Zinc binding drives the folding and association of the homo-trimeric {gamma}-carbonic anhydrase from Methanosarcina thermophila

B.Robert Simler1, Brandon L. Doyle2,3 and C.Robert Matthews1,4

1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605 and 2Department of Chemistry and Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park, PA 16802, USA 3Present address: Eli Lilly and Company, Indianapolis, IN 46285, USA

4 To whom correspondence should be addressed. e-mail: c.robert.matthews{at}umassmed.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric enzyme, the left-handed ß-helical subunits of which bind three catalytic Zn2+ ions at symmetry-related subunit interfaces. The observation of activity for holo-Cam at nanomolar concentrations provides a minimal estimated free energy of folding and assembly of the trimeric holo-complex of ~70 kcal (mol trimer)–1 at standard state. Although the direct measurement of stability by chemical denaturation was precluded by the irreversible unfolding of the holo-enzyme, the reversible unfolding of metal-free apo-Cam is well described by a three-state model involving the folded apo-trimer, the folded monomer and the unfolded monomer. The monomer is estimated to have a stability of 4.0 ± 0.3 kcal (mol monomer)–1. The association to form apo-trimer contributes 13.2 ± 0.4 kcal (mol trimer)–1, a value confirmed by analytical ultracentrifugation measurements. Far- and near-UV circular dichroism data show a progressive increase in secondary and tertiary structure as the apo-monomer is converted to holo-trimer. The literature value for the free energy of binding of one Zn2+ ion to a canonical active site, 16.4 kcal mol–1, is consistent with the presumption that the >45 kcal (mol trimer)–1 generated by the binding of three ions represents the major contribution to the stability of the holo-trimeric Cam.

Keywords: ß-helix/carbonic anhydrase/trimeric protein assembly/trimeric protein folding/zinc binding


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carbonic anhydrase (CA), which catalyzes the hydration of carbon dioxide to a bicarbonate ion and a proton, is ubiquitous in nature. Although the mechanism of catalysis by a hydroxylated Zn2+ ion is common among all forms of life (Lindskog, 1997Go; Northrop and Simpson, 1998Go; Christianson and Cox, 1999Go), at least three different structural classes of enzymes, {alpha}, ß and {gamma}, have evolved to achieve the well-known dramatic enhancement in rate over the spontaneous process. Mammals can possess multiple CA genes of the same class and prokaryotes can possess genes from two or three classes. These findings suggest that although the interconversion of carbon dioxide and bicarbonate may be its primary function, CA is likely to have ancillary physiological roles, such as acetate transport across the cell membrane (Smith and Ferry, 2000Go).

The {alpha}, ß and {gamma} classes each exhibit sequence and structural diversity (Hewett-Emmett and Tashian, 1996Go; Liljas and Laurberg, 2000Go). Additionally, a {delta} class of CA has recently been proposed although no structural data on this class are currently available (Tripp et al., 2001Go). CAs of the {alpha} class are typically monomeric enzymes whose predominant structural characteristic is a core comprised of a 10-stranded, primarily antiparallel ß-sheet (Lindskog, 1997Go). Three histidines and a water molecule coordinate the catalytic zinc ion. The core of the ß class of CA is characterized by a ß-sheet comprised of four parallel strands with {alpha}-helices forming crossover connections; the oligomerization state can vary from 2 to 8 (Kimber and Pai, 2000Go). By contrast with the {alpha} class, the ß class CAs coordinate the zinc ion with two cysteines and one histidine, and also a fourth ligand which varies between members of the class (Bracey et al., 1994Go; Kimber and Pai, 2000Go; Mitsuhashi et al., 2000Go). The CA isolated from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric, 70 kDa protein whose individual subunits contain 213 residues and which is the only identified member of the {gamma} class of enzymes. Sequence homologies with CA candidates in several bacterial and archaeal species imply that the enzyme from M.thermophila is likely to be a prototype for other members of the {gamma} class (Smith et al., 1999Go). Phylogenetic studies place the date of evolution of the {gamma} class near the origin of life (Smith et al., 1999Go).

The most striking feature of Cam is the left-handed parallel ß-helical structure adopted by the monomers (Figure 1A). Each face of the helix is comprised of a sheet of 2–3 residue parallel ß-strands connected to the subsequent face by a 120° turn to form an equilateral prism (Kisker et al., 1996Go). A pair of {alpha}-helices, which cap and border the ß-helices, have been postulated to prevent aggregation during the folding and assembly of these proteins (Richardson and Richardson, 2002Go). Each trimeric complex contains three symmetrically arranged catalytic sites at the interface between the monomers. Zinc ions are bound in these catalytic sites by three histidine residues donated by two different monomer subunits: His81 and His122 from one monomer and His117 from the neighboring monomer (Figure 1B). Although the platform is unique in the {gamma} class, the utilization of three histidines to bind the zinc ion is shared with all the {alpha} class CA enzymes.



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Fig. 1. A ribbon diagram of M.thermophila carbonic anhydrase, based on the X-ray crystal structure (Kisker et al., 1996Go). (A) An end-on view of the holo-trimeric complex, highlighting the equilateral prism shape of the ß-helices, the ligating histidines (ball-and-stick) and the zinc ions (gray balls) binding in the active site. (B) The three-dimensional structure of the isolated monomer with the active-site histidines portrayed as ball-and-stick figures. His81 and His122 coordinate the zinc ion with the displayed monomer and His117 coordinates from a neighboring monomer.

 
CAs normally serve to remove excess CO2 from the cytoplasm. However, Cam from M.thermophila has been hypothesized to be translocated across the cell membrane and play a role in the growth on acetate (Alber and Ferry, 1994Go). Consistent with this conjecture is the observation that the sequence of the cloned gene encodes an additional 34 amino acids on the N-terminus which are not present in the purified enzyme. These 34 residues contain two hydrophilic regions surrounding a hydrophobic stretch and the first hydrophobic stretch contains a positively charged arginine. These characteristics are shared by signal sequences in both bacterial and eukaryotic secretory proteins (von Heijne, 1985Go, 1986Go), leading to the possibility that Cam may function on the exterior of the cell. The transport of the protein from the cytoplasm where it is synthesized to the external surface of the cell raises the issue of how the folding, assembly and addition of the catalytic zinc ions are coordinated with transport. Although chaperones and proteins involved in the export apparatus undoubtedly play essential roles in expediting this process, the underlying thermodynamic properties of this homo-trimeric enzyme ultimately dictate its behavior.

Chemical denaturation analysis of Cam revealed that zinc binding is the principal determinant of stability. Presumably, the marginally stable monomeric polypeptides are transported to their final destination before assembling and acquiring the catalytic zinc ions required for their active, stable homo-trimeric conformation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To eliminate potential problems with the oxidation of the single cysteine residue at position 148, the cysteine was mutated to serine using a QuikChange kit from Stratagene (La Jolla, CA). Following DpnI digestion, the PCR product was transformed into DH5{alpha} Escherichia coli cells (Stratagene) for plasmid purification. Incorporation of the mutant codon was confirmed by fluorescence sequencing (Penn State Nucleic Acid Facility).

Protein production

The plasmid encoding C148S Cam was transformed into E.coli BL21(DE3) cells by standard procedures and was expressed without its signal sequence. The N-terminal methionine was cleaved during protein synthesis. Cells containing this plasmid were grown in a 20 l fermenter at 37°C in an LB medium containing 100 µg/ml ampicillin. At an optical density of 8.0 at 600 nm, Cam production was induced by the addition of isopropyl thiogalactoside to a concentration of 1.0 mM. The cells were harvested after 3 h using a Sharples continuous centrifuge and were resuspended in 50 mM HEPES, pH 7.0. The resuspended cells were stored at –70°C until further use.

Protein purification

Cells were lysed by sonication and centrifuged for 30 min at 20 000 g. The supernatant was loaded on to a Source 15Q column (Amersham Pharmacia, Piscataway, NJ) equilibrated with 50 mM HEPES, pH 7.0, at room temperature. After washing with equilibration buffer, a linear 0–1.0 M NaCl gradient was applied to the column. Fractions containing Cam were identified by SDS–PAGE and pooled. Finely powdered (NH4)2SO4 was slowly added to the pooled fractions at 4°C to a concentration of 1.5 M. The pooled fractions were loaded on to a high-performance phenyl-Sepharose column (Amersham Pharmacia) equilibrated with 50 mM HEPES, 1.5 M (NH4)2SO4, pH 7.0. After washing with equilibration buffer, a linear 1.5–0 M (NH4)2SO4 gradient was developed over the column. Fractions containing Cam were pooled and concentrated using an Amicon stirred cell with a YM-10 filter at 4°C. Residual (NH4)2SO4 was removed by dialysis using 10 mM phosphate, pH 7.0. The concentration of purified Cam was determined by absorbance at 280 nm using an extinction coefficient of 15 990 M–1 cm–1 (Tripp et al., 2002Go). Purified Cam was frozen as aliquots at –70°C for future use.

Apo-Cam was prepared by unfolding purified Cam in 10 mM phosphate, pH 7.0, 8 M urea and 1 mM K2EDTA at room temperature. After stirring for 4 h, the unfolded protein was dialyzed against a buffer containing 4 M urea, 10 mM phosphate and 0.1 mM K2EDTA, at pH 7.0. Following the 4 M urea dialysis step, the apo-protein was dialyzed into 2 M urea followed by two exchanges into buffer containing 10 mM phosphate and 0.1 mM EDTA. Reconstitution of holo-Cam was achieved by dialyzing apo-Cam against a buffer containing 10 mM phosphate, pH 7.0, and 750 µM ZnSO4 at 4°C for 4 h. Excess Zn2+ was removed by dialyzing the reconstituted holo-Cam against buffer without ZnSO4.

Thermodynamic analysis

Urea denaturation experiments were monitored on a Jasco J-810 circular dichroism (CD) spectrophotometer (Jasco, Easton, MD). A singular value decomposition analysis was performed in order to fit the signal globally at all of the wavelengths in the collected spectra (Ionescu et al., 2000Go); fitting was done with the in-house software package Savuka 5.2. Two thermodynamic models were used to analyze the data. For a monomeric intermediate, the following three-state model would apply:

K1K2

N3 {leftrightharpoons} 3I {leftrightharpoons} 3U(1)

where K1 = [I]3/[N3] and K2 = [U]/[I]. The total protein can be expressed in terms of monomers by PT = 3N3 + I + U. [N3] and [I] can be expressed in terms of [U]:

[I] = [U]/K2(2)

[N3] = [I]3/K1(3)

By substituting Equation 2 into Equation 3:

[N3] = [U]3/K1K23(4)

By substituting Equations 2 and 4 into the equation for total protein concentration and rearranging that equation, the following is obtained:

Although an analytical expression can be obtained for this equation, it can be solved for [U] numerically with an in-house fitting program, Savuka. The result can then be substituted into the expression for Fapp, the fraction apparent of unfolded protein, along with [I], which can be solved in terms of [U] (Equation 2), for this three-state model:

where Z is a factor which represents the similarity between the intermediate with the unfolded and trimeric states, i.e. if Z = 1 the intermediate resembles the unfolded state and if Z = 0 the intermediate resembles the native trimer.

If the intermediate is trimeric, the following three-state model would apply:

K1K2

N3 {leftrightharpoons} I3 {leftrightharpoons} 3U(7)

where K1 = [I3]/[N3] and K2 = [U]3/[I3]. The total protein can be expressed in terms of monomers by PT = 3N3 + 3I3 + U. [N3] and [I3] can be expressed in terms of [U]:

[I3] = [U]3/K2(8)

[N3] = [I3]/K1(9)

By substituting Equation 8 into Equation 9:

[N3] = [U]3/K1K2 (10)

By substituting Equations 8 and 10 into the equation for total protein concentration and rearranging, the following equation is obtained:

Again, an analytical expression can be obtained for this equation, but it can be solved for [U] numerically with Savuka. The result can then be substituted into the expression for Fapp, along with [I3], which can be solved in terms of [U] (Equation 8), for this three-state model:

The urea-denaturation experiments were performed at a variety of protein concentrations to provide a robust test of these models. A global fit of all these concentrations provides more precise values for the thermodynamic parameters. To obtain convergence, the folded and unfolded baselines were treated as adjustable parameters whereas the thermodynamic values were constrained to initial estimates. After obtaining fits for the baselines, the thermodynamic parameters were treated as adjustable parameters. The Z value proved to be the most sensitive parameter and, therefore, had a large estimated error. However, the thermodynamic parameters were largely insensitive to the fluctuations in the Z value.

In order to generate a species plot (see Figure 4), a partition function, Q[D], was solved at varying urea concentrations;



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Fig. 4. Calculated fractions of the trimer (solid line), folded monomeric intermediate (dotted line) and unfolded monomer (dashed line) of apo-Cam as a function of urea and monomer concentration. Fapp is calculated as the fraction of monomeric apo-Cam. Simulated concentrations of apo-Cam are 5 µM (squares), 20 µM (circles) and 50 µM (diamonds).

 

where [D] is the urea concentration. The fractions of each species as a function of urea concentration can then be determined by

where fN3, fI and fU are the fractions of native trimer, monomeric intermediate and unfolded Cam, respectively.

Equilibrium analytical ultracentrifugation was performed using a Beckman Model XL-I ultracentrifuge (Beckman Coulter, Fullerton, CA). Initial protein concentrations ranged from 2 to 80 µM. The absorbance at three wavelengths, 235, 280 and 295 nm, was used to monitor the radial distribution of the protein. Apo-Cam was centrifuged at 26 000, 28 000 and 30 000 r.p.m. for 24, 16 and 16 h, respectively. Holo-Cam was centrifuged at 14 000, 16 000 and 18 000 r.p.m. for 24, 16 and 16 h, respectively. The temperature for all experiments was 25°C and the buffer was 50 mM phosphate, pH 7.0. Establishment of equilibrium was verified by the coincidence of the final two scans at each speed. Data were fit to a variety of models using Savuka 5.2, including monomer {leftrightharpoons} dimer, monomer {leftrightharpoons} trimer, monomer {leftrightharpoons} tetramer, monomer {leftrightharpoons} dimer {leftrightharpoons} trimer and monomer {leftrightharpoons} dimer {leftrightharpoons} tetramer.

Cam activity was assayed using the method of Khalifah utilizing a KinTek SF-2001 stopped-flow instrument (KinTek, Austin, TX). A stock solution of enzyme was diluted to 1.75 µM in 87.5 mM HEPES, pH 7.5, 233 mM Na2SO4 and 350 µM p-nitrophenol and then loaded into one syringe. Various mixtures of CO2- and N2-saturated water were loaded into a second syringe. The two solutions were rapidly mixed in a ratio of 2:5 to obtain final enzyme concentrations in the nanomolar range and in buffer conditions of 25 mM HEPES, 66.6 mM Na2SO4 and 100 µM p-nitrophenol. The reaction was followed by monitoring the change in absorbance at 400 nm, reflecting the protonation of p-nitrophenol as H+ was produced by the catalytic event. To account for the spontaneous production of H+, the rate of the uncatalyzed reaction, in which enzyme was replaced with water, was also determined. The initial slope of the absorbance versus time at a series of CO2 concentrations was fitted to the Michaelis–Menten equation, after conversion of the raw absorbance data to H+ concentration via calibration.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Thermodynamic properties of Cam

The most direct method to obtain thermodynamic parameters for the folding and assembly of holo-Cam is to perform chemical denaturation studies of the complex. The far-UV CD spectrum of Cam provides a convenient probe of the global secondary structure (Figure 2A); the broad minimum at 218 nm is characteristic of a ß-sheet structure. Because the fluorescence of the single solvent-exposed tryptophan residue near the N-terminus does not offer a correspondingly useful probe of the tertiary structure (data not shown), it was not possible to monitor independently the disruption of tertiary structure for holo-Cam. Surprisingly, the unfolding of holo-Cam was found to be irreversible under a broad range of conditions. The chemically induced unfolding of holo-Cam (by urea and guanidine hydrochloride) was assayed in combination with several salts (NaCl, KCl, Na2SO4 and sodium acetate), at several different pH values, a range of protein concentrations (500 nM–25 µM) and a range of temperatures (5–40°C). A typical result is shown in Figure 3A, in which the sigmoidal unfolding transition was not mimicked by the refolding transition beginning with unfolded protein. The absence of a recovery of signal by the unfolded protein at low urea concentrations suggests that the unfolded form is stabilized under these conditions. This result is puzzling because >95% of the enzymatic activity can be recovered by dialysis of the apo-Cam, i.e. metal-free Cam, against buffers containing ZnSO4 (data not shown). By contrast, direct addition of ZnSO4 to folded apo-Cam resulted in massive precipitation.




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Fig. 2. (A) Far-UV CD spectra of holo-Cam at 10 µM (solid line), apo-Cam at 140 µM (dotted line), apo-Cam at 2 µM (dashed line) and unfolded apo-Cam at 8 µM in 8 M urea (dot-dashed line). Protein concentrations refer to the monomer unless specified otherwise. (B) Near-UV CD spectra of holo-Cam at 5 µM (solid line), apo-Cam at 140 µM (dotted line), apo-Cam at 2 µM (dashed line) and unfolded apo-Cam at 8 M in 8 µM urea (dot-dashed line). Conditions: 25°C, 10 mM potassium phosphate, pH 7.6.

 


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Fig. 3. Urea dependence of the unfolding of holo- and apo-Cam monitored by CD spectroscopy. (A) Urea-induced unfolding (filled circles) and refolding (open circles) of holo-Cam at 10 µM. (B) Urea-induced unfolding of apo-Cam at 10 µM (circles), 20 µM (triangles) and 50 µM (diamonds) measured in the concentration of monomer. The solid lines represent global fits of the data to the monomeric intermediate model (Equation 1). Open circles represent refolded apo-Cam at 10 µM to illustrate reversibility of the urea-induced folding reaction. Conditions: 25°C, 10 mM potassium phosphate, pH 7.6.

 
Speculating that the Zn2+ may be responsible for the irreversible urea denaturation of holo-Cam through chelation by multiple acidic side chains in the unfolded form, the urea-denaturation of apo-Cam was investigated by far-UV CD spectroscopy. As can be seen in Figure 3B, the urea-induced unfolding of apo-Cam was fully reversible. Also noteworthy is the dependence of the shape of the unfolding transition curve on the protein concentration. At 10 µM protein, a broad transition from ~1 to 4 M urea was observed. When the protein concentration was increased to 20 µM, the ellipticity in the absence of denaturant increased and the transition shifted to higher urea concentration. The ellipticity again increased in the absence of urea at 50 µM protein and a shoulder appears at 2 M urea. The shoulder implies the presence of an intermediate.

These data were globally fitted to two different three-state models to determine the most likely equilibrium folding model.

N3 {leftrightharpoons} 3I {leftrightharpoons} 3UModel 1

N3 {leftrightharpoons} I3 {leftrightharpoons} 3UModel 2

Details of this analysis can be found in Materials and methods. Note that in these models N3 refers to the trimeric, native state of the apo-enzyme, which is shown in Figure 2A to have a reduced amount of structure compared with that of the trimeric, native state of the holo-enzyme. The better fit to the data was provided by Model 1, which involves a monomeric intermediate, I, in addition to a trimeric native form of apo-Cam, N3, and a monomeric unfolded form, U. Global fits to Model 2 did not converge to a realistic set of thermodynamic parameters. The estimated stability of the monomeric intermediate, relative to the unfolded state, is 4.0 ± 0.3 kcal (mol monomer)–1 and the standard state free energy of association of this intermediate to form the native, trimeric apo-Cam, is 13.2 ± 0.4 kcal (mol trimer)–1. The urea dependence of these stabilities, i.e. the m values (Myers et al., 1995Go), are 1.2 kcal (mol monomer)–1 M–1 and 0.96 kcal (mol trimer)–1 M–1, respectively, indicating that the majority of the buried surface area in the apo-Cam is sequestered in the folding of the monomer. The Z value was determined as 0.54 ± 0.24. Although the error in the Z value is substantial, its uncertainty had little effect on the values of the other thermodynamic parameters. Although the very small change in the fluorescence and near-UV CD signal precluded an independent assessment of these two models, the application of SVD methodology to the entire far-UV CD spectra collected at several dozen urea concentrations ensures that the analysis is robust.

The free energy changes associated with the folding and association steps in Model 1 can be used to predict the populations of the three thermodynamic states as a function of denaturant concentration and to predict the population of monomeric intermediate and trimeric folded Cam as a function of protein concentration (see Materials and methods). Figure 4 shows the percentage of native apo-trimer, monomeric intermediate and unfolded monomer as a function of protein and urea concentration. These predictions provide an explanation of the unfolding curves shown in Figure 3B. In the absence of denaturant, the increase in the percentage of trimer with increasing protein concentration results in the decrease in ellipticity at 222 nm. At 50 µM protein concentration, the trimer is sufficiently populated that the transition from native trimer to the monomeric intermediate can be distinguished by the shoulder in the unfolding curve around 2 M urea. The unfolding curves all coalesce at a urea concentration slightly above 4 M urea where the percentages of monomeric intermediate and unfolded protein are equal for all protein concentrations (Figure 4).

Examining the distribution of apo-trimer and folded monomer along the protein concentration axis in the absence of urea (Figure 5), it can be seen that the trimer is 50% dissociated at 17 µM Cam. At 5 µM Cam the monomer comprises 90% and at 100 µM Cam the trimer comprises 80% of the population. Hence it is possible to define conditions that favor either of the two thermodynamic states which are present in the absence of denaturant.



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Fig. 5. Relative fractions of monomeric (solid line) and trimeric (dashed line) apo-Cam as a function of the protein concentration in the absence of denaturant, based on a free energy of association of 13.2 kcal (mol trimer)–1. Fapp is calculated as the fraction of monomeric apo-Cam.

 
An independent measurement of the dissociation constant of the trimeric apo-Cam was obtained by equilibrium analytical ultracentrifugation in the absence of denaturant. Data were collected and globally fitted at a variety of initial protein concentrations, wavelengths and rotor speeds. A representative curve describing the dependence of the absorbance as a function of radial position in the cell (Figure 6) was best fitted to a monomer–trimer equilibrium with a dissociation constant of 2.0x10–10 M2. Converting to free energy by {Delta}G° = –RTlnKd, the estimated free energy difference, 13.3 ± 0.6 kcal (mol trimer)–1, is in excellent agreement with the value from the urea denaturation measurement, 13.2 ± 0.4 kcal (mol trimer)–1. This convergence of values for Kd provides further support for the choice of the equilibrium folding model and validates the quantitative assessment of the thermodynamic properties of apo-Cam.



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Fig. 6. Representative trace of analytical ultracentrifugation experiments on apo-Cam. The initial concentration of enzyme was 20 µM. Conditions: 30 000 r.p.m., 10 mM potassium phosphate, 25°C, pH 7.6. The fit to a monomer–trimer equilibrium with Kd = 2.0x10–10 M2 is shown by the solid, bold curve and the residuals are plotted in the lower panel.

 
Solution structural properties of holo- and apo-Cam

To obtain insight into the secondary and tertiary structure of the monomeric and trimeric forms of apo-Cam, far- and near-UV CD spectra were collected under conditions which favor each species. The far-UV CD spectrum of 2 µM apo-Cam, where the protein exists primarily as a monomer (95%), features a minimum at 206 nm and a shoulder at ~220 nm (Figure 2A). The increase in the signal at 220 nm as the protein concentration is increased to 140 µM suggests that the association reaction to form the trimer (~85%) results in the stabilization of secondary structure in Cam. The addition of Zn2+ to form holo-Cam induces a dramatic increase in the magnitude of the CD signal near 220 nm and the loss of the minimum at 206 nm (Figure 2A). The loss in the minimum at 206 nm probably reflects the folding of unstructured protein into ß-helix. The spectrum of holo-Cam is very similar to that of pectate lyase C (Kamen et al., 2000Go), a right-handed ß-helical protein, which also has a broad minimum around 218 nm. Although X-ray structures exist for several other left-handed ß-helical proteins (Raetz and Roderick, 1995Go; Beaman et al., 1997Go; Olsen and Roderick, 2001Go), CD spectra are not available to make a direct comparison.

Near-UV CD spectra were collected to probe the packing of the aromatic side chains in monomeric and trimeric apo-Cam. For comparison, the near-UV CD spectrum of holo-Cam is dominated by a broad minimum, from 260 to 290 nm, signifying a well-packed tertiary structure involving the eight tyrosine, five phenylalanine and one tryptophan residues (Figure 2B). The small maximum at 295 nm implies that the sole Trp residue is restrained in a chiral environment. Similarly to the far-UV CD spectra, the near UV-CD spectra for holo-Cam are independent of protein concentration over the range from 5 to 100 µM (data not shown). At 140 µM apo-Cam, where the apo-trimer dominates the population, the spectrum is similar in shape to, but reduced in amplitude from that for holo-Cam between 260 and 290 nm; the tryptophan signal near 295 nm is unchanged. For monomeric apo-Cam at 2 µM protein, the further reduction of the signal between 260 and 290 nm and the decrease at 295 nm shows that a fraction of the tertiary structure is lost by the dissociation of the trimeric species. The near-UV CD spectrum of unfolded apo-Cam in 8 M urea indicates that the tertiary packing around the single tryptophan has been eliminated. However, the small negative ellipticity observed between 260 and 290 nm under these highly denaturing conditions suggests that non-random residual structure persists in the unfolded form. Although both secondary and tertiary structure are disrupted by the loss of the Zn2+ and/or the dissociation of the subunits, the retention of a substantial fraction of these structures in both monomeric and trimeric apo-Cam implies that the amino acid sequence alone favors partial ß-helix formation in Cam.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The thermodynamic parameters for the folding and assembly of the holo-Cam trimeric complex can potentially provide insight into the process by which the protein complex forms on the surface of M.thermophila in vivo. Although the irreversibility of the urea-induced unfolding of the holo-Cam precluded a direct measurement of the stability, it is possible to obtain the desired information through the construction of an equivalent thermodynamic cycle (Scheme 1). This cycle also has the advantage of dissecting the process into plausible elementary steps whose individual free energy changes can be measured or estimated from model compound data. The three steps in the cycle involve (1) the folding of the monomeric species, (2) the association of the monomers to form the trimeric apo-Cam and (3) the binding of three Zn2+ ions to the inter-subunit active sites.

Reversible urea denaturation experiments on apo-Cam revealed that monomeric apo-Cam possesses 4.0 kcal (mol monomer)–1 of stability and thus contributes a total of 12.0 kcal (mol trimer)–1 to the final holo-Cam complex. The free energy for the association reaction to form trimeric apo-Cam contributes an additional 13.2 kcal (mol trimer)–1. The value for the minimum stability at standard state required for enzymatic activity at a Cam concentration of 100 nM is estimated to be ~70 kcal (mol trimer)–1. Thus, at least ~45 kcal (mol trimer)–1 would have to be contributed from zinc binding, indicating that the incorporation of zinc into the apo-Cam complex is the major source of stability for the system.

A contribution of this magnitude is plausible considering that the free energy of binding of Zn2+ to a canonical three-histidine active site in human carbonic anhydrase II (CAII) has been reported to be 16.4 kcal mol–1 (DiTusa et al., 2001Go). The similarity of the zinc-binding site for CAII and that for Cam implies that value should represent a good estimate for the free energy of metal binding to Cam.

The CD data also provide insight into the progressive development of secondary structure for Cam accompanying folding, assembly and metal ion binding. The increase in the far-UV CD signal as the concentration of the protein is increased shows that apo-trimer formation induces the formation of additional secondary structure. Given the inter-subunit location of the Zn2+ ions and their dramatic induction of secondary structure upon binding to form holo-Cam, it is reasonable to propose that the docking surfaces for the apo-trimer do not include the Zn2+-binding region.

The observation that approximately two-thirds of the total free energy of the folding and assembly of holo-Cam is derived from the three metal-binding reactions has direct implications for the process by which Cam is translocated to and assembled on the surface of M.thermophila. The individual subunits are only weakly folded and fractionally associated in the absence of Zn2+ and at micromolar concentrations of protein. Presumably, the signal sequence rapidly targets the apo-Cam chains to the export apparatus, which would encounter little thermodynamic challenge in unfolding and translocating them to the surface of the archea bacterium. The crucial thermodynamic step in the assembly of the trimeric holo-Cam is the incorporation of the three Zn2+ ions, events which must occur after the monomer is resident on the surface and has access to other monomers. The very weak association constant for trimeric apo-Cam implies that the monomers may be tethered to the surface to increase their local concentration and, thereby, enhance the formation of the holo-Cam. The final step, addition of the Zn2+ ions, would dramatically increase the stability of the complex and produce active enzyme.

Although the actual mechanism by which holo-Cam appears on the surface of M.thermophila is undoubtedly modulated by interactions between the monomeric Cam chains and the export apparatus, chaperones (Diamond and Randall, 1997Go), etc., the thermodynamic properties of the oligomeric complex and its components suggest a progressive series of folding and assembly steps to reach this target. From another perspective, one could conclude that the formation of holo-Cam is under thermodynamic, not kinetic, control in vivo.


    Acknowledgements
 
We thank Drs Brian C.Tripp and James G.Ferry for helpful discussions. We also thank Kim Crowley and Dr Craig L.Peterson for their operation of the analytical ultracentrifuge. We appreciate the efforts of Dr Osman Bilsel in developing in-house global fitting routines for data analysis. This work was supported by NIH grant GM 54836 to C.R.M.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alber,B.E. and Ferry,J.G. (1994) Proc. Natl Acad. Sci. USA, 91, 6909–6913.[Abstract]

Beaman,T.W., Binder,D.A., Blanchard,J.S. and Roderick,S.L. (1997) Biochemistry, 36, 489–494.[CrossRef][ISI][Medline]

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Received March 2, 2004; accepted March 4, 2004 Edited by Lynne Regan





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