Mavis Agbandje-McKenna, Robert McKenna* Department of Biochemistry and Molecular Biology and Department of

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1 Ultra-High Resolution X-Ray Diffraction from Crystals of the Kinetic Mutant of Human Carbonic Anhydrase II, His 64 Ala, and its Complexes with Proton Acceptor/Donors. David Duda, Chingkuang Tu, David. Silverman, A. Joseph Kalb (Gilboa) Mavis Agbandje-McKenna, Robert McKenna* Department of Biochemistry and Molecular Biology and Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL Permanent Address: Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel *Author to whom correspondence should be addressed Abstract Crystals of human carbonic anhydrase II with a specific point mutation, His 64 to Ala, have been grown in a solution of ammonium sulfate in the presence of mercury chloride. The crystals appear in approximately two weeks and belong to the monoclinic space group P2 1, with unit cell parameters of a = 42.2 Å, b = 41.4 Å, c = 71.9 Å, β = o and one carbonic anhydrase molecule in the asymmetric unit. The crystals diffract X-rays beyond 1.0 Å resolution. These crystals, soaked with exogenous proton acceptor/donors, will be used in X-ray and neutron diffraction studies to map the fine water structure proton wire in the active site of carbonic anhydrase and to assign the intra- and intermolecular proton transfer pathway(s) from the zinc-bound water out to the bulk solvent. Introduction Carbonic anhydrase II (CA II), one of the most efficient of the isozymes in the α class of carbonic anhydrases [1], catalyzes the hydration of CO 2 in two stages [2,3]. The first is the conversion of CO 2 into bicarbonate by reaction with the zinc-bound hydroxide; the dissociation of bicarbonate leaves a water molecule bound to the zinc (Eq. 1). [H 2 O] CO 2 + EZnOH EZnHCO 3 EZnH 2 O + HCO 3 (1) EZnH 2 O + B EZnOH - + BH + (2)

2 The second step is the transfer of a proton to solution to regenerate the zinc-bound hydroxide (Eq. 2); here B designates a proton acceptor, either an exogenous proton acceptor from solution or a residue of the enzyme itself, although ultimately the proton must be transported to bulk solvent. A considerable number of studies have shown that His 64 in human CA II (HCA II) functions as a proton shuttle in this manner [4-6]. A site-specific mutant of HCA II in which His64 is replaced with Ala (H64A HCA II), has been shown to decrease the rate of the proton transfer step (Eq. 2) by more than 10-fold compared with wild type HCA II [7]. This loss of catalytic activity of H64A HCA II can be rescued by 4-methylimidazole (4-MI), an exogenous proton acceptor/donor (Fig. 1), in a saturable process with a maximum activity of 40% of wild-type HCA II [7]. The crystal structure of the rescued complex at 1.6 Å resolution shows 4-MI bound in the active-site cavity of H64A HCA II, through π stacking interactions with residue Trp5 and H-bonding interactions with water molecules [7]. Crystals of H64A HCA II that diffract X-rays beyond 1 Å resolution have been grown to study the fine structural changes in the activite site associated with the loss and rescue by exogenous acceptor/proton donors (Fig. 1) of the proton transfer step (Eq. 2). Because of the exceptionally high quality of these crystals, the structures obtained from X-ray and neutron diffraction studies can be used to elucidate the detailed structure of intervening water channel(s) proton wire(s) by which a proton can be transferred from the zinc-bound hydroxide to the bulk solvent (Eq. 2). Preliminary experiments conducted at the Laue diffraction (LADI) experimental station at the Institut Laue-Langevin (ILL) confirms the feasibility of low temperature (12K) neutron crystallography of these crystals. 4-Methylimidazole (4-MI) Pyridine (PYR) 3-Methylpyridine (3-MP) Figure 1. Chemical structure of exogenous acceptor/proton donors of proton shuttling in Human Carbonic Anhydrase Mutant His64Ala. Methods Expression and purification: H64A HCA II was prepared and expressed in E. coli as described in previous publications [5,8] and purified by affinity chromatography [9]. The mutation was confirmed by sequencing the DA of the entire coding region for H64A HCA II in the expression vector. Crystallization and X-ray diffraction data collection: Crystals of H64A HCA II (Fig. 2) were produced by the hanging drop method [10], using a 10 mg/ml enzyme solution in 50 mm Tris HCl, ph = 7.8, containing 1

3 mm HgCl 2. The drops were obtained by mixing 5 µl of the enzyme solution with 5 µl of precipitant solution, consisting of M (H 4 ) 2 SO 4 in 50 mm Tris HCl, ph = 7.8, and 1 mm HgCl 2. The drop was equilibrated by vapor diffusion against 1 ml of precipitant solution at 4 0 C [11]. H64A CA II complexed with the exogenous proton acceptor/donors (Fig. 1) were obtained by soaking crystals in 50mM Tris HCl, ph = 7.8, containing 3.0 M (H 4 ) 2 SO 4 and 0.5 M of the exogenous acceptor/donor. The crystals were left to soak at 4 o C for 1 week prior to data collection and were prepared for cryogenic data collection by a quick immersion in a solution of 3.0 M (H 4 ) 2 SO 4 and 30% glycerol in 50 mm Tris HCl, ph = 7.8, prior to flash freezing in liquid nitrogen. Figure 2. Crystal of Human Carbonic Anhydrase II Mutant His64Ala. Preliminary X-ray diffraction data sets have been collected at 100K for crystals of H64A HCA II alone and complexed with exogenous proton acceptor/donors 4-MI, 3-methlypyridine (3-MP), and pyridine (PYR) (Fig.1) at the F1 station at CHESS (λ = Å). Frames were collected with an exposure time of 60 s/frame, using a 0.3 mm collimator, crystal to detector distances of mm, and 1.0 o oscillation angles (Fig. 3). The frames were recorded on an ADSC Quantum-4 CCD detector. The unit cell parameters (Table 1) were determined using the DEZO software and scaled with SCALEPACK [12]. Results and discussion The diffraction images (Fig. 3) of H64A HCA II are consistent with the monoclinic P2 1 space group with unit cell parameters a = 42.7 Å, b = 41.7 Å, c = 73.0 Å, and β = o that has been reported previously for wild

4 type HCA II [13]. Wild type crystals were reported to diffract to a maximal resolution of 1.54 Å [13], whereas the H64A HCA II diffraction images obtained at the Cornell High Energy Synchrotron Source (CHESS) show diffraction to 1.2 Å resolution consistently (Fig. 3) Four preliminary data sets have been collected for this study, H64A HCA II alone and soaked with the exogenous proton acceptor/donors 4-MI, 3-MP, and PYR (Fig. 1). A summary of the current data sets is given in table 1. The diffraction quality of the crystals of the H64A HCA II complexed with 4-MI (1.05 Å resolution) and 3-MP (1.10 Å resolution) is better than that of H64A HCA II (1.20 Å resolution), whereas for the PYR soaked crystals (1.45 Å resolution) the diffraction quality has been reduced. The 4-MI soaked H64A HCA II crystals diffract X-rays under to at least 0.97 Å in the corners of the detector. This resolution limit was imposed by the mechanical geometry of the optical bench since the minimum detector-to-crystal distance is 80 mm. Improved instrument desgn should allow data collection of complete data to this resolution or higher. It should also be noted that all the soaked data sets are isomorphous with the H64A HCA II crystals, with the exception of those soaked with 3-MP. These crystals showed a color transition from colorless to dark blue and cell dimension changes, the most noticeable being a doubling of the a axis (Table 1), suggesting that 3-MP may have caused conformational changes in the H64A HCA II structure, but did not degrade the crystal quality. Table 1. X-ray diffraction data set statistics for Human Carbonic Anhydrase II Mutant His64Ala in the absence and presence of the exogenous proton acceptor/donors 4-methylimidazole (4-MI), 3-methylpyridine (3- MP), and pyridine (PYR). Crystal Cell dimensions (Å, o ) # Of unique % Rsym* Resolution a b c β reflections Completeness Å H64A HCA II , H64A HCA II , MI H64A HCA II , MP H64A HCA II , PYR *Rsym is defined as, Rsym = SUM ( ABS(I - <I>)) / SUM (I), where I is the intensity of an individual reflection and <I> is the average intensity for this reflection; the summation is over all intensities.

5 Figure 3. X-ray diffraction pattern of Human Carbonic Anhydrase II Mutant His64Ala extending to 1.0 Å resolution. Data were collected on the F1 beamline at Cornell High Energy Synchotron Source (CHESS). These structures will provide the first insights into the binding of enhancers to H64A HCA II at ultra-high resolution. Acknowledgments The authors thank the staff at the Cornell High Energy Synchotron Source (CHESS) for their help and support at the F1 station during X-ray data collection. This work was supported by grants from the ational Institutes of Health GM25154 (DS) and the University of Florida, College of Medicine start-up funds (RM). References [1] Hewett-Emmett, D., and Tashian, R. E. (1996) Molecular Phylogen. and Evol. 5, [2] Lindskog, S. (1997) Pharmacol. Ther. 74, [3] Christianson, D.W. and Fierke, C. A. (1996) Accounts of Chem. Res. 29, [4] Steiner, H., Jonsson, B.-H., and Lindskog, S. (1975) Eur. J. Biochem. 59, [5] Tu, C. K., Silverman, D.., Forsman, C., Jonsson, B. H., and Lindskog, S. (1989) Biochemistry, 28, [6] Silverman, D.., and Lindskog S. (1988) Accts. Chem. Res. 21, [7] Duda, D., Tu, C., Qian, M., Laipis, P., Agbandje-McKenna, M., Silverman, D.., and McKenna, R. (2000) Biochemistry, submitted. [8] Tanhauser, S. M., Jewell, D. A., Tu, C. K., Silverman, D.., and Laipis, P. J. (1992) Gene, 117, [9] Khalifah, R. G., Strader, D. J., Bryant, S. H., and Gibson, S. M. (1977) Biolchemistry 16, [10] McPherson, A. (1985) Methods Enzymol., 114, [11] Tilander,B, Strandberg, B. and Fridborg,K. (1965) J. Mol. Biol. 12, [12] Otwinowski, Z. and Minor, W. (1997) Methods Enzymol., 276, [13] Hakansson, K., Carlsson, M., Svensson, L. A., Liljas, A. (1992) J. Mol. Biol. 227,