From the
Crystallographic analysis indicated that Clostridium
thermocellum endoglucanase CelD contained three
Ca
Clostridium thermocellum synthesizes a multienzymatic
cellulase complex with a molecular mass of 2-4 MDa, termed
cellulosome
(1, 2) . Endoglucanase CelD is a component
of the cellulosome, which can be easily purified in large amounts from
inclusion bodies produced in recombinant Escherichia coli (3) . CelD belongs to the family E of cellulases
(4, 5) . The three-dimensional structure of
CelD
We
have previously shown that Ca
This paper reports
the structural analysis of the Zn
The pCT6540 and
pCT6547 plasmids were harbored by E. coli TG1
(12) = K-12,
Enzyme activity was
assayed at 60 °C in 50 mM Na-MOPS buffer, pH 6.3,
containing CaCl
Proteins
were incubated at 75 °C at a concentration of 3-5
10
The protein
loop forming site C is completely exposed to the solvent, with three
out of the six oxygen ligands donated by water molecules
(Fig. 2 C). Main chain carbonyls at positions 520 and 525
and the carboxylate group of Asp-523 complete the calcium coordination
polyhedron. Unlike sites A and B, the protein loop forming binding site
C is partially involved in intermolecular interactions in the crystal.
The side chain of Arg-314 from a neighbor molecule is stacked against
Trp-526, and the carbonyl group at position 524 forms an intermolecular
hydrogen bond with the guanido group of Arg-416 (data not shown).
Sites B and C are close to either end of the substrate-binding
groove and are expected to have some influence on the catalytic
activity of CelD. On the opposite site of the
Overall, only
small structural differences were observed for the structure of CelD at
0 and 300 mM calcium. The coordination geometry of the three
sites was essentially the same within experimental error
(). Only the temperature factors of the calcium atoms bound
at sites A and C were different in the two crystal forms (the
temperature factors for the three calcium atoms were 27, 25, and 32
Å
The D246A, D361A, and D523A mutations were chosen to inactivate
Ca
The presence of one Zn
Previous interpretation of Ca
Mutagenesis of site A or B seemed to abolish binding to site
C, as if site C could form only when both sites A and B are occupied.
Why this should be the case is not obvious from structural analysis.
Investigation of the kinetic parameters of CelD indicated that the
change in K
The stabilization
of wild type CelD occurred at concentrations that were an order of
magnitude higher than those required to affect catalytic parameters.
This may be explained by the fact that changes in catalytic properties
induced by Ca
The fact that Ca
The
self-association of monomeric CelD-A* and CelD-B* into a high
M
Unlike catalytic residues, none of the residues involved in
Ca
We thank Raphal de Lecubarri for performing cultures
in 15-liter fermentors and Marie-Kim Chaveroche for skillfultechnical
help. We are grateful to Jean-Paul Aubert and Maxime Schwartz for
interest and support.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-binding sites, termed A, B, and C, and one
Zn
-binding site. The protein contributed five, six,
and three of the coordinating oxygen atoms present at sites A, B, and
C, respectively. Proteins altered by mutation in site A
(CelD
), B (CelD
), or C
(CelD
) were compared with wild type CelD. The
Ca
-binding isotherm of wild type CelD was compatible
with two high affinity sites ( K
=
2
10
M
) and one low
affinity site ( K
< 10
M
). The Ca
-binding
isotherms of the mutated proteins showed that sites A and B were the
two high affinity sites and that site C was the low affinity site.
Atomic absorption spectrometry confirmed the presence of one tightly
bound Zn
atom per CelD molecule. The inactivation
rate of CelD at 75 °C was decreased 1.9-fold upon increasing the
Ca
concentration from 2
10
to 10
M. The
K
of CelD was decreased 1.8-fold upon
increasing the Ca
concentration from 5
10
to 10
M. Over similar
ranges of concentration, Ca
did not affect the
thermostability nor the kinetic properties of CelD
.
These findings suggest that Ca
binding to site C
stabilizes the active conformation of CelD in agreement with the close
vicinity of site C to the catalytic center.
(
)
has been determined by x-ray
crystallography
(6) . The protein contains two distinct
structural domains that are closely associated: a small amino-terminal
-barrel domain and a larger, mostly
-helical domain, whose
amino acid sequence is similar in all catalytic domains of family E
cellulases
(4, 7) . The COOH terminus of CelD consists
of a duplicated segment of 23 residues that is involved in anchoring
the protein to the scaffolding component of the cellulosome
(8, 9) . The part of the protein visible in the electron
density map terminates 10 residues upstream from the beginning of the
COOH-terminal duplication. A cleft on the surface of the
-helical
domain constitutes the active site. According to structural analysis
(6) and mutagenesis data
(10) , the two residues
participating in acid-base catalysis are Asp-201 and Glu-555.
binds to CelD, thereby
stabilizing the enzyme against thermal denaturation and increasing its
substrate binding affinity
(11) . Three putative
Ca
-binding sites and one putative
Zn
-binding site were identified in the catalytic
domain of the CelD crystal structure
(6) .
-binding site and of
the three Ca
-binding sites of C. thermocellum CelD. The presence of Zn
in CelD was assayed by
atomic absorption spectrometry. CelD proteins carrying mutations in
each of the Ca
-binding sites were purified and
characterized to assess the contribution of each site to Ca
binding. The rate of inactivation at 75 °C and the kinetic
parameters of wild type CelD were determined in the presence of varying
Ca
concentrations to correlate changes in these
parameters with the occupancy of high or low affinity
Ca
-binding sites. The same assays were performed with
CelD mutated in the low affinity Ca
-binding site.
Crystallographic Analysis
Two
isomorphous crystal forms of CelD were grown using ammonium sulfate
( i.e. no added calcium) or 300 mM calcium chloride as
precipitants. Structure determination and independent refinement of the
two forms at 2.3 Å resolution have been described elsewhere
(6) . The present models comprise residues 36-574 and
include three calcium ions, one zinc ion, and 221 (ammonium sulfate) or
204 (calcium chloride) water molecules. The final agreement factors
between observed and calculated structure factor amplitudes in the
resolution range 6-2.3 Å were 17.0% for 33,211 observed
reflections with F > 5 (F) (ammonium sulfate) and 17.4% for
29,797 observed reflections (calcium chloride). Root mean squares
deviations of bond lengths and angles from ideality were 0.007 Å
and 1.6°, respectively, in both crystal structures.
Bacterial Strains and Plasmids
Plasmids
pCT6523, pCT6525, and pCT6527, encoding the catalytic domain of CelD
and carrying the D246A, D361A, and D523A mutations, respectively, were
previously obtained
(10) . Each of the mutations was inserted
into a plasmid whose sequence included the 3`-end of celD, as
previously described
(10) . The resulting plasmids, carrying the
D246A, D361A, and D523A mutations, were termed pCT6543, pCT6545, and
pCT6547, respectively. The isogenic plasmid, pCT6540, encoding the wild
type enzyme, has been described
(10) .
( lac-proAB), thi,
supE, hsdD5 (F` traD36,
proAB
, lacI
,
lacZ
M15). pCT6543 and pCT6545 were harbored by E.
coli JM101
(13) = K-12,
( lac-proAB),
thi, supE (F` traD36,
proAB
, lacI
,
lacZ
M15).
Purification of Wild Type and Mutant Forms of
CelD
E. coli cells harboring the appropriate
plasmids were grown to stationary phase at 37 °C in Luria Bertani
broth
(14) containing 100 µg/ml ticarcillin. Wild type and
mutant forms of CelD were purified from inclusion bodies as previously
described
(3) . Low and high Mforms of
CelD
(CelD-A*) and CelD
(CelD-B*) were
separated on a Mono-Q anion exchange column using a fast performance
liquid chromatography system (Pharmacia Biotech Inc.). Up to 4 mg of
purified protein was loaded on a Mono-Q HR5/5 anion exchange column (1
ml) equilibrated with 20 mM Tris-HCl, pH 7.7, at a rate of 1
ml/min. Elution was performed at 0.7 ml/min using a linear gradient
from 100 to 220 mM NaCl in the same buffer. The low
M
and high M
peaks were
eluted at 150 and 180 mM NaCl, respectively, and concentrated
by ultrafiltration using a YM10 Amicon membrane. All samples were
dialyzed against 40 mM Tris-HCl, pH 7.7.
Protein Electrophoresis
SDS-PAGE was
performed according to Laemmli
(15) . Samples were boiled for 5
min in 2% SDS, 10% glycerol, 5% -mercaptoethanol, 62.5 mM
Tris-HCl, pH 6.8. Non-denaturing PAGE was performed using the same
procedure, omitting SDS and
-mercaptoethanol and the heat
treatment of the samples.
Zinc Assay
The zinc content of wild type
CelD was assayed by flame atomic absorption spectroscopy at 213.9 nm
using a Varian AA-1275 spectrophotometer (Varian Techtron, Springvale,
Australia), with a single element hollow-cathode lamp for zinc
(16) .
Ca
Binding of -binding
Assay
Ca to purified proteins was
assayed by monitoring the release of
Ca from Chelex-100
(Bio-Rad) previously equilibrated with various concentrations of
Ca
(11) .
Enzyme and Protein Assays
All reagents
used in assays performed in the presence of controlled concentrations
of Cawere kept in disposable plasticware (Sterilin)
and were handled with disposable plastic pipettes or pipette tips.
Divalent metals were removed from 50 mM Na-MOPS buffer, pH
6.3, and from 20 mM p-NPC, dissolved in the same
buffer, by shaking with 10% (w/v) Chelex-100. The resin was removed by
centrifuging at 1,000
g for 2 min. Ca
was removed from CelD by shaking in the presence of 10%
Chelex-100 followed by decantation. Alternatively, the enzyme was
diluted in Chelex-treated buffer so that the contribution of
protein-bound Ca
in the assay medium was less than 5
10
M, assuming 3 mol of
Ca
bound/mol of CelD. No difference was observed
between the results obtained with either procedure, even when no
Ca
was added (data not shown).
, EGTA, or ZnCl
as indicated for
each experiment and 0.5-20 mM p-NPC as
substrate. The reaction was stopped after less than 5% of the substrate
had been hydrolyzed by adding vol 1 M
Na
CO
. 1 unit of activity is defined as the
amount of enzyme liberating 1 µmol of p-nitrophenol (
= 1.61
10
cm
mol
) per min. Protein concentration was measured
using the Coomassie Blue reagent supplied by Bio-Rad
(17) , with
bovine serum albumin as a standard.
Thermostability
Proteins were either
treated with Chelex-100 or diluted so that their contribution to the
concentration of Cain the inactivation reaction was
less than 1.5
10
M. No difference
was observed between the results obtained with either procedure, even
when no Ca
was added (data not shown).
M in 50 mM MOPS buffer, pH 6.3,
containing CaCl
, EGTA, or ZnCl
as indicated for
each experiment. Temperature control was ascertained by checking the
temperature inside of a plastic vial similar to those in which the
inactivation reaction was performed. Samples were withdrawn at several
time intervals and chilled on ice, and ZnCl
and CaCl
were added to a final concentration of 1 mM (2
mM CaCl
in the case of samples containing 1
mM EGTA). Residual activity was assayed as described above,
using 0.9 mM p-NPC.
Computations
Kinetic constants (including
the 95% confidence interval) for the rate of inactivation were computed
from linear regressions of log (residual activity) versus time, using the Instat Macprogram (version 2.0, GraphPad
Software). Kand k
values were calculated by non-linear regression using the
KaleidaGraphprogram (version 2.1, Abelbeck Software).
Crystallographic Analysis of
Ca
The
three-dimensional structure of CelD revealed four metal-binding sites
occupied by atoms heavier than water in the crystal. A first internal
site is located immediately behind a protein loop involved in substrate
binding and catalysis ( Zn sphere in Fig. 1).
The tetrahedrical coordination by two Cys and two His side chains and
the displacement by Hg suggests that this site is occupied by a
Zn-binding Sites in CelD
ion
(6) . The three other metal binding
sites are located close to the molecular surface in different regions
of the protein ( spheres A, B, and C in Fig. 1). From the coordination geometry, these three
positions could be identified as Ca
-binding sites.
Figure 1:
Metal-binding sites of endoglucanase
CelD. The polypeptide chain is indicated by a ribbon diagram.
-Helices are indicated by wound ribbons, and
-strands are
indicated by arrows. Metal ions are shown as white spheres. A, B, and C,
Ca
-binding sites; Zn,
Zn
-binding site. The diagram was drawn with MOLSCRIPT
(21).
The coordination of the Caion bound at site A
appears as a slightly distorted octahedral arrangement with a water
molecule at one of the vertices (Fig. 2 A). Protein
groups donate the five other oxygen ligands: two main chain carbonyls
at positions 236 and 241 and the side chains of residues Asn-239,
Asp-243, and Asp-246. The loop forming this site protrudes into the
solvent and appears to be stabilized by calcium.
Figure 2:
Coordination polyhedra of the three
Ca-binding sites. The course of the polypeptide chain
is indicated by a smooth tracing. Liganded groups (side chain residues,
main chain carbonyls, water molecules) are indicated explicitly. Oxygen
and nitrogen atoms are in gray. Ca
is drawn
as a larger sphere inside of the coordination polyhedron. A,
site A; B, site B; C, site C. Diagrams were drawn
with MOLSCRIPT (21).
Seven oxygen atoms
chelate the Caion at site B. In this case, the
coordination polyhedron appears as a distorted pentagonal bipyramid
with Asp-362 and a main chain carbonyl at position 401 on the vertices,
or alternatively as a distorted octahedral arrangement with one
bidentate ligand, Asp-361 (Fig. 2 B). In addition to the
aspartate residues, protein oxygens involved in Ca
binding include the side chain of Thr-356 and the main chain
carbonyl groups at positions 358 and 401. As shown in
Fig. 2B, this site appears to have a structural role in
linking together two different regions of the protein.
-barrel, the
Ca
ion bound at site A stabilizes a helix-connecting
loop with no obvious role in enzymatic activity. As a general rule, the
conformation of the loops forming the three
Ca
-binding sites does not follow the EF-hand pattern
observed in many Ca
-binding proteins
(18) .
Moreover, they differ significantly from each other in loop
conformation as well as in the side chains and the number of water
molecules involved in the coordination polyhedra.
, respectively, at 300 mM CaCl, and 43, 28,
and 47 Å
at 0 mM CaCl), suggesting partial
calcium occupancy of sites A and C in ammonium sulfate-grown crystals.
-binding sites A, B, and C, respectively. The
corresponding proteins will be termed CelD-A*, CelD-B*, and CelD-C*,
respectively.
Separation of High and Low M
SDS-PAGE analysis
indicated that the wild type and the three mutant proteins were mainly
composed of 65-kDa CelD, with 68- and 63-kDa CelD being present as
minor species in some of the preparations (Fig. 3 A).
Previous work has shown that proteolysis accounts for some
heterogeneity of the COOH terminus of CelD. However, cleavage does not
affect the catalytic domain of the protein, and the 68-, 65-, and
63-kDa species were shown to share very similar catalytic properties
(9, 11, 19) .
Forms of CelD-A* and CelD-B*
Figure 3:
Electrophoretic analysis of wild type and
mutant forms of CelD. 4 µg of each purified protein were analyzed
by SDS-PAGE ( panel A)and by non-denaturing PAGE ( panel
B). Lane 1, CelD; lane 2, CelD-C*; lane
3, CelD-A*; lane 4, monomeric form of CelD-A*; lane
5, high Mform of CelD-A*; lane 6,
CelD-B*; lane 7, monomeric form of CelD-B*; lane 8,
high M
form of CelD-B*. The migration and
molecular mass of rabbit myosin (200 kDa), E. coli
-galactosidase (116 kDa), rabbit phosphorylase B (97 kDa),
bovine serum albumin (66 kDa), ovalbumin (45 kDa), soybean trypsin
inhibitor (21 kDa), and hen egg white lysozyme (14 kDa) are indicated
on the right of panel A.
In non-denaturing
electrophoresis (Fig. 3 B), CelD-C* displayed the same
mobility as wild type CelD, which is a monomeric protein
(3) .
However, CelD-A* and CelD-B* could be separated into a form with a
mobility similar to that of the wild type monomer and a slower
migrating, higher Mform, presumably resulting
from self-association. The two forms could be separated by ion exchange
chromatography on a Mono-Q column (Fig. 3 B) or by gel
filtration on a TSK G2000 column (data not shown) but tended to
reequilibrate over a period of a few days. This explains the partial
contamination of one form by the other seen in Fig. 3 B.
Presence of Zn
Atomic
absorption spectroscopy showed the presence of 1.0 ± 0.2 mol of
Zn/mol of wild type CelD. No change in Zn
content was detected when the enzyme was incubated for 15 min at
room temperature or at 60 °C in the presence of 10% (w/v)
Chelex-100, but incubation with Chelex at 75 °C for 9 min resulted
in total loss of detectable enzyme-bound Zn
(data not
shown). Dissociation of Zn
was correlated with an
increase in the inactivation rate of the protein (see below).
Ca
Fig. 4
shows the Scatchard analysis of
Ca-Binding
Parameters
binding to the wild type and various mutant forms
of CelD. The binding isotherm of wild type CelD was compatible with the
presence of two high affinity sites ( K
= 2
10
M
)
and one low affinity site ( K
=
0.66
10
M
) per
molecule. CelD-A* (Fig. 4 A) and CelD-B*
(Fig. 4 B) each displayed one high affinity site with
K
= 5.1
10
M
and K
= 3.2
10
M
, respectively. CelD-C* displayed
two high affinity sites with K
=
3.1
10
M
(Fig. 4 C). By contrast to the wild type, no low
affinity site was detected in any of the mutant CelD proteins.
Ca
-binding isotherms were the same for the low and
high M
forms of CelD-A* and CelD-B* (Fig. 4,
A and B).
Figure 4:
Scatchard analysis of Ca
binding to wild type and mutant forms of CelD. Binding of
Ca (5
10
M <
(CaCl
) < 2.5
10
M)
to 1.7
10
M purified protein is
shown. Each point was the average of a duplicate determination.
Closed circles, wild type CelD; open circles, monomeric mutant CelD; x, high
M
form of mutant CelD. Panel A,
CelD-A*; panel B, CelD-B*; panel C,
CelD-C*. The curve fitting the data for the wild type was drawn
assuming that the enzyme contained two sites with a K of 2
10
M
and one site with
a K of 0.66
10
M
.
Effect of Ca
Previous data indicated that Caon Kinetic
Parameters
decreased the K
but had little
effect on the k
of CelD
(11) .
Fig. 5A confirms that addition or removal of
Ca
had little effect on the k
of CelD and indicates that the strongest decrease in
K
(from 6.2 to 3.5 mM) occurred
when the Ca
concentration was increased from 5
10
to 10
M. As a
consequence, there was a concomitant increase in catalytic efficiency
k
/ K
. Addition of 1
mM EGTA had little effect on CelD after Ca
ions had been removed by dilution in Ca
-free
buffer. Fig. 5 B shows that the kinetic parameters of
CelD-C* were not affected by EGTA nor by Ca
in the
range of concentrations tested.
Figure 5:
Kinetic parameters of wild type CelD and
CelD-C* as a function of divalent metal concentration. Results are
presented using double logarithmic scales. Panel A,
wild type CelD; panel B, CelD-C*. Open circles, k; closed circles, K; closed squares,
k
/ K. The lowest Ca
concentration was calculated from the contribution of
Ca
initially bound to the enzyme added to the assay.
Except for the EGTA-treated samples, all samples contained 1
µM ZnCl
in addition to the Ca
concentrations indicated.
Thermostability
Fig. 6
shows the
kinetic rate of inactivation kof wild type
CelD and of CelD-C* incubated at 75 °C in the presence of 1
mM EGTA or various Ca
concentrations.
Addition of 1 mM EGTA in the inactivation reaction after
Ca
ions had been removed by dilution in
Ca
-free buffer or by Chelex treatment at room
temperature resulted in a 2.4-fold increase in the rate of inactivation
of both enzymes. Addition of Chelex-100 at 75 °C produced a similar
effect (data not shown).
Figure 6:
Inactivation rate kat 75 °C of wild type CelD and CelD-C* as a function of
divalent metal concentration. The first-order inactivation rate was
determined as described under ``Materials and Methods.''
Results are presented using a double logarithmic scale. Closed circles, wild type CelD; open circles,
CelD-C*. Error bars indicate the 95% confidence
interval for each determination. Ca
was removed from
wild type CelD by treating with Chelex-100 and from CelD-C* by diluting
into Chelex-100-treated buffer (contribution of Ca
initially bound to the enzyme added to the assay was < 1.5
10
M). Except for the EGTA-treated
samples, all samples contained 1 µM ZnCl
in
addition to the Ca
concentrations
indicated.
For the wild type enzyme, increasing the
concentration of Caup to 5
10
M had no significant effect on the rate of inactivation.
However, a 1.8-fold decrease in k
was observed
upon increasing the concentration of Ca
from 5
10
to 10
M. Over
the same range of Ca
concentration, the inactivation
rate of CelD-C* was not affected.
ion/mol of CelD,
predicted from the crystallographic analysis of the protein, was
confirmed by biochemical analysis. Zn
binding
appeared quite stable at room temperature and at 60 °C, and
dissociation of Zn
at 75 °C was accompanied by
rapid denaturation of the enzyme. By contrast, Ca
could be dissociated from CelD without denaturing the protein.
binding data had led
to the conclusion that CelD contained two high affinity
Ca
-binding sites
(11) . Points extending
beyond two sites/molecule in the Scatchard plots were not considered in
the analysis. However, crystallographic analysis revealed the presence
of three putative Ca
-binding sites in CelD
(6) . The presence of three functional
Ca
-binding sites was confirmed by the analysis of
CelD-C*, whose mutation affects site C. The
Ca
-binding isotherm of CelD-C* displayed two high
affinity sites similar to those of the wild type, but, in contrast to
the wild type, binding did not exceed 2.1 mol of Ca
bound/mol of protein. This suggests that in the wild type, points
extending between 2 and 3 mol of Ca
bound/mol of
protein were due to the presence of site C, which behaved like a low
affinity site. High affinity Ca
binding to sites A
and B was confirmed by analysis of CelD-A* and CelD-B*. The
Ca
-binding isotherms of both proteins showed that
each mutation abolished high affinity binding to one site. The relative
affinities of sites A, B, and C were consistent with the fact that in
sites A and B, the protein contributes five and six, respectively, of
the coordinating oxygens but only three of the coordinating oxygens of
site C.
of the enzyme as a function
of the Ca
concentration was strongest between 5
10
and 10
M.
This range is most likely accounted for by the increased occupancy of
the low affinity site C rather than the high affinity sites A and B.
The fact that the kinetic parameters of CelD-C* were not affected by
Ca
confirms this interpretation.
dissociation are reversible, whereas
thermal denaturation is not. The Ca
concentrations at
which stabilization was observed were consistent with a requirement for
occupancy of site C rather than site A and B. Accordingly, inactivation
of site C abolished Ca
-induced stabilization of
CelD.
binding to site C enhanced
the substrate binding affinity and stabilized the conformation of the
catalytic site is consistent with the close vicinity of the two sites.
The loop containing the Ca
-coordinating residues
Ser-520, Asp-523, and Ile-525 is connected to the substrate-binding
residues His-516 and Arg-518. His-516 and Arg-518 formed hydrogen bonds
with hydroxyl groups of the inhibitor
o-iodobenzyl-
-D-cellobioside in the crystal
structure of the enzyme-inhibitor complex
(6) . In addition,
chemical modification and mutagenesis studies identified His-516 as an
important residue of the catalytic center
(20) .
, presumably dimeric form was not correlated with
the occupancy of Ca
-binding sites. For both proteins,
addition of Ca
or EGTA during non-denaturing
electrophoresis failed to alter the proportion of the two forms (data
not shown). Both forms displayed very similar
Ca
-binding isotherms. Self-association did not seem
to influence thermostability nor kinetic parameters (data not shown).
However, the compound effects of site A and B mutations on site C
precluded a straightforward analysis of the influence of Ca
on the stability and kinetic properties of the mutant enzymes.
binding is strictly conserved among all catalytic
domains of family E cellulases. At present, it is difficult to predict
from sequence analysis which of the other members of family E may be
stabilized in a similar manner by Ca
. It would be of
interest to know whether the presence of functional
Ca
-binding sites is correlated with the
thermostability of the enzymes.
Table: Interatomic distances in Å between
Caions and protein oxygen atoms in
CelD crystals grown in the presence of ammonium sulfate (form I) or
calcium chloride (form II) as precipitants
-D-cellobioside.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.