I-chemical
,	I-chemical
3	I-chemical
-	I-chemical
1	I-chemical
,	I-chemical
4	I-chemical
-	I-chemical
glucan	I-chemical
hydrolysis	O
(	O
BhCel5B	B-protein
is	O
about	O
60	O
-	O
fold	O
less	O
active	B-protein_state
without	B-protein_state
CBM46	B-structure_element
),	O
the	O
truncated	B-protein_state
enzyme	O
is	O
completely	O
active	B-protein_state
against	O
xyloglucan	B-chemical
,	O
suggesting	O
that	O
the	O
CBM46	B-structure_element
,	O
in	O
this	O
case	O
,	O
is	O
necessary	O
for	O
the	O
binding	O
to	O
specific	O
substrates	O
.	O

A	O
closer	O
inspection	O
of	O
results	O
of	O
the	O
phylogenetic	B-experimental_method
analysis	I-experimental_method
,	O
more	O
specifically	O
of	O
the	O
clade	O
composed	O
by	O
GH5_4	B-protein_type
enzymes	O
with	O
trimodular	B-protein_state
architecture	O
(	O
Supplementary	O
Fig	O
.	O
4C	O
),	O
reveals	O
subclades	O
whose	O
main	O
characteristic	O
is	O
the	O
varying	O
length	O
of	O
the	O
loop	B-structure_element
located	O
between	O
residues	O
161	B-residue_range
and	I-residue_range
163	I-residue_range
(	O
BlCel5B	B-protein
residue	O
numbering	O
).	O

Therefore	O
,	O
our	O
results	O
show	O
that	O
BlCel5B	B-protein
represents	O
a	O
smaller	O
group	O
of	O
enzymes	O
that	O
are	O
completely	O
dependent	O
on	O
its	O
AMs	B-structure_element
for	O
hydrolysis	O
of	O
plant	B-taxonomy_domain
cell	O
wall	O
polysaccharides	B-chemical
,	O
and	O
that	O
the	O
underlying	O
mechanism	O
may	O
rely	O
on	O
large	O
-	O
scale	O
interdomain	O
motions	O
.	O

The	O
amino	O
acid	O
sequence	O
of	O
the	O
BlCel5B	B-protein
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
is	O
recognized	O
by	O
BLASTP	B-experimental_method
as	O
belonging	O
to	O
CBM_X2	B-structure_element
,	O
a	O
poorly	O
described	O
group	O
that	O
has	O
been	O
compared	O
with	O
CBM	B-structure_element
-	I-structure_element
like	I-structure_element
accessory	I-structure_element
modules	I-structure_element
without	O
a	O
defined	O
function	O
.	O

Despite	O
the	O
similarity	O
of	O
BlCel5B	B-protein
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
to	O
CBMs	B-structure_element
,	O
it	O
lacks	O
an	O
identifiable	O
aromatic	O
residue	O
-	O
rich	O
carbohydrate	B-site
-	I-site
binding	I-site
site	I-site
.	O

Nonetheless	O
,	O
according	O
to	O
our	O
results	O
,	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
seems	O
to	O
play	O
an	O
important	O
function	O
as	O
a	O
structural	B-structure_element
hinge	I-structure_element
,	O
dynamically	O
holding	O
the	O
CBM46	B-structure_element
and	O
CD	B-structure_element
in	O
positions	O
that	O
are	O
appropriate	O
for	O
enzymatic	O
activity	O
.	O

Based	O
on	O
the	O
results	O
of	O
our	O
crystallographic	B-experimental_method
,	I-experimental_method
computer	I-experimental_method
simulation	I-experimental_method
,	O
and	O
SAXS	B-experimental_method
structural	I-experimental_method
analyses	I-experimental_method
,	O
as	O
well	O
as	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
and	O
activity	B-experimental_method
assays	I-experimental_method
,	O
we	O
propose	O
a	O
molecular	O
mechanism	O
for	O
BlCel5B	B-protein
substrate	O
binding	O
,	O
which	O
might	O
apply	O
to	O
other	O
GH5_4	B-protein_type
subfamily	O
enzymes	O
that	O
share	O
this	O
tri	B-structure_element
-	I-structure_element
modular	I-structure_element
architecture	O
.	O

BlCel5B	B-protein
can	O
be	O
found	O
in	O
several	O
different	O
conformational	O
states	O
ranging	O
from	O
CBM46	B-structure_element
/	O
CD	B-structure_element
closed	B-protein_state
(	O
or	O
occluded	B-protein_state
)	O
to	O
extended	B-protein_state
conformations	O
(	O
Fig	O
.	O
7	O
).	O

In	O
extended	B-protein_state
configurations	O
,	O
the	O
substrate	O
may	O
dock	O
at	O
the	O
shallow	O
substrate	B-site
binding	I-site
site	I-site
of	O
CD	B-structure_element
in	O
one	O
of	O
the	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
conformations	O
of	O
the	O
enzyme	O
;	O
however	O
,	O
its	O
binding	O
is	O
properly	O
stabilized	O
for	O
hydrolysis	O
only	O
with	O
the	O
aid	O
of	O
induced	O
-	O
fit	O
repositioning	O
mediated	O
by	O
CBM46	B-structure_element
.	O

After	O
cleavage	O
,	O
the	O
intrinsic	O
dynamics	O
of	O
BlCel5B	B-protein
would	O
eventually	O
allow	O
the	O
opening	O
of	O
the	O
active	B-site
site	I-site
for	O
product	O
release	O
.	O

The	O
proposed	O
mechanism	O
is	O
consistent	O
with	O
our	O
mutagenesis	B-experimental_method
and	I-experimental_method
enzymatic	I-experimental_method
activity	I-experimental_method
assays	I-experimental_method
,	O
which	O
show	O
that	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
module	I-structure_element
and	O
CBM46	B-structure_element
are	O
indispensable	O
for	O
BlCel5B	B-protein
catalytic	O
activity	O
and	O
,	O
together	O
with	O
the	O
CD	B-structure_element
,	O
form	O
the	O
unique	B-protein_state
catalytic	B-structure_element
domain	I-structure_element
of	O
the	O
enzyme	O
.	O

These	O
experiments	O
reveal	O
a	O
novel	O
function	O
for	O
CBMs	B-structure_element
in	O
which	O
they	O
are	O
intimately	O
involved	O
in	O
the	O
assembly	O
of	O
the	O
active	B-site
site	I-site
and	O
catalytic	O
process	O
.	O

Computer	B-experimental_method
simulations	I-experimental_method
suggest	O
that	O
large	O
-	O
scale	O
motions	O
of	O
the	O
CBM46	B-structure_element
and	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
domains	I-structure_element
mediate	O
conformational	O
selection	O
and	O
final	O
induced	O
-	O
fit	O
adjustments	O
to	O
trap	O
the	O
substrate	O
at	O
the	O
active	B-site
site	I-site
and	O
promote	O
hydrolysis	O
.	O

SAXS	B-experimental_method
data	O
support	O
the	O
modeling	B-experimental_method
results	O
,	O
providing	O
compelling	O
evidence	O
for	O
highly	B-protein_state
mobile	I-protein_state
domains	O
in	O
solution	O
.	O

Crystal	B-evidence
models	I-evidence
of	O
BlCel5B	B-protein
.	O

Complete	O
structure	B-evidence
is	O
shown	O
as	O
a	O
cartoon	O
illustration	O
in	O
(	O
a	O
)	O
and	O
a	O
van	O
der	O
Waals	O
surface	O
in	O
(	O
b	O
).	O

The	O
CD	B-structure_element
module	O
(	O
red	O
)	O
has	O
a	O
typical	O
TIM	B-structure_element
-	I-structure_element
barrel	I-structure_element
fold	I-structure_element
,	O
and	O
its	O
substrate	B-site
-	I-site
binding	I-site
site	I-site
is	O
adjacent	O
to	O
CBM46	B-structure_element
(	O
blue	O
).	O

Despite	O
the	O
proximity	O
of	O
the	O
binding	B-site
site	I-site
in	O
the	O
crystallographic	O
model	O
,	O
the	O
CBM46	B-structure_element
residues	O
W479	B-residue_name_number
and	O
W481	B-residue_name_number
are	O
distant	O
from	O
the	O
substrate	O
cellotetraose	B-chemical
(	O
yellow	O
).	O

The	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
domain	I-structure_element
(	O
green	O
)	O
has	O
a	O
lateral	O
position	O
,	O
serving	O
as	O
a	O
connector	O
between	O
the	O
CD	B-structure_element
and	O
CBM46	B-structure_element
.	O
(	O
c	O
)	O
A	O
superposition	B-experimental_method
of	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
domain	I-structure_element
and	O
CBM46	B-structure_element
illustrates	O
their	O
structural	O
similarity	O
,	O
with	O
most	O
of	O
the	O
structural	O
differences	O
present	O
in	O
the	O
loop	B-structure_element
highlighted	O
by	O
a	O
red	O
circle	O
.	O
(	O
d	O
)	O
Cellotetraose	B-chemical
occupies	O
subsites	B-site
-	I-site
1	I-site
to	I-site
-	I-site
3	I-site
and	O
is	O
primarily	O
coordinated	B-bond_interaction
by	O
the	O
residues	O
represented	O
in	O
gray	O
.	O

BlCel5B	B-protein
enzymatic	B-experimental_method
activity	I-experimental_method
characterization	I-experimental_method
.	O

(	O
a	O
)	O
MALDI	B-experimental_method
/	I-experimental_method
TOF	I-experimental_method
-	I-experimental_method
MS	I-experimental_method
spectra	B-evidence
of	O
the	O
products	O
released	O
after	O
incubation	O
of	O
BlCel5B	B-protein
and	O
its	O
two	O
deletion	B-experimental_method
constructs	I-experimental_method
(	O
ΔCBM46	B-mutant
and	O
ΔIg	B-mutant
-	I-mutant
CBM46	I-mutant
)	O
with	O
the	O
substrate	O
cellopentaose	B-chemical
(	O
C5	B-chemical
).	O

The	O
first	O
three	O
spectra	B-evidence
show	O
the	O
substrate	O
,	O
enzyme	O
and	O
buffer	O
controls	O
.	O

The	O
forth	O
spectrum	B-evidence
reveals	O
that	O
full	B-protein_state
length	I-protein_state
BlCel5B	B-protein
is	O
capable	O
of	O
enzymatic	O
hydrolysis	O
of	O
C5	B-chemical
into	O
smaller	O
oligosaccharides	B-chemical
such	O
as	O
C4	B-chemical
,	O
C3	B-chemical
and	O
C2	B-chemical
.	O

The	O
last	O
two	O
spectra	B-evidence
show	O
that	O
the	O
C	O
-	O
terminal	O
deletions	O
eliminate	B-protein_state
the	I-protein_state
enzyme	I-protein_state
activity	I-protein_state
.	O

BlCel5B	B-protein
activities	O
on	O
CMC	B-chemical
as	O
functions	O
of	O
pH	O
and	O
temperature	O
are	O
shown	O
in	O
(	O
b	O
)	O
and	O
(	O
c	O
),	O
respectively	O
.	O

(	O
d	O
)	O
Michaelis	B-evidence
-	I-evidence
Menten	I-evidence
curve	I-evidence
using	O
CMC	B-chemical
as	O
a	O
substrate	O
.	O

Open	B-protein_state
-	O
close	B-protein_state
transitions	O
of	O
BlCel5B	B-protein
.	O

(	O
a	O
)	O
BlCel5B	B-protein
in	O
the	O
absence	B-protein_state
of	I-protein_state
substrate	O
and	O
(	O
b	O
)	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
cellooctaose	B-chemical
,	O
as	O
observed	O
in	O
our	O
aMD	B-experimental_method
simulations	I-experimental_method
.	O

The	O
distance	B-evidence
between	O
the	O
α	O
carbon	O
of	O
residues	O
I120	B-residue_name_number
(	O
CD	B-structure_element
)	O
and	O
E477	B-residue_name_number
(	O
CBM46	B-structure_element
),	O
illustrated	O
as	O
spheres	O
in	O
(	O
a	O
),	O
is	O
plotted	O
in	O
(	O
c	O
),	O
revealing	O
a	O
transition	O
by	O
the	O
decrease	O
in	O
the	O
distance	B-evidence
from	O
40	O
Å	O
to	O
7	O
Å	O
(	O
substrate	B-protein_state
-	I-protein_state
free	I-protein_state
)	O
or	O
20	O
Å	O
(	O
in	O
presence	B-protein_state
of	I-protein_state
cellooctaose	B-chemical
).	O

For	O
the	O
substrate	B-protein_state
-	I-protein_state
free	I-protein_state
enzyme	O
,	O
the	O
red	O
line	O
refers	O
to	O
a	O
1	O
μs	O
-	O
long	O
aMD	B-experimental_method
;	O
for	O
the	O
BlCel5B	B-complex_assembly
-	I-complex_assembly
cellooctaose	I-complex_assembly
complex	O
,	O
the	O
first	O
500	O
ns	O
refers	O
to	O
aMD	B-experimental_method
(	O
in	O
blue	O
)	O
and	O
the	O
second	O
500	O
ns	O
to	O
conventional	O
MD	B-experimental_method
(	O
in	O
turquoise	O
).	O

(	O
d	O
)	O
A	O
snapshot	O
of	O
the	O
BlCel5B	B-complex_assembly
-	I-complex_assembly
cellooctaose	I-complex_assembly
complex	O
,	O
highlighting	O
the	O
tryptophan	B-residue_name
residues	O
that	O
interact	O
with	O
the	O
glucan	B-chemical
chain	O
in	O
subsites	B-site
−	I-site
4	I-site
to	I-site
+	I-site
4	I-site
.	O

Residues	O
W479	B-residue_name_number
and	O
W481	B-residue_name_number
belong	O
to	O
CBM46	B-structure_element
and	O
only	O
become	O
available	O
for	O
substrate	O
interactions	O
in	O
the	O
closed	B-protein_state
configuration	O
of	O
BlCel5B	B-protein
.	O

Large	O
-	O
scale	O
movements	O
of	O
BlCel5B	B-protein
modules	O
and	O
superposition	B-experimental_method
of	O
their	O
representative	O
conformations	O
with	O
the	O
SAXS	B-experimental_method
envelope	B-evidence
.	O

(	O
a	O
)	O
BlCel5B	B-protein
structure	B-evidence
showing	O
the	O
distance	B-evidence
between	O
the	O
backbone	O
beads	O
of	O
residues	O
I120	B-residue_name_number
and	O
E477	B-residue_name_number
,	O
which	O
are	O
centrally	O
located	O
in	O
CD	B-structure_element
and	O
CBM46	B-structure_element
,	O
respectively	O
,	O
as	O
a	O
metric	O
for	O
the	O
relative	O
disposition	O
between	O
the	O
two	O
domains	O
.	O
(	O
b	O
)	O
Time	O
history	O
of	O
the	O
I120	B-residue_name_number
-	O
E477	B-residue_name_number
distance	B-evidence
computed	O
using	O
CG	B-experimental_method
-	I-experimental_method
MD	I-experimental_method
simulations	I-experimental_method
.	O

Different	O
colors	O
separated	O
by	O
vertical	O
lines	O
correspond	O
to	O
independent	O
simulations	B-experimental_method
of	O
approximately	O
120	O
μs	O
.	O
(	O
c	O
)	O
The	O
distance	B-evidence
distribution	I-evidence
indicates	O
three	O
major	O
peaks	O
:	O
closed	B-protein_state
or	O
occluded	B-protein_state
CBM46	B-structure_element
/	O
CD	B-structure_element
conformations	O
(	O
I	O
);	O
semi	B-protein_state
-	I-protein_state
open	I-protein_state
(	O
II	O
),	O
which	O
is	O
similar	O
to	O
the	O
crystallographic	B-evidence
structure	I-evidence
;	O
and	O
extended	B-protein_state
conformers	O
(	O
III	O
).	O

(	O
d	O
)	O
Superimposition	B-experimental_method
of	O
the	O
three	O
representative	O
molecular	O
conformations	O
of	O
BlCel5B	B-protein
with	O
the	O
SAXS	B-experimental_method
model	B-evidence
.	O
(	O
e	O
)	O
Average	O
structures	B-evidence
obtained	O
from	O
the	O
simulation	B-experimental_method
segments	O
corresponding	O
to	O
population	O
groups	O
I	O
-	O
III	O
,	O
which	O
are	O
individually	O
superposed	B-experimental_method
on	O
the	O
SAXS	B-experimental_method
envelope	B-evidence
.	O

Comparison	B-experimental_method
of	O
the	O
binding	B-site
site	I-site
shape	O
of	O
GH5_4	B-protein_type
enzymes	O
available	O
on	O
the	O
Protein	O
Data	O
Bank	O
.	O

(	O
a	O
)	O
BlCel5B	B-protein
in	O
the	O
crystallographic	B-experimental_method
and	O
closed	B-protein_state
configuration	O
;	O
(	O
b	O
)	O
Bacillus	B-species
halodurans	I-species
Cel5B	B-protein
(	O
BhCel5B	B-protein
)	O
(	O
PDB	O
id	O
:	O
4V2X	O
)	O
(	O
c	O
)	O
Piromyces	B-species
rhizinflata	I-species
GH5	B-protein_type
endoglucanase	B-protein_type
(	O
PDB	O
id	O
:	O
3AYR	O
);	O
(	O
d	O
)	O
Clostridium	B-species
cellulolyticum	I-species
GH5	B-protein_type
endoglucanase	B-protein_type
(	O
PDB	O
id	O
:	O
1EDG	O
);	O
(	O
e	O
)	O
Clostridium	B-species
cellulovorans	I-species
GH5	B-protein_type
endoglucanase	B-protein_type
(	O
PDB	O
id	O
:	O
3NDY	O
);	O
(	O
f	O
)	O
Bacteroides	B-species
ovatus	I-species
GH5	B-protein_type
xyloglucanase	B-protein_type
(	O
PDB	O
id	O
:	O
3ZMR	O
);	O
(	O
g	O
)	O
Paenibacillus	B-species
pabuli	I-species
GH5	B-protein_type
xyloglucanase	B-protein_type
(	O
PDB	O
id	O
:	O
2JEP	O
);	O
(	O
h	O
)	O
Prevotella	B-species
bryantii	I-species
GH5	B-protein_type
endoglucanase	B-protein_type
(	O
PDB	O
id	O
:	O
3VDH	O
);	O
(	O
i	O
)	O
Ruminiclostridium	B-species
thermocellum	I-species
multifunctional	O
GH5	B-protein_type
cellulase	B-protein_type
,	O
xylanase	B-protein_type
and	O
mannase	B-protein_type
(	O
PDB	O
id	O
:	O
4IM4	O
);	O
(	O
j	O
)	O
Bacteroidetes	B-taxonomy_domain
bacterium	I-taxonomy_domain
AC2a	B-protein_type
endocellulase	B-protein_type
(	O
PDB	O
id	O
:	O
4YHE	O
).	O

Comparison	B-experimental_method
of	O
the	O
binding	B-site
cleft	I-site
of	O
the	O
BlCel5B	B-protein
and	O
BhCel5B	B-protein
.	O

The	O
main	O
difference	O
between	O
BlCel5B	B-protein
and	O
BhCel5B	B-protein
is	O
that	O
the	O
latter	O
exhibits	O
a	O
deeper	O
cleft	B-site
due	O
to	O
the	O
presence	B-protein_state
of	I-protein_state
residue	O
W181	B-residue_name_number
in	O
the	O
loop	B-structure_element
between	O
F177	B-residue_name_number
and	O
R185	B-residue_name_number
.	O

We	O
conjecture	O
that	O
this	O
difference	O
in	O
the	O
binding	B-site
site	I-site
architecture	O
relates	O
to	O
the	O
importance	O
that	O
the	O
CBM46	B-structure_element
plays	O
in	O
the	O
BlCel5B	B-protein
enzymatic	O
mechanism	O
.	O

Proposed	O
molecular	O
mechanism	O
of	O
BlCel5B	B-protein
conformational	O
selection	O
.	O

As	O
suggested	O
by	O
the	O
simulations	B-experimental_method
and	O
SAXS	B-experimental_method
data	O
,	O
BlCel5B	B-protein
spans	O
multiple	O
conformations	O
ranging	O
from	O
closed	B-protein_state
to	O
extended	B-protein_state
CBM46	B-structure_element
/	O
CD	B-structure_element
states	O
.	O

In	O
a	O
given	O
open	B-protein_state
state	O
,	O
the	O
substrate	O
may	O
reach	O
the	O
active	B-site
site	I-site
and	O
become	O
entrapped	O
by	O
the	O
capping	O
of	O
CBM46	B-structure_element
onto	O
CD	B-structure_element
and	O
induced	O
-	O
fit	O
conformational	O
adjustments	O
.	O

After	O
hydrolysis	O
,	O
the	O
reaction	O
product	O
is	O
released	O
to	O
yield	O
apo	B-protein_state
-	O
BlCel5B	B-protein
,	O
which	O
becomes	O
ready	O
for	O
a	O
new	O
cycle	O
.	O

Activity	O
of	O
BlCel5B	B-protein
constructs	O
against	O
tested	O
substrates	O
.	O

Substrate	O
(	O
1	O
%)	O
Relative	O
Activity	O
(%)	O
WT	B-protein_state
*	O
W479A	B-mutant
W481A	B-mutant
ΔCBM46	B-mutant
ΔIg	B-mutant
-	I-mutant
CBM46	I-mutant
β	B-chemical
-	I-chemical
glucan	I-chemical
100	O
79	O
.	O
1	O
63	O
.	O
6	O
nd	O
nd	O
CMC	B-chemical
25	O
.	O
5	O
12	O
.	O
2	O
2	O
.	O
4	O
nd	O
nd	O
Lichenan	B-chemical
52	O
.	O
4	O
41	O
28	O
.	O
6	O
nd	O
nd	O
Xyloglucan	B-chemical
45	O
.	O
2	O
41	O
.	O
2	O
30	O
.	O
8	O
nd	O
nd	O
Azo	B-chemical
-	I-chemical
Avicel	I-chemical
nd	O
**	O
nd	O
nd	O
nd	O
nd	O
Arabinoxylan	B-chemical
nd	O
nd	O
nd	O
nd	O
nd	O
Galactomannan	B-chemical
nd	O
nd	O
nd	O
nd	O
nd	O
1	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
β	I-chemical
-	I-chemical
mannan	I-chemical
nd	O
nd	O
nd	O
nd	O
nd	O

*	O
WT	B-protein_state
=	O
wild	B-protein_state
type	I-protein_state
.	O