short communications

Acta Crystallographica Section D

Biological
Structure of bovine carbonic anhydrase II at 1.95 AÊ
Crystallography resolution
ISSN 0907-4449

Ryuta Saito, Takao Sato, Atsushi Carbonic anhydrase (CA) is a zinc-containing enzyme that catalyzes Received 21 September 2003
the reversible hydration of CO2 to HCOÿ Accepted 10 February 2004
Ikai* and Nobuo Tanaka 3 . In eukaryotes, the enzyme
plays a role in various physiological functions, including interconver-
sion between CO2 and HCOÿ 3 in intermediary metabolism, facilitated
PDB Reference: bovine
Department of Life Science, Graduate School of diffusion of CO2, pH homeostasis and ion transport. The structure of
carbonic anhydrase II, 1v9e,
Bioscience and Biotechnology, Tokyo Institute bovine carbonic anhydrase II (BCA II) has been determined by r1v9esf.
of Technology, Japan molecular replacement and re®ned to 1.95 A Ê resolution by simulated-
annealing and individual B-factor re®nement. The ®nal R factor for
the BCA II structure was 19.4%. BCA II has a C-terminal knot
Correspondence e-mail: aikai@bio.titech.ac.jp
structure similar to that observed in human CA II. It contains one
zinc ion in the active site coordinated to three histidines and one
putative water molecule in a tetrahedral geometry. The structure of
BCA II reveals a probable alternative proton-wire pathway that
differs from that of HCA II.

1. Introduction An earlier X-ray structural study of HCA II

Carbonic anhydrases (CAs) are zinc metallo- revealed it to have a knot-forming topology in
enzymes that can be grouped into three broad its C-terminal region (Eriksson et al., 1988).
classes called , and , with very little or no More recently, crystal structures of murine CA
amino-acid sequence homology between them isozymes IV and V and BCA isozyme III have
(Hewett-Emmett & Tashian, 1996). The class also been determined (Stams et al., 1998; Heck
includes mammalian enzymes such as the et al., 1996; Eriksson & Liljas, 1993). The
bovine carbonic anhydrases (BCAs) used in atomic structure of BCA II is required for full
this study. Class CAs are monomeric proteins interpretation of the data from the single-
with a molecular weight of around 30 kDa. At molecule stretching experiments and to
least 14 CA isozymes have been identi®ed so con®rm the C-terminal knot structure. There
far (Parkkila, 2000). These catalyse the same have been reports of BCA II crystallization
reversible reaction CO2 + H2O ! HCOÿ + (Carlsson et al., 1973; Kumar et al., 1986) but no
3 +H ,
but differ in catalytic ef®ciency and inhibitor- structural coordinates have been made avail-
binding properties. The kinetic properties of able, even though the BCA II structure was
determined at 2.8 A Ê resolution (Kumar et al.,
cytosolic CA II, one of seven isozymes (CA I to
VII), show it to be one of the most ef®cient 1989). Therefore, we set to work on solving the
catalysts known, along with the catalases crystal structure of BCA II.
(Stainer et al., 1975).
Since the report by Wong & Tanford (1973) 2. Materials and methods
of intermediate states in the transition from the
native to the denatured state or vice versa, 2.1. Crystallization of commercial BCA II
intensive investigations have focused on BCA Bovine erythrocyte BCA II was purchased
II and human CA II (HCA II) as models for from Biozyme Laboratories (London,
characterizing the partially folded or unfolded England) and puri®ed by gel-®ltration chro-
states of globular proteins (McCoy & Wong, matography on a Superdex-75 column
1981; Cleland & Wang, 1990; Hammarstorm et (Amersham Biosciences, Buckinghamshire,
al., 1997). BCA II is also a good model for its England). BCA II crystals were grown by the
human homologue, with which its sequence batch method at 277 K using 17 mg mlÿ1
homology is 79.5%. The structural stability of protein solution and a precipitant solution
BCA II has also been investigated by atomic containing 50 mM Tris buffer pH 7.5 and 2.4 M
force microscopy. In this case, the protein was ammonium sulfate. Crystals of typical dimen-
mechanically unfolded by pulling its N- and sions 0.2  0.3  0.3 mm appeared after two
C-termini apart, revealing the presence of a weeks. These crystals belonged to the hexa-
tight core structure that showed a remarkable gonal space group P61, with unit-cell para-
# 2004 International Union of Crystallography resistance towards tensile stretching (Alam & Ê and two BCA
meters a = b = 66.7, c = 240.0 A
Printed in Denmark ± all rights reserved Ikai, 2001; Wang et al., 2002). II molecules per asymmetric unit. The unit-cell

792 DOI: 10.1107/S0907444904003166 Acta Cryst. (2004). D60, 792±795

 short communications
Table 1 containing 25%(v/v) glycerol as a cryopro- Of the 573 solvent molecules identi®ed in
Crystallographic data and re®nement statistics. tectant followed by freezing in an N2 stream the electron-density map, all were within
Values in parentheses are for the outermost shell.
at 100 K. The diffraction data were collected proper hydrogen-bonding distances of the
using synchrotron radiation ( = 1.00 A Ê) protein (<3.4 A Ê or >2.4 A
Ê ) or other solvent
Unit-cell parameters
Ê)
a = b, c (A 66.7, 240.0
with an ADSC Quantum 4R CCD detector molecules, with re®ned B factors of less than
, , ( ) 90, 90, 120 (ADSC, California, USA) on beamline 6A 50 AÊ 2. The average B factors of the main-
Space group P61 at the Photon Factory, Tsukuba, Japan. The chain atoms of individual residues varied
Z value 12 Ê 2, compared with
VM (A Ê 3 Daÿ1) 2.66 data were processed, integrated and scaled between 20.2 and 21.9 A
No. protein atoms 4114 with MOSFLM and SCALA from the CCP4 22.2 A Ê 2 for the overall molecule.
No. heteroatoms 2 program package (Collaborative Computa-
No. water molecules 573
Resolution range (A Ê) 20.0±1.95 (2.06±1.95) tional Project, Number 4, 1994). Integrated 2.5. Semi-empirical quantum-mechanical
Total No. measurements 213573 intensities were converted into structure calculation
No. unique re¯ections 35487
I/(I) 6.4 (2.3)
factors using TRUNCATE from the CCP4
To analyse the chemistry in the catalytic
Completeness (%) 90.6 (90.6) suite. The data-collection and processing
Rsym 0.076 (0.284) sites, theoretical computations were
statistics are given in Table 1.
R factor 0.194 performed using v.3.5 of WinMOPAC Pro
Rfree 0.239 (Fujitsu Ltd, Tokyo, Japan), based on semi-
Average B factors (A Ê 2) 22.16
Zn atoms 15.79 empirical quantum-mechanical calculation.
Water atoms 29.79 2.4. Structure solution and refinement Optimizations were performed with the
R.m.s. deviation from ideal geometry
Bond lengths (A Ê) 0.005
eigenvalue-following method (EF) and an
Initial phases for BCA II were obtained
Bond angles ( ) 1.42 SCF ®eld was achieved. The model for the
Dihedrals ( ) 24.7 by molecular replacement using the EPMR
catalytic site was studied, including the
Impropers ( ) 0.832 program (Kissinger et al., 1999). The coor-
Luzzati coordinate error (A Ê) 0.22 central Zn2+ ion. The ®rst coordinate shell
dinates of the HCA II structure (protein
 A coordinate error (A Ê) 0.16 ligands consisted of residues His93, His95
Ramachandran outliers 1 molecule only without inhibitor or water
and His118 and the oxygen of water mole-
molecules; PDB code 2cbb) were used as an
cule 419 (W419), the second-shell coordi-
initial search model. The amino-acid
volume was 9.25  105 A Ê 3 which, on the nation sphere consisted of Gln91, Glu105,
sequences of HCA II and BCA II were
basis of two molecules of molecular weight Glu116, Thr197 and W33 and the outer shell
compared and 52 non-identical residues
29 kDa each, gives a VM value of consisted of Tyr6, Asn61, His63, Asn66,
were replaced with alanines or serines. The
Ê 3 Daÿ1.
2.66 A Thr198, Arg244 and nine water molecules.
initial R factor and correlation coef®cients
Ê The system has an overall charge of +1
for data in the resolution range 15.0±4.0 A
(MOPAC keyword CHARGE = +1). We
2.2. Dynamic light scattering were 0.508 and 0.357, respectively, when a
performed EF geometry optimizations on all
single 2cbb molecule was inserted and 0.412
12 samples of BCA II were prepared possible protonated forms and atomic
and 0.609, respectively, when two molecules
varying the pH and the precipitant and charges (MOPAC keyword = AM1).
were inserted.
protein concentrations around the condi-
The structural model of BCA II was then
tions described above and were examined as
re®ned within the resolution range 20.0± 3. Results and discussion
possible inducers of protein aggregation in Ê using CNS (BruÈnger et al., 1998) by
2.3 A
mother liquor. Each sample was made up to 3.1. Comparison of BCA II structure with
iterative cycles of simulated annealing and
40 ml volume in a standard ¯uorescence HCA II
individual B-factor re®nement, with manual
cuvette and was kept at 281 K. To monitor
rebuilding using XtalView (McRee, 1992). The re®ned structure of BCA II is a
the crystallization process in these solutions,
The model was initially re®ned with strict complete protein model that includes
a dynamic light-scattering system was used
non-crystallographic symmetry (NCS) several residues that were not observed in
consisting of an ALV-5000 system (ALV,
relating the two molecules in the asymmetric HCA II (PDB code 1ca2; Eriksson et al.,
Langen, Germany) coupled with a He±Ni
unit. The 52 alanines or serines in the initial 1988; PDB code 1moo; Duda et al., 2003).
laser ( = 633 nm). The total scattering
BCA II model were replaced with their The N-terminal residues Ser1 and His2 were
intensities were measured and integrated
correct amino-acid residues when their clearly located in BCA II, whereas they were
®ve times in succession for each sample.
positions were clearly indicated by electron in ¯exible terminal regions and were dif®cult
Each autocorrelation function was analysed
density in 2|Fo| ÿ |Fc| or |Fo| ÿ |Fc| difference to locate in HCA II. The root-mean-square
using the CONTIN program to obtain the
Fourier maps. The ®nal model contained (r.m.s.) difference in C -atom positions
average hydrodynamic radius and its distri-
residues 1±259 for two molecules, with no between the two BCA II monomers was
bution. In the evolution of a supersaturated Ê , showing that the two
chain breaks. All residues except the 258th found to be 0.11 A
solution of BCA II, monomers ®rst assem-
have main-chain dihedral angles that fall molecules in the asymmetric unit are almost
bled to form aggregates, which then led to
within the allowed regions of the Rama- identical. The number of bound water
crystal growth. Only when the aggregate size
chandran diagram as de®ned by the program molecules was different, however, being 277
was larger than approximately 3000±
PROCHECK (Laskowski et al., 1993). The for the A monomer and 300 for the B
4000 nm in diameter did nucleation of
asymmetric unit also contained two Zn2+ monomer. The r.m.s. difference in backbone
crystal growth follow. Ê between HCA II
ions and 573 water molecules (Table 1). R atom positions is 0.58 A
and Rfree were reduced to 0.194 and 0.239, and BCA II, showing that the structure of
2.3. Data collection and processing respectively, in the resolution range 20.0± BCA II is very similar to that of HCA II.
Crystals of BCA II were ¯ash-frozen by 1.95 AÊ . The free R value was calculated with The typical motifs of HCA II are preserved:
transfer into an arti®cial mother liquor 5% (2515) of the total re¯ections. the central fold of BCA II consists of ten

Acta Cryst. (2004). D60, 792±795 Saito et al.  Carbonic anhydrase II 793
short communications
strands of parallel or antiparallel -sheet the presumed `knot' topology in BCA II af®nity can be estimated, showing net gains
forming a core structure, as shown in (Wang et al., 2001, 2002; Alam & Ikai, 2001; of 8.8±82 kJ molÿ1 per imidazole when the
Fig. 1(a). Alam et al., 2002). tetracoordinated aqua complex of Zn2+ ion
BCA II was found to contain three cis- is compared with structures containing one,
prolines, namely Pro29, Pro200 and Pro258, two and three imidazoles. The spontaneous
in 19 proline residues (Kumar et al., 1989), 3.2. Active site deprotonation of the zinc water is more
compared with two cis-prolines in HCA II The active site of BCA II forms a funnel- exothermic than the deprotonation of the
(Eriksson et al., 1988). The extra cis-proline, shaped channel with an outer diameter of imidazole N atoms of His93, His118 and
Pro258, interacts with the side chain of trans- 13 A Ê that extends from the molecular His95 in descending order. We think that
Pro41 on the turn intersection to the surface to the centre, with the zinc ion this stability results from the fact that the
C-terminus. located at the bottom of the channel at a three histidyl residues have their second
The C-terminal region of HCA II is depth of 10 A Ê from the surface. In Fig. 1(b) imidazole N atoms hydrogen bonded to
characterized by a unique `knot' topology, the active site Zn2+ ion is coordinated to other residues: His93 N1 to Gln91 O"1,
meaning that the polypeptide chain crosses three histidyl residues (His93, His95 and His95 N1 to Glu105 O"2 and His118 N"2 to
itself in such a manner that one chain His118). The fourth ligand coordinated to Glu116 O"2.
segment goes `below±above±below' the the Zn2+ ion is a water molecule (W419 in The number of hydrogen bonds formed
other segment. We have con®rmed that the molecule A and W420 in molecule B), the suggests that the carboxylic groups of
`knot' topology is maintained in BCA II. In so-called `zinc water' (Eriksson et al., 1988). Glu105 and Glu116 are negatively charged
BCA II, some new interactions related to No electron density could be found for a as predicted by MOPAC, with a partial
the `knot' topology were found, including water molecule corresponding to the so- charge of ÿ0.53. With this assumption, it
a salt bridge between Arg255 N1 and called `deep water' in the HCA II molecule, is possible to assign the donor±acceptor
Asp40 O1 and a hydrogen bond between however. relationships of several hydrogen bonds
Pro258 and Pro41. We suggest that the The zinc coordination is almost tetra- in the active site. The O 1 proton of
`knot' topology of BCA II is thus tighter and hedral and the zinc coordination distances, Thr197 is hydrogen bonded to Glu105 O"1;
more stable than that of HCA II. The angles and deprotonation energies are Thr197 O 1 can thus only be a hydrogen-
consequence of this `knot' structure is that if shown in Table 2. The three histidines have bond acceptor for the proton of the zinc-
the polypeptide chain is pulled from both very stable conformations, as indicated by bound hydroxyl ion.
ends, the molecule will become tightened their low thermal B factors and formation In addition to the zinc-bound water
rather than loosened and, after the structure energies. After optimization of the structure molecule, nine and ten water molecules are
is completely destabilized, the chain will by molecular mechanics, the ligand-binding found in the active sites of the two inde-
remain as a straight chain with a single knot.
Table 2
This structure provides de®nite support for Coordinate structure of the zinc environment in BCA II.
the interpretation of the experimental
results of mechanical stretching in terms of Bond angle ( )
Deprotonation Bond
Active site Ligand X (kJ molÿ1) Ê)
distance (A XÐZnÐHis93 XÐZnÐHis95 XÐZnÐHis118

Zn W204 OHÿ 1000 2.19 101.3 123.0 109.2

His93 N"2 ÿ432 2.11 Ð 109.5 115.7
His95 N"2 143 2.15 Ð Ð 99.0
His118 N1 ÿ374 2.26 Ð Ð Ð

Figure 1
Crystal structure of BCA II molecule. (a) Ribbon diagram showing the BCA II monomer. The colour-coding of the secondary elements is as follows: red, -strands; blue,
-helices; grey, coil. The position of the zinc ion (lilac) with three histidine ligands (sky blue) and the N- and C-termini are indicated. The core of the monomer consists of ten
-sheet strands. (b) Active-site residues and bound water molecules within 10 A Ê of the Zn2+ ion. Water molecules are shown as small red spheres, with hydrogen bonds shown
by dotted lines. The zinc-bound water ligand is W419 in the A monomer and W420 in the B monomer. The hydrogen-bond network of water molecules differs near Gln91,
with the number of bound water molecules in the active sites being nine and ten, respectively, for the A and B monomers. Figure created using BOBSCRIPT (Esnouf, 1997)
and RASMOL.

794 Saito et al.  Carbonic anhydrase II Acta Cryst. (2004). D60, 792±795
 short communications
pendent BCA II molecules, respectively, as
shown in Fig. 1(b). These water molecules
were predicted by MOPAC to have zero or
slightly positive electric charges and to form
the electric slope required for proton
transfer. Only the oxygen of the zinc-bound
hydroxyl ion is negatively charged, with a
partial charge of ÿ0.50 as predicted by
MOPAC (see Fig. 2). Several potential
proton wires can be envisaged leading from
the zinc-bound H2O. One leads to His63 via
W33 and W162 (molecule A) or W66 and
W168 (molecule B) in a manner similar to
that in HCA II (Eriksson et al., 1988). A
second involves a water molecule bound to
Gln91 (W100 in molecule A, W154 in
molecule B) which is part of a potential
proton wire consisting of W434-W100-
W482-W33-W419. Switching may occur
between the various proton wires during the
proton-transfer process, making use of the
extensive water network leading away from
the zinc ion.

4. Conclusions Figure 2
We report here the structure of BCA II at Schematic drawing of interactions and distances around the active site determined from the crystal structure of
BCA II at pH 7.5. The net charges assigned by theoretical calculations using MOPAC are shown. The side-chain
1.95 AÊ resolution, representing the highest
atom Gln91 O"1 (net charge ÿ0.30) accepts a hydrogen bond from of His93 N1 (net charge ÿ0.21) and
resolution structure of BCA II reported contributes to His93 ligand stabilization. The side-chain atom of Gln91 N"2, with a net charge of ÿ0.40, has a
to date. As expected, the overall re®ned dominant role in the binding of water molecule W482, with net charge of +0.02, in its slightly acidic or cationic
form. This interaction is likely to be more hydrogen-bonding in character than the interaction between W162 (net
structure of this protein is very similar to charge +0.01) and His63 N"2 (net charge ÿ0.14) that has been a biological focus in the case of HCA II. This
that of HCA II and con®rms the presence of ®nding suggests that the dipole donor group of Gln91 may also participate in processes that require relatively
the C-terminal knot structure in BCA II. rapid proton movement or release.
This is important for interpretation of their
mechanical responses. The high level of the Japanese Ministry of Education, Culture, Heck, R. W., Boriack-Sjodin, P. A., Qian, M., Tu,
activity for BCA II is most likely to be a Sports, Science and Technology. C., Christianson, D. W., Laipis, P. J. & Silverman,
D. N. (1996). Biochemistry, 35, 11605±11611.
consequence of another water pathway in
Hewett-Emmett, D. & Tashian, R. E. (1996). Mol.
the active site forming a `secondary' proton Phylogenet. Evol. 5, 50±77.
wire. We have also performed theoretical References Kissinger, C. R., Gehlhaar, D. K. & Fogel, D. B.
calculations on the active-site cavity and Alam, M. T. & Ikai, A. (2001). Appl. Phys. A, 72, (1999) Acta Cryst. D55, 481±491.
carried out automated docking studies of S121±S124. Kumar, V., Kannan, K. K. & Chidambaram, R.
Alam, M. T., Yamada, T. & Ikai, A. (2002). FEBS (1989). Curr. Sci. 58, 344±348.
inhibitor binding. The structure gives insight Lett. 519, 35±40. Kumar, V., Sankaran, K. & Kannan, K. K. (1986).
into the existence of a secondary proton BruÈnger, A. T., Adams, P. D., Clore, G. M., J. Mol. Biol. 190, 129±131.
wire (W482, W100 and W434). DeLano, W. L., Gross, P., Grosse-Kunstleve, Laskowski, R. A., MacArthur, M. W., Moss, D. S.
R. W., Jiang, J.-S., Kuszewski, J., Nilges, N., & Thornton, J. M. (1993). J. Appl. Cryst. 26,
Pannu, N. S., Read, R. J., Rice, L. M., Simonson, 283±291.
The present research was undertaken with T. & Warren, G. L. (1998). Acta Cryst. D54, 905± McCoy, L. F. Jr & Wong, K. P. (1981). Biochem-
the approval of the Photon Factory Advisory 921. istry, 20, 3062±3067.
Committee, Japan. The authors wish to Carlsson, U., Lindskog, S., Andersson, U., Lind- McRee, D. E. (1992). J. Mol. Graph. 10, 44±47.
express their sincere thanks to the staff at qvist, O. & Olsson, G. (1973). J. Mol. Biol. 80, Parkkila, S. (2000). The Carbonic Anhydrases:
373±375. New Horizons, edited by W. R. Chegwidden,
the Photon Factory for their help with the Cleland, J. L. & Wang, D. I. (1990). Biochemistry, N. D. Carter & Y. H. Edwards, pp. 79±94. Berlin:
data collection. This work was partly 29, 11072±11078. Birkhauser Verlag.
supported by grants-in-aid to AI from the Collaborative Computational Project, Number 4 Stainer, H., Jonsson, B. H. & Lindskog, S. (1975).
Japan Society for the Promotion of Science (1994). Acta Cryst. D50, 760±763. Eur. J. Biochem. 59, 253±259.
Duda, D., Govindasamy, L., Agbandje-McKenna, Stams, T., Chen, Y., Boriack-Sjodin, P. A., Hurt,
(Research for the Future Program No. J. D., Liao, J., May, J. A., Dean, T., Laipis, P.,
M., Tu, C., Silverman, D. N. & McKenna, R.
99R16701) and the Japanese Ministry of (2003). Acta Cryst. D59, 93±104. Silverman, D. N. & Christianson, D. W. (1998)
Education, Culture, Sports, Science and Eriksson, A. E., Jones, T. A. & Liljas, A. (1988). Protein Sci. 7, 556±563.
Technology [Scienti®c Research on Priority Proteins, 4, 274±282. Wang, T., Arakawa, H. & Ikai, A. (2001).
Areas (B) No. 11226202] and by a grant Eriksson, A. E. & Liljas, A. (1993). Proteins, 16, Biochem. Biophys. Res. Commun. 285, 9±14.
29±42. Wang, T., Arakawa, H. & Ikai, A. (2002).
from the 21st Century COE Program and Esnouf, R. M. (1997). J. Mol. Graph. 15, 132±134. Ultramicroscopy, 91, 253±259.
Grant-in-Aid to TS for Scienti®c Research Hammarstrom, P., Kalman, B., Jonsson, K. P. & Wong, K. P. & Tanford, C. (1973). J. Biol. Chem.
on Priority Areas (A) (No. 13033011) from Carlsson, U. (1997). FEBS Lett. 420, 63±68. 248, 8518±8523.

Acta Cryst. (2004). D60, 792±795 Saito et al.  Carbonic anhydrase II 795