Interactions between immunoglobulin-like and catalytic modules in Clostridium thermocellum cellulosomal cellobiohydrolase CbhA

Irina A. Kataeva1,2, Vladimir N. Uversky3,4, John M. Brewer1, Florian Schubot5, John P. Rose1, B.-C. Wang1 and Lars G. Ljungdahl1

1Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA, 3Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA, 4Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia and 5Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA

2 To whom correspondence should be addressed. E-mail: kataeva{at}uga.edu


    Abstract
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Cellobiohydrolase CbhA from Clostridium thermocellum cellulosome is a multi-modular protein composed starting from the N-terminus of a carbohydrate-binding module (CBM) of family 4, an immunoglobulin(Ig)-like module, a catalytic module of family 9 glycoside hydrolases (GH9), X11 and X12 modules, a CBM of family 3 and a dockerin module. Deletion of the Ig-like module from the Ig–GH9 construct results in complete inactivation of the GH9 module. The crystal structure of the Ig–GH9 module pair reveals the existence of an extensive module interface composed of over 40 amino acid residues of both modules and maintained through a large number of hydrophilic and hydrophobic interactions. To investigate the importance of these interactions between the two modules, we compared the secondary and tertiary structures and thermostabilities of the individual Ig-like and GH9 modules and the Ig–GH9 module pair using both circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). Thr230, Asp262 and Asp264 of the Ig-like module are located in the module interface of the Ig–GH9 module pair and are suggested to be important in ‘communication’ between the modules. These residues were mutated to alanyl residues. The structure, stability and catalytic properties of the native Ig–GH9 and its D264A and T230A/D262A mutants were compared. The results indicate that despite being able to fold relatively independently, the Ig-like and GH9 modules interact and these interactions affect the final fold and stability of each module. Mutations of one or two amino acid residues lead to destabilization and change of the mechanism of thermal unfolding of the polypeptides. The enzymatic properties of native Ig–GH9, D264A and T230A/D262A mutants are similar. The results indicate that inactivation of the GH9 module occurs as a result of multiple structural disturbances finally affecting the topology of the catalytic center.

Keywords: fold/glycosidase/interactions/module protein/stability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial extracellular glycoside hydrolases active against heterogeneous plant cell wall material are mostly modular enzymes. In the simplest case they are composed of only a catalytic module, but in most cases they bear other ‘accessory’ modules including carbohydrate-binding modules (CBM) which bind the enzymes to insoluble carbohydrates (Tomme et al., 1995Go) and at least in some cases direct the carbohydrate chain into the catalytic site (Sakon et al., 1997Go; Mandelman et al., 2003Go), surface layer homology modules mediating the attachment to the microbial cell wall (Fuchs et al., 2003Go), modules with no significant binding to cellulosic material but shown to modify its surface (Kataeva et al., 2002Go) and modules with unknown biological functions. The last category, in addition to numerous modules designated ‘X’ (Coutinho and Henrissat, 1999Go; Davies and Henrissat, 2002Go), also includes the Fn3-like and Ig-like modules which display sequence similarities to a type 3 human fibronectin and an immunoglobulin, respectively, but with no biological functions established. The long-term existence of these modules in bacterial genomes implies that they play more important biological roles than simple linking of neighboring modules (Little et al., 1994Go; Bayer et al., 2000Go).

As modules display more prominent interactions inside their structural units than with other parts of the modular polypeptide, they possess relatively independent folds and when expressed individually usually their folds and biological functions are preserved (Janin and Wodak, 1983Go; Jaenicke, 1999Go; Kataeva et al., 2001Go). In particular, the catalytic modules of glycoside hydrolases are able to hydrolyze substrates and carbohydrate-binding modules bind to carbohydrates (Tomme et al., 1995Go; Kataeva et al., 2001Go; Arai et al., 2003Go). In many cases the modules are separated by linker sequences of different lengths which are rich in proline and the hydroxylic amino acid residues serine and threonine (Black et al., 1996Go; Janecek et al., 2003Go). In some cases the characteristic linkers are absent, suggesting more intimate interactions between neighboring modules (Zverlov et al., 1998Go; Kataeva et al., 1999Go).

Little is known about module interactions in glycoside hydrolases although the importance of such interactions has been demonstrated in various mammalian, fungal and bacterial proteins (Wenk et al., 1998Go; Jaenicke, 1999Go; Wassenberg et al., 1999Go; Berr et al., 2000Go; Clout et al., 2000Go). It has been noted that some modules in glycoside hydrolases are not randomly combined. They often are located in a particular place within a protein molecule and are associated with a particular type of neighboring module(s) (Tomme et al., 1995Go; Meissner et al., 2000Go; Reeves et al., 2000Go).

Catalytic modules and carbohydrate-binding modules of glycoside hydrolases (GH) have been classified into over 80 GH families and over 30 CBM families, respectively, based entirely on amino acid sequence similarity (Coutinho and Henrissat, 1999Go). A large family, GH9 includes mostly endoglucanases, two endo-/exoglucanases and two highly homologous exoglucanases, CelK and CbhA. Representatives of this family are characterized by different modular arrangements of the catalytic module. Based on these differences, the GH9 family can be divided into four groups (Bayer et al., 2000Go). Group A represents mainly plant enzymes having only a catalytic module and an endoglucanase Cel9M from Clostridium cellulolyticum. The latter contains besides the catalytic module a dockerin module at the C-terminus (Parsiegla et al., 2002Go); group B includes enzymes in which GH9 is joined at its C-terminus with a CBM of family 3a (Sakon et al., 1997Go; Mandelman et al., 2003Go); group C includes enzymes with an Ig-like module attached to the N-terminus of GH9 (Juy et al., 1992); and group D contains enzymes starting from an N-terminus of CBM4, followed by an Ig-like module connected to the GH9 module (Zverlov et al., 1998Go; Kataeva et al., 1999Go). The deletion of the Ig-like module from the N-terminus of GH9 of endoglucanase Cel9A (former CelD) (Béguin and Alzari, 1998Go), CelK (I.A.Kataeva, unpublished work) or CbhA results in the complete inactivation of the catalytic GH9 module.

The Clostridium thermocellum cellobiohydrolase CbhA is composed, starting from the N-terminus, of a CBM4, an Ig-like module, a GH9 module, X11 and X12 modules, a CBM3 and a dockerin module (Zverlov et al., 1998Go). In the present work, we investigated the importance of the interactions between the Ig-like and GH9 modules of CbhA by comparison of the properties of individual modules, the Ig–GH9 module pair and mutants with a modified module interface.


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 Materials and methods
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Bacterial strains, culture conditions and plasmids

Clostridium thermocellum JW20 was used as a source of genomic DNA. The bacterium was grown anaerobically under an N2 atmosphere at 60°C in a pre-reduced medium with 1% (w/v) cellobiose (Kataeva et al., 1999Go). Escherichia coli BL21(DE3)pLys (Stratagene Cloning Systems, La Jolla, CA) was used as cloning host for the T7 RNA polymerase expression vector pET-21b(+) (Novagen, Madison, WI). It was grown in Luria–Bertani medium supplemented with ampicillin (100 µg/ml).

Primer design, PCR and cloning

Flanking primers containing Nhe I and NotI restriction sites for cloning of the module combination Ig–GH9 and the individual modules Ig and GH9 were designed according to the DNA sequence of cbhA (accession number X80993) (Table I) and synthesized with an Applied Biosystems DNA synthesizer. DNA fragments were amplified by PCR using the primers in combination with purified genomic DNA as a template. PCRs were carried out on a 480 Thermal Cycler (Perkin-Elmer, Norwalk, CT). The reactions were carried out with Taq polymerase (New England Biolabs, Beverly, MA). The annealing temperature was 54°C and the extension time depended on the length of the fragment. PCR products were separated by 1% agarose gel electrophoresis and extracted from the gel using a Geneclean II Kit (Bio 101, La Jolla, CA). The extracted DNA fragments were digested with restriction enzymes and ligated into the pET-21b(+) vector linearized with the same enzymes. The ligation products were used to transform BL21(DE3)pLys competent cells. Each construct was verified by both restriction analysis and DNA sequencing.


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Table I. Oligonucleotides used for cloning and site-directed mutagenesisa

 
Mutagenesis

Site-directed mutagenesis was done using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). In a single D264A and a double mutant T230A/D264A aspartic and threonine amino acid residues were replaced by alanyl residues using the primers shown in Table I. The replacements were confirmed by DNA sequencing.

Protein purification

All polypeptides were 6x His tagged at the C-terminus. They were purified from BL21(DE3)pLys cultures harboring pET-21b(+) containing the DNA fragment of interest. Harvest was 5 h after induction with 1 mM isopropyl-ß-D-thiogalactopyranoside. All purification steps were done at 4°C, except for FPLC column chromatography, which was carried out at room temperature. After collection, the cells were washed with 20 mM sodium phosphate buffer, pH 7.5, containing 0.5 M NaCl (buffer A) and disintegrated with a French press. Cell debris was removed by centrifugation at 20 000 g for 30 min. The clear supernatant was applied to an Ni-NTA agarose (Qiagen, Valencia, CA) column equilibrated with buffer A. The column was then washed with 20 mM sodium phosphate, 0.5 M NaCl, pH 6.0. Proteins were eluted with a gradient of 0.0–0.5 M imidazole in 20 mM sodium phosphate, 0.5 M NaCl, pH 6.0. Fractions containing imidazole-eluted proteins were combined, concentrated by precipitation with (NH4)2SO4 and dialyzed against 20 mM Tris–HCl buffer, pH 7.5, containing 0.1 M NaCl. Dialyzed proteins were further purified by gel filtration on a TSK 3000SW column (TosoHaas, Montgomeryville, PA). Eluted samples were concentrated using Centricon 3 concentrators (Amicon, Beverly, MA) and stored at 4°C. All proteins were assayed for homogeneity as ascertained by SDS–PAGE.

Protein determination

During early stages of purification, protein concentrations were determined using Coomassie Protein Assay Reagent (Pierce, Rockford, IL). Concentrations of purified proteins were determined on the basis of A278 values. Each protein was extensively dialyzed against 20 mM sodium phosphate buffer, pH 6.0, and then the solution was centrifuged at 20 000 g for 60 min. The absorbance was read using the dialysis buffer as a blank. Molar absorption coefficients calculated from tryptophan and tyrosine contents were (M–1 cm–1) 21 650 for Ig, 139 690 for GH9 and 161 340 for Ig–GH9 and its mutants.

Circular dichroism (CD) spectra

CD measurements were carried out using a Jasco J-710 spectropolarimeter with a jacketed quartz cell with a 1.0 mm (far-UV region) or 10 mm (near-UV region) pathlength. The cell temperature was controlled to within ±0.1°C by circulating (15 l/min) a mixture of water and antifreeze (1:1) through the cell jacket using a Neslab R-111 water-bath (Neslab Instruments, Portsmouth, NH). Protein samples were in 20 mM sodium phosphate buffer, pH 6.0, at a concentration of 10 µM for the far-UV and at an A278 of 0.5 for the near-UV CD. The results were expressed as mean residue ellipticity [{Theta}]mrw. The spectra obtained were averages of five scans. The spectra were smoothed using an internal algorithm in the Jasco software package J-170 for Windows.

Thermal denaturation of proteins was monitored by CD in both the near- (190–250 nm) and far-UV (250–320 nm) regions. For the analysis of thermostability, the temperature was increased from 25 to 100°C. The CD spectra were measured at intervals of 10°C (from 25 to 45°C) and at 5°C at higher temperatures. Before recording, the sample was incubated for 15 min at each temperature. At the end of each thermal denaturation experiment, the sample was cooled for 10 min to 25°C and the spectra were again recorded to determine the extent of refolding.

Analysis of spectroscopic data (phase diagrams)

The phase diagram method of analysis of spectroscopic data is extremely sensitive to the detection of intermediate steps (Permyakov et al., 1980Go; Bushmarina et al., 2001Go; Kuznetsova et al., 2002Go, 2004Go; Ahmad et al., 2003Go). Only extensive parameters (i.e. those parameters whose value is proportional to the amount of the analyzed matter in a system) should be used for such an analysis. The essence of the phase diagram method is to construct the diagram of I{lambda}1 versus I{lambda}2, where I{lambda}1 and I{lambda}2 are the spectral intensity values measured at wavelengths {lambda}1 and {lambda}2 under different experimental conditions for a protein undergoing structural transformations. In application to protein unfolding, the relation I{lambda}1 = fI{lambda}2 will be linear if changes in the protein environment lead to an all-or-none transition between two different conditions. In contrast, non-linearity of this function reflects the sequential character of structural transformations, where each linear portion of the I{lambda}1 = fI{lambda}2 plot describes an individual all-or-none transition. In principle, {lambda}1 and {lambda}2 are arbitrary wavelengths of the spectrum, but in practice, such diagrams are more informative if {lambda}1 and {lambda}2 are on different slopes of the spectrum. If the wavelengths are from one slope or near the maximum, some transitions may remain undetected.

Differential scanning calorimetric (DSC) measurements and data analysis

DSC measurements were carried out using a Calorimetry Sciences (Spanish Fork, Utah) MS-DSC system with three identical chambers so that up to three samples could be heated at a time. All samples were intensively dialyzed against 20 mM sodium phosphate buffer, pH 6.0, before the measurements. Protein solutions at a concentration of 6 mg/ml and volume of 0.5 ml were heated from 15 to 100°C at 60°C/h. Disposable 50 µl glass pipettes (Fischer Scientific, Tampa, FL) were broken into short segments and added to the protein solutions before heating to reduce precipitation artifacts. A buffer scan was used as a baseline. After heating, the samples were cooled to 15°C and re-heated to check the reversibility of thermal denaturation. Use of either baseline yields the same results. Hence the denaturation processes described here are irreversible. Data were processed using Cp Calc Data Analysis Software (Calorimetry Sciences). Apparent (van't Hoff) denaturation enthalpies were calculated using the following equation (Kozhevnikov et al., 2001Go):

where Td is the denaturation temperature (K), Cp(Td) is the heat capacity at the denaturation temperature (kcal/mol) and {Delta}dHcal is the calorimetric denaturation enthalpy (kcal/mol).

The number of cooperative units r was calculated from the ratio {Delta}dHcal/{Delta}dHv.H.. Note that the DSC data were obtained at much higher protein concentrations and under a different heating protocol (continuous as opposed to discontinuous heating) than the CD data. Note also that the equations employed to calculate the above denaturation parameters are derived assuming reversible processes. However, these equations are routinely used for this purpose even when the denaturations studied are irreversible; see Kozhevnikov et al. (2001)Go for an example of this; see also the Discussion section.

Activity assay

Activities of truncated and mutated variants of cellobiohydrolase CbhA were assayed using p-nitrophenyl(PNP)-cellobioside as a substrate. Hydrolysis of PNP-cellobioside was determined by the release of p-nitrophenol. Samples in 1 ml of 50 mM sodium citrate buffer, pH 6.0, with 2 mM substrate were incubated at 65°C for 5 min. Then the reactions were stopped by addition of 1 ml of 2% Na2CO3 and the optical density at 405 nm was read. One enzyme unit was defined as an amount of the enzyme releasing 1 µmol of p-nitrophenol per minute. Temperature optima of proteins were determined within the range 25–80°C at 5°C intervals following incubation at each temperature for 10 min before addition of protein. Kinetic parameters of the proteins were determined at 65°C and short-term reactions (1 min). The values of Vmax and KM were determined from the Lineweaver–Burk (or double reciprocal) plot. The catalytic constants kcat were calculated from the equation kcat = Vmax/[E]. The efficiency of catalysis was expressed as kcat/KM.

Metal content

The metal contents of the proteins were determined using inductively coupled plasma emission spectrophotometry (EPA Method 610) using a VG Plasma Quad 3 ICP ICP-MS system. Samples (1 ml) containing 50–200 µmol of proteins were quantitatively analyzed for elements of interest using multi-element calibration solutions. A single calibration set was prepared and run at the beginning of the analysis procedure. Before analysis, the proteins were extensively dialyzed against 1 l of 20 mM Tris–HCl buffer, pH 7.5, which was used as a blank. Each data point is an average of three replicates.


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Modifications of module interface

The deletion of the Ig-like module results in a complete inactivation of the GH9 module of CbhA (I.A.Kataeva, unpublished work). The crystal structure of the Ig–GH9 module pair shows folds of the modules and an extensive interface between them composed of over 40 amino acid residues from both domains which are involved in numerous hydrophobic and hydrophilic interactions (Figure 1A and B). Comparison of the structure of Ig–GH9 of CbhA and that of Cel9D, the only other available structure with the same domain arrangement (Juy et al., 1992), reveals that only three of 10 hydrogen-bonding pairs are conserved in both proteins, suggesting that they play a crucial role in the maintenance of stable domain interactions and affect overall fold of the protein. The Thr230 of the Ig-like module and G221 of the GH9 module form one of these pairs. This interaction helps to stabilize an otherwise flexible loop, which in turn interacts with another loop that is part of the catalytic site of the enzyme. Residues D262 and D264 of the Ig-like module form two other conserved hydrogen bonds with G221 and Y676, respectively, of the GH9 module (Figure 1B). Y676 in turn stabilizes the position of highly conserved W678, which directly interacts with the substrate near the cleavage site (Schubot et al., 2004Go). To investigate the importance of inter-domain interactions for the fold, stability and activity of Ig–GH9, we constructed two mutants with the modified module interface (Figure 1B). In the single mutant D264A Asp264 was replaced with an alanyl residue. In double mutant T230A/D262A, Thr230 and Asp262 were mutated to alanyl residues.



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Fig. 1. (A) Crystal structure of Ig–GH9 module pair in complex with cellotetraose. The rainbow coloring starts at the N-terminus, giving the all-ß-sheet Ig-like module a dark-blue taint. A larger catalytic module GH9 folds into the ({alpha}/{alpha})6-barrel. The two calcium ions are displayed as dark spheres. (B) Surface plot showing strictly conserved hydrogen-bonding interactions. Asp264, Thr230 and Asp262 involved in these interactions were replaced by alanyl residues giving D264A and T230A/D262A mutants.

 
Purity of proteins

All proteins were soluble. They were purified to near homogeneity as revealed by SDS–PAGE. As they bound to Ni-NTA affinity columns and their molecular masses corresponded closely to those calculated from the deduced amino acid sequence, we assume that the proteins were purified in the intact form without significant proteolysis.

Metal content

The crystal structure of module pair Ig–GH9 reveals two conserved calcium-binding sites located in the GH9 module (Schubot et al., 2004Go). According to metal analysis, the individual Ig-like module does not contain any metals. The Ig–GH9 module pair, its mutants D264A and T230A/D262A and the individual GH9 module bind 2.45 ± 0.21, 2.21 ± 0.19, 2.46 ± 0.13 and 2.31 ± 0.15 mol of calcium, respectively. These data are in good agreement with the three-dimensional structure of Ig–GH9 (Figure 1A) and indicate that both the mutants and the inactive GH9 module contain the same amount of calcium as the Ig–GH9 construct. The detection of more than 2 mol of calcium in these proteins might be a result of unspecific binding of the ion or the presence of a low-affinity calcium center not detected in the 3D structure of Ig–GH9.

CD spectra of individual modules and module combination

The near-UV CD spectra of proteins reflect asymmetric environments in the vicinity of aromatic amino acid residues, i.e. the structural integrity of the proteins. The Ig-like module (residues 210–309) contains four phenylalanine, seven tyrosine and two tryptophan residues. The GH9 module (residues 310–815) contains 16 phenylalanine, 29 tyrosine and 18 tryptophan amino acid residues. The near-UV CD spectra of the individual Ig-like and GH9 modules and Ig–GH9 module pair differ according to their contents of aromatic amino acid residues (Figure 2A).



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Fig. 2. (A) Near-UV and (C) far-UV CD spectra of individual Ig-like and GH9 modules, Ig–GH9 module pair and spectra of Ig–GH9 calculated from the spectra of the individual modules. (B) Near-UV and (D) far-UV CD spectra of native Ig–GH9 module pair and its D264A and T230A/D262A mutants. Spectra were recorded at 25°C. Protein samples were in 20 mM sodium phosphate buffer, pH 6.0, at a concentration of 10 µM for the far-UV and at an A278 of 0.5 for the near-UV CD.

 
Far-UV CD spectroscopy allows the characterization of the secondary structures of proteins and is a good criterion of the proper fold of individually expressed modules. The crystal structure of the Ig–GH9 module pair (Figure 1A) shows that the N-terminal Ig-like module consists of two-layered ß-sheets that form the anticipated ß-sandwich structure. The large catalytic module consists of an ({alpha}/{alpha})6-barrel, which is characteristic of numerous members of family 9 glycoside hydrolases (Coutinho and Henrissat, 1999Go).

The far-UV CD spectra of the individual Ig-like and GH9 modules recorded at 25°C are shown in Figure 2C (see also Figure 3A and B for details). The shapes and intensities of the spectra of both modules are rather unusual. It is known that in some cases the shapes of the far-UV CD spectra can be considerably distorted owing to the contribution of aromatic side groups (Bolotina et al., 1987; Perczel et al., 1991; Chaffotte et al., 1992Go; Chakrabarty et al., 1993; Uversky and Ptitsyn, 1996Go). Such an effect is usually more pronounced for ß-proteins and the largest deviations from the ‘normal’ spectrum have been reported for human carbonic anhydrase B (Jagannadham and Balasubramanian, 1985Go; Rodionova et al., 1989Go) and capsular protein Caf1 (Abramov et al., 2001Go). It is interesting that the far-UV CD spectrum of the Ig-like module resembles these unusual spectra and clearly shows a considerable contribution of aromatic amino acid residues. Despite this irregularity, the spectrum of the Ig-like module is typical of an all-ß protein with one negative and one positive maximum at 226 and 203 nm, respectively (Figure 3A). This is consistent with the crystal structure of the Ig–GH9 discussed above and with the fold-based classification of Ig-like modules into an immunoglobulin superfamily, members of which display a typical fold of two antiparallel ß-sheets often called a ß-sandwich (Fischer et al., 1999Go; Kelley et al., 1999Go, 2000Go). The atypical far-UV CD spectra are a characteristic feature of members of the immunoglobulin superfamily (Tetin et al., 1992Go; O'Connor et al., 2000Go; Soulilac et al., 2002a,b).



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Fig. 3. Far-UV CD spectra of individual (A) Ig-like module and (B) GH9 module recorded at different temperatures. The temperature was increased from 25 to 100°C at 5 or 10°C intervals. The spectra were measured at each temperature after incubation for 15 min.

 
The far-UV CD spectrum of the individual GH9 module recorded at 25°C (Figures 2C and 3B) has two prominent negative maxima at 210 and 222 nm with signal amplitudes of about –9000°/cm.dmol and a positive maximum at wavelengths below 200 nm characteristic of all-{alpha} proteins. This is also consistent with the fold of the GH9 module within the Ig–GH9 construct (Figure 1A). The spectrum of the Ig–GH9 module pair (Figure 2C) is a spectrum of an ({alpha} + ß) protein where the {alpha}-helix predominates over the ß-sheet.

To reveal possible interactions between the modules in the Ig–GH9 construct, we compared recorded near-UV (Figure 2A) or far-UV (Figure 2C) CD spectra of the module pair with the corresponding spectra calculated as a simple weighted sum of the recorded spectra of individual Ig-like and GH9 modules. In the absence of module interactions we would expect the recorded and calculated spectra to coincide. However, the spectra differ significantly. This is evidence that the two modules in the Ig–GH9 module pair interact, affecting both secondary and tertiary structures of the individual modules. This is not surprising, as there is no visible linker sequence between the Ig-like and GH9 modules and contacts between these modules seem to be more intimate.

CD spectra of mutants

The near- and far-UV CD spectra of the D264A and T230A/D262A mutants are shown in Figure 2B and D, respectively. In comparison with the spectra of native Ig–GH9, the spectra of the mutants display obvious differences, which are more prominent in the near-UV region (Figure 2B). The near-UV CD spectrum of the Ig–GH9 possesses a well-defined fine structure characteristic of a protein with a rigid structure. In contrast, the near-UV spectra of the mutants, especially that of the double mutant T230A/D262A, are smoothed, suggesting partial loss of rigidity. The far-UV CD spectra of the mutants show apparently greater helical contents then wild-type protein, probably owing to alterations in the environment(s) of their aromatic residues.

Thermal unfolding monitored by CD spectroscopy

The thermal unfolding of the Ig-like and GH9 modules recorded in the far-UV region occurs as a cooperative process gradually resulting in loss of secondary structure. The CD spectra of these proteins recorded at 100°C are characteristic of unfolded polypeptides with one negative maximum at ~200 nm. The specific temperatures at which the spectra were recorded are shown in Figure 3A and 3B.

Heat denaturation of all the proteins described here is irreversible. In the case of individual Ig-like and GH9 modules and the double mutant T230A/D262A, the thermal unfolding is complicated by visible aggregation and precipitation affecting the CD spectra at high temperatures. Otherwise, the dependences of the molar ellipticity [{Theta}]mrw signals at 275 and 222 nm on temperature are shown in Figure 4A and B, respectively, for all the proteins. Only curves of the Ig–GH9 module pair and the D264A mutant shown in Figure 4A fit the equation of a sigmoid curve. The S-curves of the Ig-like and GH9 modules and the double mutant T230A/D262A are incomplete and do not permit determination even of their melting (denaturation) temperatures (Td) because of precipitation. The origins of the curves start differently, indicating different mechanisms and cooperativities of thermal transitions of the proteins.



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Fig. 4. Temperature-induced denaturation of individual Ig-like and GH9 modules, Ig–GH9 module pair and its D264A and T230/D262A mutants monitored by changes at (A) 275 and (B) 222 nm.

 
Phase diagram analysis

To elucidate the mechanisms of the temperature-induced structural transformations we used the method of ‘phase diagrams’. Figure 5 shows phase diagrams for the temperature-induced denaturation of the Ig-like module (A), GH9 module (B), native Ig–GH9 module pair (C) and its two mutants, D264A (D) and T230/D262A (E). Analysis of these diagrams emphasizes three important points: (a) the mechanisms of denaturation are different for the different constructs; (b) intermodular interactions affect the denaturation mechanism; and (c) amino acid substitutions at the intermodular interface modulate the mechanism of Ig–GH9 thermal denaturation. The phase diagram of the Ig-like module consists of two linear parts, whereas that of the GH9 module has four linear parts. This means that the denaturation of the individual Ig-like and GH9 modules involves two and four independent structural transitions, respectively (Figure 5A and B). The combination of these two modules dramatically changes the shape of the phase diagram. The plot has three linear parts, indicating the existence of at least three independent transitions linking four different conformations (Figure 5C). Figure 5D and E show that the introduction of single or double amino acid substitutions into the modular interface significantly changes the shape of the phase diagram; however, these mutations do not affect the denaturation mechanism, which is still characterized by a combination of three independent transitions. Overall, these data show that modular interactions have a dramatic effect on the conformational stability of the individual modules.



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Fig. 5. Phase diagrams based on [{theta}]{lambda}1 vs [{theta}]{lambda}2 (see text for details). These characterize heat denaturation of proteins based on the temperature-induced changes in the CD spectra of individual (A) Ig-like module and (B) GH9 module, (C) Ig–GH9 module pair and its (D) 264A and (E) T230A/D262A mutants.

 
Thermal unfolding monitored by DSC

DSC heating thermograms of individual modules, the module combination and its mutants are shown in Figure 6A. The thermogram peaks, except for that of the Ig-like module, look asymmetric. This suggests that the heat denaturation of these proteins is not a simple two-state process. Some thermodynamic parameters calculated from these data are given in Table II. The Ig–GH9 module pair displays the highest denaturation temperature (77.9°C). The individual Ig-like and GH9 modules are not as stable as their transition temperatures are lower by 4 and 10°C, respectively. Interactions between modules are clearly seen in the comparison of calculated calorimetric enthalpies of individual modules and the module combination: the sum of {Delta}Hcal values of the Ig-like and GH9 modules (350.6 kcal/mol) is significantly lower than the {Delta}Hcal of the Ig–GH9 module pair (530.5 kcal/mol). The D264A and T230A/D262A mutants possess Tds lower by 6 and 8.7°C, respectively, than that of Ig–GH9. The same tendency is observed for the calculated {Delta}Scal values (Table II).



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Fig. 6. (A) DSC thermograms of Ig–GH9 module pair (1), its D264A (2) and T230/D262A (3) mutants and individual GH9 module (4) and Ig-like module (5). (B) DSC thermograms of individual GH9 module in buffer (1), in the presence of 2 mM EDTA (2) and 2 mM cellobiose (3). 20 mM sodium phosphate buffer, pH 6.0, was used in all experiments. The protein concentrations and scan rate were 6 mg/ml and 60°C/h, respectively. All thermal transitions were completely irreversible, so that second scans of the proteins were used as baselines.

 

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Table II. Calculated thermodynamic denaturation parameters of individual Ig-like and GH9 modules, Ig–GH9 module pair and its single D264A and double T230A/D262A mutants

 
The calculated van't Hoff enthalpy of heat denaturation of the individual Ig-like module is close to the calculated calorimetric enthalpy (Table II). This means that the Ig-like module denatures according to a simple two-state model and possesses a well-organized rigid structure. In all other cases, the calculated van't Hoff enthalpies are significantly lower than the corresponding calorimetric enthalpies, suggesting that thermal denaturation of these proteins occur according to a more complex mechanism. The calculated number of cooperative units (r) of the individual GH9 module is 4.0. The corresponding values for Ig–GH9, D264A and T230/D262A mutants are 3.4, 3.2 and 2.0, respectively (Table II). The highly asymmetric thermogram of the GH9 module and large difference between calculated {Delta}Hcal and {Delta}Hv.H. values indicate that this module is significantly destabilized without the N-terminal Ig-like module. According to these calculations, the individual GH9 module is not a highly cooperative unit and is composed of at least four subdomains in terms of its thermal denaturation properties.

The thermal denaturation of the individual GH9 module in buffer and in the presence of 2 mM EDTA or 2 mM cellobiose reveals some interesting features (Figure 6B). EDTA significantly destabilizes the protein as it decreases its denaturation temperature Td and calculated calorimetric enthalpy {Delta}Hcal by 3.4°C and almost 4-fold, respectively, in comparison with the corresponding values of the GH9 module in buffer. The presence of 2 mM cellobiose stabilizes the GH9 module, increasing its Td to 78.5°C, which is higher than that of the Ig–GH9 construct. The calculated {Delta}Hcal of GH9 in the presence of cellobiose is 1.8 times higher than that of GH9 in buffer. In contrast, in the presence of 2 mM EDTA, the calculated {Delta}Hv.H. is twice as large as {Delta}Hcal (Table II). Such differences are strong evidence for non-two-state behavior, i.e. the system is not an equilibrium system. A similar situation was described for the complex irreversible thermal unfolding of cutinase from Fusarium solani (Petersen et al., 2001).

The thermogram of the GH9 in the presence of cellobiose is still highly asymmetric (Figure 6B). Deconvolution of this thermogram yields at least three calculated independent transitions with Tds of 72.7, 78.4 and 83.2°C. Note that these values are higher than that of GH9 in buffer (67.6°C). The deletion of the Ig-like module reduces the thermostability of the GH9 module, so that even in the presence of cellobiose this module is not converted into a polypeptide with a presumed homogeneous rigid structure.

Kinetic analysis of the Ig–GH9 module pair and its mutants

The temperature optimum (Topt) of the Ig–GH9 was at 65.1 ± 1.42°C (not shown). The Topt values of D264A and T230A/D262A mutants were 60.8 ± 1.1 and 58.6 ± 2.31°C. Hence mutations of the residues located in the module interface exhibit shifts of temperature optima of the protein to lower temperatures. To compare kinetic parameters of the Ig–GH9 construct and the mutants, we used short-term (1 min) incubations at 65°C. In this case no differences in Topt values were observed. Both mutants were catalytically active. The values of KM and kcat for Ig–GH9, D264A and T230A/D262A were 2.0 ± 0.15, 2.3 ± 2.25 and 2.0 ± 1.73 mM and 18.4 ± 2.11, 16.7 ± 2.42 and 19.0 ± 2.35 s–1, respectively. Correspondingly, the efficiencies of catalysis were similar. Hence the intermodular interactions affected the thermal stability of the mutants but they did not affect the kinetic parameters on short-term incubation.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Module interactions have been intensively studied using several proteins of different origins (Jaenicke, 1999Go). It has been concluded that in many cases these interactions stabilize module proteins; in other cases module(s) are actually destabilized upon combination into one polypeptide (Wenk et al., 1998Go; Jaenicke, 1999Go; Douglas et al., 2002). In the case of glycoside hydrolases, it has been noted that in the five-modular xylanase A from the hyperthermophilic bacterium Thermotoga maritima, the modules are coupled together so that the thermal unfolding of the whole enzyme is a highly cooperative process (Wassenberg et al., 1997Go). Recently we demonstrated that three internal modules in thermophilic cellobiohydrolase CbhA X11, X12 and CBM3, interact. These interactions are mediated by calcium, a component of each module. In the presence of calcium, the modules are essentially independent, but in the absence of calcium, module interactions play an important role in stabilization of the rigid structure of the protein (Kataeva et al., 2003Go). In contrast, the two-modular xylanase A from the mesophilic fungus Streptomyces lividans, which is composed of a catalytic and a carbohydrate-binding module, does not show interactions between the two modules. It displays two thermal transitions very close to the transitions of the individual modules (Roberge et al., 2003Go). These observations imply that in (hyper)thermophilic proteins, interactions between modules are an important stabilizing factor, but not in mesophilic polypeptides.

In the present work we continued our studies on the interactions between modules in cellobiohydrolase CbhA. This protein is composed of seven modules and is one of the largest (Zverlov et al., 1998Go) and most abundant (Choi and Ljungdahl, 1996Go) components of the C.thermocellum cellulosome. Gradual deletions of N- and C-terminal modules from the catalytic module of CbhA (or the highly similar CelK) reveal that the smallest enzymatically active construct is the Ig–GH9 module pair (Kataeva et al., 1999Go, 2002Go). Further deletion of the Ig-like module results in the complete inactivation of the catalytic module GH9. The crystal structure of the Ig–GH9 indicates that the two modules have different folds. The far-UV CD spectra of the individual Ig-like and GH9 modules are spectra of all-ß and all-{alpha} proteins, respectively, in agreement with the crystal data. The thermal unfolding of individual modules monitored by both CD spectroscopy and DSC occurs as a cooperative process. This means that both modules expressed individually mostly preserve their native folds. Although the CD and DSC experiments were conducted under very different conditions, the qualitative interpretation of both sets of data is the same: these modules interact tightly when combined into one polypeptide and adopt a final structure which is more stable than those of the individual modules. This conclusion is based on the comparison of the recorded and calculated CD spectra, numbers of spectral transitions revealed by the phase diagram method, denaturation temperatures and calculated calorimetric and van't Hoff enthalpies of the individual modules and the Ig–GH9. In addition, mutations of two or even one amino acid residues located in the modular interface of Ig–GH9 construct clearly demonstrate the importance of the modular interactions for the fold and stability of protein moiety. Both mutants although enzymatically active possess less rigid structure and lower thermostabilities than the native Ig–GH9 construct.

There is no doubt that the inactivation of the individual GH9 module is a result of the loss of multiple module interactions destabilizing its rigid fold and finally affecting the topology of the active site. The crystal structure of Ig–GH9 reveals two conserved calcium-binding sites in the catalytic module GH9, one of which is located close to the active center (Schubot et al., 2004Go). In previous experiments we showed that removal of Ca2+ by EDTA resulted in a partial (~40%) inactivation of Ig–GH9. However, the lower thermostability of the individual GH9 module is not due to loss of calcium as it binds the same amount of Ca2+ as the Ig–GH9 construct. In the individual GH9 module, bound Ca2+ still plays a structural role as treatment with EDTA causes further destabilization of the GH9. A possible mechanism of the inactivation of the GH9 module is a disruption of module interactions in the line D264Ig–Y676GH9–W678GH9 suggested stabilization of the position of tryptophan 678 directly involved in the binding of substrate (Schubot et al., 2004Go). However, the enzymatic properties of Ig–GH9 and its D264A mutant are the same. An interesting conclusion can be drawn from the comparison of thermograms of individual GH9 module in buffer and in the presence of cellobiose. This carbohydrate, composed of two glucose residues (G2), is the end product of substrate hydrolysis and is a powerful competitive inhibitor of CbhA. The presence of cellobiose stabilizes GH9 significantly, increasing its denaturation temperature. This shows that the active site of the catalytically inactive GH9 is still able to bind this substrate analog. The crystal structure of the inactive proton-donor mutant of Ig–GH9(D172A) in the presence of cellotetraose shows that the active site of GH9 accommodates as many as four glucose residues which occupy subsites –2 to +2 (Schubot et al., 2004Go). Presumably, cellobiose in the active site of individual the GH9 module binds to the subsites +1 and +2 (Parsiegla et al., 2002Go; Schubot et al., 2004Go). Correspondingly, the loss of enzymatic activity by the individual GH9 module can be explained by the inability of the active center to bind properly carbohydrates composed of more than two glucose residues. Alternatively, the active site might not be able to perform a hydrolytic attack or to release the hydrolysis product. Studies on binding of carbohydrates of different length in the active center of the individual GH9 module and further modification of the module interface of the Ig–GH9 construct are in progress.


    Acknowledgments
 
Support of this research was provided in parts by US Department of Energy grant DE-FG02-93ER20127/A009 to L.G.L. and by funds to B.-C. Wang from the University of Georgia Research Foundation, the Georgia Research Alliance.


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Received May 27, 2004; revised November 15, 2004; accepted November 30, 2004.

Edited by Daniel Raleigh