Selective recognition of carbonic anhydrase using transition metal complexes

Article abstract of DOI:10.1039/a909001k

A metal complex was designed to bind to the protein carbonic anhydrase strongly and selectively in aqueous medium.

Full text of DOI:10.1039/a909001k

Selective recognition of carbonic anhydrase using transition metal complexes†
Bidhan C. Roy, Md. Abul Fazal, Shuguang Sun and Sanku Mallik*
Department of Chemistry, North Dakota State University, Fargo, ND 58105, USA.
E-mail: sanku_mallik@ndsu.nodak.edu
Received (in Bloomington, IN, USA) 15th November 1999, Accepted 17th February 2000
Published on the Web 17th March 2000
A metal complex was designed to bind to the protein
carbonic anhydrase strongly and selectively in aqueous
medium.
Strong and selective binding to a pre-determined protein by
synthetic receptors is of paramount importance for drug
design,1 protein purification2 and protein sensing.3 The recogni-
tion of active sites of enzymes leading to very effective
inhibitors has been developed to a high level of sophisti-
cation. 4 Recognition of protein surface patterns by polypeptides
has been used as a model system for studying protein–protein
Fig. 1 Structures of the tris–Cu2+ complexes 2, 3 and the control 1 used for
interactions5 or to design enzyme inhibitors.6 These studies
recognition of carbonic anhydrase.
employed multiple hydrogen bonding or ionic interactions as
the basis of recognition. Herein, we report our results on
(CA) but their distributions are different. If the recognition is
selective recognition of a protein, carbonic anhydrase (CA,
based on the contents of surface exposed histidine residues (e.g.
bovine erythrocyte) based on its surface histidine pattern,
IMAC), then CEA is expected to bind more strongly compared
employing Cu2+–histidine interactions in water (25 mM HEPES
to CA. Since the studies reported here rely on the histidine
buffer, pH 7.0, 25 °C). To our knowledge, this is the first report
pattern, CEA was chosen as a control protein.
Complexes 2 and 3 were synthesized from the corresponding
bromides and diethyl iminodicetate, using K2CO3 as the base in
of protein surface recognition using the surface histidine pattern
of the protein. Metal–histidine interactions have been used for
protein purification using immobilized metal affinity chroma-
acetonitrile solvent. The ester groups were hydrolyzed by LiOH
tography (IMAC).2 However, in IMAC, the proteins are
in methanol–water at 25 °C.12 The complexes were found to be
distinguished based on their surface histidine contents.
stable in aqueous buffer solution (25 mM HEPES buffer,
Metal–ligand interactions have several advantages in recog-
pH 7.0), at room temperature, in air for more than a month. It is
nition compared to hydrogen bonding, ion-pair or other weak
reported in the literature that the IDA–Cu2+ complex binds to
interactions. Metal–ligand interactions are stronger especially
proteins (pH 7.0) primarily through the surface exposed
in aqueous media.7 A variety of transition metal ions are
histidine residues of proteins. Primary amino groups and
available and are used by nature in biological systems for
carboxylate residues on the protein surface only play weak,
molecular recognition and catalysis.8 This allows fine tuning of
secondary roles.13
each of the interactions and opens the possibility of catalysis,
following recognition. The spectroscopic properties of the metal
Binding studies were conducted in water (25 mM HEPES
buffer, pH 7.0; [protein] = 100 mM, [1] = 0–1.2 mM; [2] or [3]
ions can be utilized to monitor the binding process and to get
= 0–500 mM, 25.0 °C) and were followed by isothermal
titration microcalorimetry14 (ITC-4200, Calorimetry Scientific
Corporation, Provo, UT). The software provided by the
instrument manufacturer (Bindworks 3.0) was used for data and
error analysis. The raw data were corrected for the heat of
dilution of the appropriate Cu2+-complex before the curve
fitting process (data for titrations are available as electronic
supplementary information†).
structural information about the resultant complex.9
CA has five histidines (3, 10, 15, 17, 64) exposed on the
surface (determined using the modeling software insight-II and
discover, version 98.0, Molecular Simulations Inc., Burlington,
MA).10 The distance amongst the histidines 3, 10, 17 (or 15) are
ca. 16 Å. This pattern was mapped into the pattern of Cu2+ ions
by the tris–Cu2+ complex 2. Another shorter tris–Cu2+ complex
3 (distance amongst the Cu2+ ions: ca. 12 Å) was synthesized
for comparative studies. A mono-Cu2+ complex 1 served as the
control for the recognition studies. Structures of the metal
complexes 1, 2 and 3 are shown in Fig. 1. These complexes were
modeled using the software Spartan (version 5.0.3, Wavefunc-
tion Inc., Irvine) employing the Merck molecular mechanics
force field. After energy minization, the resultant structures
were subjected to systematic conformational searches to
identify the lowest energy structures.
Two other proteins with different surface histidine patterns
were used as controls for these studies, chicken egg albumin
(CEA) and chicken egg lysozyme. CEA has six histidines on the
surface (22, 23, 329, 332, 363, 371); lysozyme (chicken egg)
has only one histidine (15) exposed to surface.10 All of these
three proteins are known to interact with transition metal ions
through the surface exposed histidines.11 CEA has a higher
number of surface exposed histidines than the target protein
Results for the binding experiments (stoichiometry, binding
constant and enthalpy) are shown in Table 1. The control 1
showed low affinity (K < 4000 M21) for the proteins tested.
With the complex 2, CA was found to bind tightly (K ≈
300 000 M21). CEA showed weak binding with complex 2 (K
< 1000 M21) under the experimental conditions. Lysozyme
was found to precipitate when [2] > 25 mM. With the shorter
complex 3, the affinity for CA was found to decrease (K ≈
75 000 M21) compared to that of 2. Both lysozyme and CEA
bound weakly to complex 3 (no precipitation). Complex 2
bound CA more strongly compared to the control 1 (90+1) or the
shorter tris–Cu2+ complex 3 (4+1). This slight selectivity of CA
for 2 (over 3) may be due to greater strain in binding for 3 (DH
= 215.1 kcal mol21 for 2; 212.2 kcal mol21 for 3) to the
protein. Complex 2 also showed very good selectivity for CA
when compared to CEA (300+1).
In order to demonstrate selective binding of 2 to CA, a
mixture of the protein and the control 1 ([CA] = 100 mM; [1] =
100 mM) was titrated with 2 ([2] = 0–500 mM). No change in
the binding constant of 2 with CA was observed. When a
† Electronic supplementary information (ESI) available: scheme for the
synthesis of 2, 3 and ITC titration data (raw and processed) for the results
reported in Table 1. See http://www.rsc.org/suppdata/cc/a9/a909001k/
DOI: 10.1039/a909001k
Chem. Commun., 2000, 547–548
This journal is © The Royal Society of Chemistry 2000
547

Table 1 Binding parameters for the receptors 2, 3 and control 1 with carbonic anhydrase, chicken egg albumin and lysozyme
Protein
Stoichiometry (n)
K/M21
2DH/kcal mol21
Lysozyme
1: 1.2 ± 0.3
2: nd
3: —
(1.6 ± 0.2) 3 103
Precipitation
< 103
7.0 ± 0.5
—
—
Carbonic anhydrase
1: 0.90 ± 0.03
2: 0.94 ± 0.006
2: 0.81 ± 0.02 (pH 6.0)
2: 0.82 ± 0.01 (pH 8.0)
3: 1.3 ± 0.01
3: 1.3 ± 0.04 (pH 6.0)
3: 1.3 ± 0.02 (pH 8.0)
1: —
(3.5 ± 0.7) 3 103
(299 ± 30) 3 103
(199 ± 35) 3 103 (pH 6.0)
(97 ± 16) 3 103 (pH 8.0)
(75 ± 6) 3 103
(27.5 ± 4) 3 103 (pH 6.0)
(45 ± 5) 3 103 (pH 8.0)
< 103
39.2 ± 2.0
15.1 ± 0.3
10.8 ± 0.5 (pH 6.0)
10.2 ± 0.3 (pH 8.0)
12.7 ± 0.2
12.2 ± 0.7 (pH 6.0)
20.6 ± 0.6 (pH 8.0)
—
Albumin
2: —
3: 1.6 ± 0.07
< 103
—
(16 ± 2.5) 3 103
20.9 ± 1.5
2 S. Vunnum, V. Natarajan and S. Cramer, J. Chromatogr. A, 1998, 818,
31; K. M. Muller, K. M. Arndt, K. Bauer and A. Pluckthun, Anal.
Biochem., 1998, 259, 54; J. Mahiou, J. P. Abastado, L. Cabanie and F.
Godeau, Biochem. J., 1998, 330, 1051.
3 F. Loscher, T. Ruclestuhl and S. Seeger, Adv. Mater., 1998, 10, 1005; S.
Okada, S. Peng, W. Spevak and D. Charych, Acc. Chem, Res., 1998, 31,
229; I. Vikham and W. M. Albers, Langmuir, 1998, 14, 3865; D.
Elbaum, S. K. Nair, M. W. Patchan, R. B. Thompson and D. W.
Christainson, J. Am. Chem. Soc., 1996, 118, 8381.
4 A. C. Bishop, C. Kung, K. Shah, L. Witucki, K. M. Shokat and Y. Liu,
J. Am. Chem, Soc., 1999, 121, 627; G. F. Short, M. Lodder, A. L.
Laikhter, T. Arslan and S. M. Hecht, J. Am. Chem. Soc., 1999, 121, 478;
M. Beuck, Angew. Chem., Int. Ed., 1999, 38, 631; R. Zutshi, M.
Brickner and J. Chmielewski, Curr. Opin. Chem. Biol., 1998, 2, 62.
5 S. Hirota, M. Endo, K. Hayamizu, T. Tsukazaki, T. Takake, T.
Kohzuma and O. Yamaguchi, J. Am. Chem. Soc., 1999, 121, 849.
6 H. S. Park, Q. Lin and A. D. Hamilton, J. Am. Chem. Soc., 1999, 121,
8; Y. Hamuro, C. Calama, H. S. Park and A. D. Hamilton, Angew.
Chem., Int. Ed. Engl., 1997, 36, 2680.
7 P. A. Frey and W. W. Cleland, Bioorg. Chem., 1998, 26, 175.
8 S. J. Lippard and J. M. Berg, Principles of Bioorganic Chemistry,
University Science Books, Mill Valley, CA, 1994, pp. 349–376; H.
Dugas, BioOrganic Chemistry: A Chemical Approach to Enzyme
Action, Springer, New York, NY, 1996, pp. 388–460.
mixture of 2 and CA ([CA] = 100 mM; [2] = 100 mM) was
titrated with control 1 ([1] = 0–1 mM), no binding was
observed. Similar results were obtained with the complex 3.
When a mixture of the three tested proteins (100 mM each) was
titrated with complex 2, the binding constant remained
unchanged (280 000 ± 30 000). If carbonic anhydrase was not
included in the mixture (i.e. CEA and lysozyme, 100 mM each),
very weak affinity ( < 1000) was detected. The binding
selectivity reported here (for complex 2) is distinctly different
compared to that observed with a random distribution of
copper(II) ions (on chelating Sepharose fast flow with iminodia-
cetate–Cu2+, pH 7.0, 22 °C, either in equilibrium binding
experiments or in chromatograhy).15
To demonstrate the role of Cu2+–histidine interactions in the
recognition process, we have studied the binding between CA
and complex 2 by EPR spectroscopy in the solution phase
(9.4 GHz, [CA] = 1.2 mM; [2] = 600 mM, 25.0 °C). Upon
addition of the protein, the g|| value of Cu2+ ions of 2 decreased
from 2.290 to 2.261. These values match well with the reported
g|| values of the iminodiacetate–Cu2+ complex, free and bound
to myoglobin through the histidine residues (2.288 and 2.264,
respectively).16 Also ITC titrations failed to detect any binding
between the metal-free ligands (for 1, 2 and 3) and the
proteins.
9 M. J. Kendrick, M. T. May, M. J. Plishka and K. D. Robinson, Metals
in Biological Systems, Ellis Horwood, New York, NY, 1992, pp. 17–
56.
The binding of CA with the metal complexes 2 and 3 were
followed by UV–VIS spectrometry. Upon addition of CA to a
solution 2 (or 3, [2 or 3] = 0.5 mM; [CA] = 0.8 mM, 25 mM
HEPES buffer, pH 7.0, 25 °C, 10 3 100 mL additions), the
absorption maxima progressively shifted from 727 to 660 nm.
This indicated the coordination of one imidazole group (of
histidine residues) per copper(II) ion of the complexes.17
Analyses18 of the resultant titration curves corroborated the ITC
results. Circular dichroism studies (80 mM protein, 0–500 mM of
2 or 3, 25 mM phosphate buffer, pH 7.0, 23.0 °C) indicated that
the proteins were not unfolding in presence of the control 1 or
receptors 2 or 3.
10 The pdb files were obtained from the pdb server. The URL on the
internet is: http://www.rcsb.org/pdb/. The files used for modeling are:
1can.pdb (for carbonic anhydrase), 1ova.pdb (for albumin) and 1azf.pdb
(for lysozyme).
11 P. J. Sadler and J. H. Viles, Inorg. Chem., 1996, 35, 4490; A. Singh, M.
Markowitz, L.-I. Tsao and J. Deschapms, ACS Symp. Ser., 1993, 556,
252.
12 The synthetic scheme for the synthesis of 2 and 3 is available as
electronic supplementary material.† The tribromides were prepared
following reported procedures. Y. Kim and R. Beekerbauser, Macro-
molecules, 1994, 24, 1968; W. P. Cochrane, P. L. Pauson and T. S.
Stevens, J. Chem. Soc. C, 1968, 630. All new compounds gave
satisfactory 1H and 13C NMR spectra. The Cu2+ complexes were
characterized by elemental analysis. For 1: Anal. Calc. for
C11H11CuNO4: C, 46.39; H, 3.89; N, 4.92. Found: C, 46.06; H, 3.58; N,
4.62. For 2: Anal. Calc. for C39H33Cu3N3O12·3H2O: C, 49.60; H, 3.74;
N, 4.45. Found: C, 49.68; H, 3.90; N, 4.52. For 3: Anal. Calc. for
C21H21Cu3N3O12·3H2O: C, 33.54; H, 3.62; N, 5.59. Found: C, 33.53; H,
3.59; N, 5.54%.
13 W. Jiang, B. Graham, L. Spiccia and M. T. W. Hearn, Anal. Biochem.,
1998, 255, 47.
14 M. Doyle, Curr. Opin. Biotech., 1997, 8, 31; I. Wadso, Chem. Soc. Rev.,
1997, 79.
15 T. W. Hutchens, T.-P. Yip and J. Porath, Anal. Biochem., 1988, 170,
168.
16 D. R. Schnek, D. W. Pack, D. Y. Sasaki and F. H. Arnold, Langmuir,
1994, 10, 2382.
Thus these studies demonstrate that the tris–Cu2+complex 2
binds carbonic anhydrase strongly and with good selectivity
compared to two other proteins with different histidine patterns
on the surface. It should be noted that the reported method of
protein recognition is applicable to proteins of known structures
possessing histidine residues on the surface. With rapidly
increasing number of solved protein structures, the method has
wide applicability.
This work was supported by NSF-CAREER award (CHE-
9896083) and an NIGMS - AREA grant (1R15 59594-01, NIH).
The microcalorimeter was purchased through an NSF-EPSCoR
award to North Dakota. We thank Professor Kenton Rodgers for
help with the EPR experiments.
17 P. K. Dhal and F. H. Arnold, Macromolecules, 1992, 25, 7051.
18 K. A. Connors, Binding Constants, Wiley, New York, 1987.
Notes and references
1 H.-J. Bohm and G. Klebe, Angew. Chem., Int. Ed. Engl., 1996, 35, 2588;
R. E. Baine and S. L. Bender, Chem. Rev., 1997, 97, 1359.
Communication a909001k
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Chem. Commun., 2000, 547–548