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- Materials and methods
The filamentous fungus Trichoderma reesei produces two cellobiohydrolases (CBHI and CBHII). These, like most other cellulose-degrading enzymes, have a modular structure consisting of a catalytic domain linked to a cellulose-binding domain (CBD). The isolated catalytic domains bind poorly to cellulose and have a much lower activity towards cellulose than the intact enzymes. For the CBDs, no function other than binding to cellulose has been found. We have previously described the reversibility and exchange rate for the binding of the CBD of CBHI to cellulose. In this work, we studied the binding of the CBD of CBHII and showed that it differs markedly from the behaviour of that of CBHI. The apparent binding affinities were similar, but the CBD of CBHII could not be dissociated from cellulose by buffer dilution and did not show a measurable exchange rate. However, desorption could be triggered by shifting the temperature. The CBD of CBHII bound reversibly to chitin. Two variants of the CBHII CBD were made, in which point mutations increased its similarity to the CBD of CBHI. Both variants were found to bind reversibly to cellulose.
Cellulose, the major polysaccharide component of plant cell walls, is degraded in nature by the concerted action of a number of bacterial and fungal organisms . The cellulase enzyme system secreted by the filamentous fungus Trichoderma reesei catalyses the hydrolysis of insoluble crystalline substrate to glucose . This system can be separated by various procedures into three major types of catalytic activity: cellobiohydrolase, endoglucanase and β-glucosidase. Two cellobiohydrolases (also called exoglucanases), CBHI [3,4] and CBHII , account for ≈ 80% of the secreted protein. Like many other cellulases, CBHI and CBHII have a tripartite structure with a catalytic core domain linked by a relatively long O-glycosylated polypeptide to a small cellulose-binding domain (CBD). CBDs from different organisms can be grouped into several families . The CBDs of CBHI (CBDCBHI) and CBHII (CBDCBHII) belong to family I. This family is distinct from the others, being the only one found in fungi and having a protein fold unrelated to the others. Their structure displays two distinct faces, one of which is remarkably flat and the other more rough. The flat face contains several conserved aromatic residues and is responsible for the binding to cellulose [7–9].
The role of CBDs in the activity of many cellulases has been studied extensively [10–13]. Removal of the CBD results in a decreased affinity and hydrolytic activity on crystalline cellulose, but does not generally affect the activity towards soluble substrates. In spite of their apparent importance for the degradation of crystalline cellulose and relatively detailed characterization of their binding properties, an acceptable mechanistic explanation for the function of CBDs and their interplay with the catalytic core during the hydrolysis of cellulose is still lacking.
Because substrate binding will also dictate the mobility of the enzyme on the cellulose surface, the reversibility of binding is a key issue for understanding CBD action. Various studies on fungal cellulases have shown that once adsorbed, the desorption of the enzyme requires drastic conditions and that the binding is at least partly irreversible [14–16]. It has been suggested that the irreversible binding is due to the CBD [17–20]. Irreversible binding has been described in particular detail for some bacterial family-II CBDs [21–23]. However, binding of CBDCBHI, which belongs to family I, has been shown to be completely reversible .
In this study we analysed the off rate of CBDCBHII and found that it differs remarkably from the desorption properties of CBDCBHI binding. We also showed that point mutations in CBDCBHII allowed a switch in desorption behaviour. The results for CBDCBHI have in principle been described previously  but are repeated here because they constitute a critical control for the experiments on CBDCBHII.
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- Materials and methods
The aim of this study was to analyse the desorption and exchange rate of CBDCBHII binding to cellulose using the techniques previously used for CBDCBHI. At first glance, CBDCBHI and CBDCBHII have similar binding properties. The values of their apparent partition coefficients are quite close, and binding to cellulose is similarly affected by temperature and pH. However, experiments in this study showed two significant differences in the interaction. (a) When a CBDCBHII–cellulose mixture was diluted at apparent equilibrium (steady-state), the system did not adjust to a new equilibrium on the isotherm. Instead, the bound CBD was not released to return to a point on the isotherm even after a long incubation period (Fig. 2). (b) When exchange rates between labelled and unlabelled protein at the cellulose surface were studied, the CBDCBHII did not show a measurable exchange rate, whereas the exchange rate of CBDCBHI allows equilibrium within 300 s (Fig. 3). From these two observations we conclude that the binding of CBDCBHII is not compatible with a reversible binding model, in contrast with CBDCBHI, as has been demonstrated previously .
The observed behaviour of the CBDCBHII presents a paradox. Binding assays were performed after incubation times that were long enough for the proportion of total CBDCBHII bound to cellulose to remain unchanged, which would suggest that equilibrium was reached. However, dilution of the free CBDCBHII indicated that bound CBDCBHII could not be dissociated. This suggests that the binding is not controlled by thermodynamic equilibrium. As binding of CBDCBHII does not continue until the unbound CBDCBHII is exhausted, it means that binding and desorption are kinetically controlled. In the equilibrium exchange experiments, not only did bound labelled CBDCBHII fail to dissociate on dilution with cold CBDCBHII, but labelled CBDCBHII also failed to bind when added to substrate that had been preincubated with cold CBDCBHII. In apparent contradiction, bound CBDCBHII was able to desorb from cellulose when the temperature of equilibrated mixtures was increased, and binding of CBDCBHII increased when the temperature was decreased.
The unusual behaviour of the CBDCBHII could perhaps be described by a two-step model. The first step would involve a simple reversible adsorption, with the rate constants k+1 for adsorption and a k–1 for desorption, as in the equation:
The first transient reversible complex would in a second step be converted into an irreversible complex with no detectable dissociation rate. The second step might involve a conformational change in the CBD or the cellulose, or some surface-penetration phenomenon, for example the CBD ‘digging’ into the cellulose. The observed temperature-induced desorption could be due to a possible temperature-induced reversal of the change occurring in the second step. The second step would not occur for CBDCBHI or the mutants of CBDCBHII on cellulose or the binding of CBDCBHII on chitin, explaining their reversible binding. This could indicate that the second component is an extra acquired specific property, implying some added benefit for the CBHII enzyme as a whole. However, a comparison of CBHI and CHBII enzymes indicates a very similar role for the CBD in both . This theory has, however, some serious shortcomings. We did not observe a continuous increase in the amount of bound CBDCBHII, which would be expected because the CBDbound, reversible should be depleted as the k2 reaction proceeds and thus drive the k1/k–1 reaction further to the right. Similarly, the theory does not explain why there is no exchange in the equilibrium exchange experiments.
An alternative hypothesis could be that a global change would occur at cellulose surface upon binding of CBDCBHII, thereby preventing any desorption or further binding of CBD. As the effect is observed at CBD concentrations well below the amount required to saturate the substrate, it would seem that some long-range interactions are involved, perhaps through perturbation of the surface layer of the crystal lattice. Whatever changes are responsible for the shift to an irreversible mode of binding, these must be labile, as reversibility is at least transiently restored upon changing the temperature. However, because the irreversibility effect was observed at a very wide concentration range, also as low as nanomolar, it is very difficult to see how this low amount of CBD could provide the necessary energy for such a large conformational change. The change should also be evident in other ways such as the shape of the binding isotherms or visible changes in the substrate, but these are not observed.
Irreversible binding to BMCC has also been reported for the CBD of the mixed function exoglucanase/xylanase Cex (CBDCex) from Cellulomonas fimi[21–23]. CBDCex which also fails to dissociate from cellulose, although the binding isotherm suggests saturable binding with a measurable affinity constant. Irreversible binding does not imply complete immobilization on the surface of the substrate, which would hardly be compatible with the efficiency of the enzyme. Fluorescence recovery after local photobleaching of cellulose-bound fluorescently labelled CBDCex showed that bound fluorescent molecules from unbleached areas were able to diffuse laterally into the bleached patches, although unbound fluorescent CBD failed to bind in the centre of the patches .
The two mutants of CBDCBHII aimed at the most obvious differences between the CBDs. Both mutations made the binding reversible. The W7Y mutation led to a dramatic loss of affinity, while the apparent partition coefficient of the ΔSS3 mutant was only twofold lower than that of the wild-type CBDCBHII. Thus, as observed with CBHI, large differences in desorption rates do not correlate with large changes in apparent binding constants. Which structural parameter is critical for irreversible binding behaviour is not known at present. A possible factor may be the rigidity of the structure. The loss of a disulfide bridge in the ΔSS3 mutant would be expected to increase the flexibility of the structure. Indeed, molecular-dynamics simulation predicted that the structure of CBDCBHII should be more rigid than that of CBDCBHI owing to the presence of an additional disulfide bridge . The W7Y mutation may have the same effect, as it affects a residue the homologue of which in CBDCBHI is known to play a role in the structural framework at the N-terminus of the polypeptide .
The two modes of binding displayed by CBDCBHI and CBDCBHII reflect a significant difference in the way they interact with the substrate. This new variable appears to be largely distinct from true or apparent binding affinity for cellulose. Thus, it will be of interest to discover how it is correlated with the functional properties of CBDs with respect to cellulose hydrolysis.