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Abstract Cellobiohydrolases are the most effective single component of fungal cellulase systems; however, their molecular mode of action on cellulose is not well understood. These enzymes act to detach and hydrolyze cellodextrin chains from crystalline cellulose in a processive manner, and the carbohydrate-binding module (CBM) is thought to play an important role in this process. Understanding the interactions between the CBM and cellulose at the molecular level can assist greatly in formulating selective mutagenesis experiments to confirm the function of the CBM. Computational molecular dynamics was used to investigate the interaction of the CBM from Trichoderma reesei cellobiohydrolase I with a model of the (1,0,0) cellulose surface modified to display a broken chain. Initially, the CBM was located in different positions relative to the reducing end of this break, and during the simulations it appeared to translate freely and randomly across the cellulose surface, which is consistent with its role in processivity. Another important finding is that the reducing end of a cellulose chain appears to induce a conformational change in the CBM.

Simulations show that the tyrosine residues on the hydrophobic surface of the CBM, Y5, Y31 and Y32 align with the cellulose chain adjacent to the reducing end and, importantly, that the fourth tyrosine residue in the CBM (Y13) moves from its internal position to form van der Waals interactions with the cellulose surface. As a consequence of this induced change near the surface, the CBM straddles the reducing end of the broken chain. Interestingly, all four aromatic residues are highly conserved in Family I CBM, and thus this recognition mechanism may be universal to this family. View from top of cellulose surface showing the numbering scheme for the glucose residues. Starting from the top going to the left, they are numbered sequentially from 1 to 60. The orange glucose residues were restrained to their initial configuration during the simulation.

Results and discussion Cellulose Before investigating the interactions of the CBM with cellulose, simulations were conducted with a singly hydrolyzed cellulose substrate alone in water. This allowed the cellulose structure to equilibrate to a solvated conformation. After the glucosidic ether linkage was hydrolyzed, there were two hydroxyl groups located in the space that formerly contained an oxygen atom and this crowding resulted in a puckering of one or both of the terminal glucose residues. A potential energy minimization was conducted to allow relaxation of this packing frustration. This forced the glucose molecule at the reducing end slightly out of the plane of the crystal. After this initial minimization, a heating and equilibration simulation was conducted, followed by an MD simulation that ran for 6 000 000 steps or 12 ns.

During this simulation, the reducing end and the non-reducing end created from hydrolysis twist out of the plane initially, but then the non-reducing end reannealed back into the plane. Figure shows a side view of the top layer cellulose at different times during the simulation. Note that at the beginning of the simulation, the reducing end is slightly puckered out of the plane.

At 4 ns, the non-reducing end is twisted out of the plane and the primary alcohol on the reducing end is pointing out of the plane. After 8 ns, the non-reducing end reannealed back into the plane and there was little change in the cellulose after this. This result suggests that the system had equilibrated. Four simulations considered for this study at the start and after 9 ns. The backbone of the protein is shown as a green ribbon and the three tyrosine residues on the hydrophobic face are shown in orange.

The angles made by these residues relative to the direction of the cellulose chains are shown by the black lines. The non-reducing end of the broken chain (g27) is also shown. Although the CBM appeared capable of significant translation on the cellulose surface, some of the simulations did not suggest a trend to this movement. For example, there did not appear to be a tendency in some of the simulations for the aromatic rings of the tyrosines to line up with the cellulose chains. If CBH I is to process along a cellulose chain, one would expect the CBM to also align with the chain to be hydrolyzed. However, Simulations I and IV showed no tendency for such alignment with the cellulose chains. On the other hand, Simulation III, which started out nearly parallel to the center cellulose chain, did roughly maintain this position.

Simulation II also showed some tendency for the tyrosines to align with the chain adjacent to the broken chain, and after ∼3.5 ns, the tyrosines were indeed aligned with this chain, so that the aromatic rings stacked on top of alternate sugar rings. The CBM maintained essentially this configuration throughout the remaining 9 ns of Simulation II. Future simulations are needed to characterize the association of the CBM with a single or multiple chains of cellulose. Induced fit of CBM In addition to aligning with the cellulose chain, Simulation II also showed that the CBM is capable of structural accommodation, possibly in response to the reducing end of a cellulose chain. This distortion is shown in Fig. As mentioned above, within 3.5 ns the CBM moves so that the three tyrosines (Y5, Y31 and Y32) on the hydrophobic surface align with the cellulose chain adjacent to the broken chain. In this conformation, Y5 stacks on glucose residue g39, which is adjacent to the non-reducing end, Y31 stacks on g41 and Y32 stacks on g43.

Figure b shows a snapshot of the simulation after 4.5 ns. Once the protein is in this position, the fourth tyrosine (Y13) unfolds from its internal location in the CBM and forms a hydrophobic or van der Waals interaction with the cellulose surface on the other side of the broken chain. The aromatic ring in Y13 stacks roughly above the aliphatic hydrogen atoms on C6 of residue g29. This transformation is complete after 5.8 ns, and Y13 remains in this position through the remaining 5.5 ns of the simulation.

The surface-induced conformational structure is shown in Fig. C, which shows the CBM structure after 7.1 ns.

As this figure shows, the CBM straddles the reducing end of the cellulose chain. This type of deformation and added van der Waals interaction with the cellulose was not observed in any of the other CBM simulations conducted for this study, but additional simulations starting with this distorted conformation retained this structure for several nanoseconds.

Furthermore, Simulation III showed a similar distortion without binding between Y13 and the surface. Structures showing the rearrangement of the CBM during Simulation II. Notice tyrosine Y13 in purple is ( a ) faced into the protein at the start and ( b ) after the three other tyrosine residues align with the cellulose chain, but faces the cellulose in ( c ). Note that in Simulation II the reducing end of the broken chain (g28) has twisted the opposite direction from the other simulations. In Simulation II, O6 on g28 remains hydrogen-bonded to the adjacent glucose residue (g40), whereas O2 and O3 of g28 have broken their hydrogen bonds with g16 and they point out of the plane. In the other CBM simulations and the simulation without the CBM, the O6 on g28 is not hydrogen-bonded to g40 and O2 and O3 on g28 remain hydrogen-bonded to g16.

The rearrangement of the CBM to encapsulate the reducing end of the broken chain strongly suggests that there may be an induced fit substrate/enzyme interaction. That is, a possible mechanism of recognition and specificity of the CBM is the rearrangement of the CBM to fit the reducing end of a cellulose chain. The odd twist of the cellulose chain further suggests that the substrate may also need to deform in order for this fit to occur.

Induced-fit substrate–enzyme interactions are well-known phenomena (;; ), although this has not been suggested for Family I CBMs. Interestingly, the four aromatic residues in this study Y13, Y5, Y31 and Y32 are highly conserved in this family. Figure shows a comparison of the sequences for a variety of Family I CBMs and shows that these four tyrosine residues match tyrosine, tryptophan or phenylalanine in the other family I CBMs. Thus, it seems likely that CBMs in this family have the ability to distort in a manner similar to that shown in Fig.

It is not clear why this rearrangement was only seen in one simulation. It is possible that this response was not observed in the other simulations due to insufficient conformational sampling (simulation time) required to bring the various initial orientations of CBM and cellulose chain into biologically relevant position. Ongoing work is aimed at further investigating this interaction and quantifying its lifetime and probability.

Amino acid sequences for several Family I CBMs: ( a ) exoglucanases, ( b ) endoglucanases and ( c ) esterases. The structural changes induced in the CBM during Simulation II are consistent with the flexibility of the loop containing Y13 in this otherwise rigid protein. Figure shows a cartoon representation of the protein structure with its three-strand β-sheet. In the β-sheet, strand β3 (residues 33–36) is hydrogen-bonded to strands β1 (residues 5–9) and β2 (residues 24–28). The rigidity of the β-sheet is further strengthened by a disulfide bridge between β1 (residue 8) and β2 (residue 25). A small loop between β2 and β3 is restricted because of hydrogen bonds formed between the backbone carbonyls and amides in this group and the side chain on an asparagine, N29. The β-sheet and this tight loop contain three tyrosine residues that make up the hydrophobic surface of CBM I.

This rigidity allows the CBM to maintain the spacing of the tyrosine rings to match the cellobiose periodicity in cellulose. A second disulfide bridge between residues 19 and 35 adds rigidity to the loop between residue 19 and the start of β2 (residue 24). The remainder of this loop (residues 10–19) is more flexible. A picture showing the beta sheets and the disulfide bonds (yellow) ( a ) before and ( b ) after (right) Y13 (purple) has unfolded. The three tyrosines on the hydrophobic face are shown in orange.

Plots of the spatial deviation of each residue during Simulations I–IV show the rigidity of different sections of the CBM. During each step of the simulation, the backbone of the protein is aligned to the starting structure and the average of the deviation of each individual amino acid for each simulation was calculated, i.e.

The root mean squared deviation (RMSD) over all atoms in each residue was determined for each time step in the simulation. Figure a and b show plots of the average RMSD for each residue during each simulation. We note that there was considerably more movement of the residues 11–17, which is in the loop between β1 and β2 and contains Y13. The flexibility of this section of the CBM allowed movement of the fourth tyrosine and enabled it to bind to the hydrophobic cellulose surface near the reducing end. The plot in Fig.

C shows the RMSD for each residue for Simulation II averaged over the starting 2 ns (red) of the simulation and the last 2 ns of the simulation (black), after the protein has distorted. From this plot, one can clearly see that the fourth tyrosine (Y13) clearly underwent a large displacement after the protein distorts. Conclusions We believe that we are the first to show, using computational simulation, that Family I CBMs in proximity to cellulose are capable of distorting so that the fourth (and remaining) tyrosine can establish hydrophobic interactions with the cellulose surface. Now that this apparent induced fit mechanism has been observed for the CBH I CBM, one may conclude, considering the high homology demonstrated by other Family I CBMs, that all type 1 CBMs are capable of this conformational change. The interaction of four tyrosines with cellulose is entirely consistent with the CBMs found in bacterial enzymes; however, this interaction has not been reported for fungal CBMs. That this rearrangement only occurs in one simulation may be an indication that in the other simulations, the sampling was not long enough to correct for less productive starting orientations of CBM and cellulose.

The observation that in Simulation II distortion occurred after the reducing end twisted in the opposite direction from the other simulations may provide an indication of the substrate conformations necessary to induce this conformational change. In more than one of the MD simulations, significant motion of the CBM was observed. The observation that the CBM translates freely and randomly on the cellulose surface is potentially of great interest for understanding the mechanism of processivity.

This translation occurred in both perpendicular and parallel directions relative to the cellulose chains and appears to be undirected. This implies that the entire enzyme complex is required for processivity.

The ease of two-dimensional translation is thus likely a requirement for efficient location of and interaction with reducing ends on the cellulose surface. Such translational freedom would be necessary in the event the enzyme adsorbs on the surface far from a reducing end. The general nature of the interactions we observed for the Family 1 CBM with the cellulose surface using computer simulations reported here are consistent with traditionally held views of the role that hydrophobic interactions play in this process (see Introduction). The hydrophobic face of the three tyrosine rings stack on the hydrophobic (1,0,0) cellulose surface and there are only occasional hydrogen-bonding interactions between amino acids of the CBM and the hydroxyl groups of the unhydrolyzed glucose residues. The weakness of these individual van der Waals interactions is compensated for by the cumulative strength of the multiple hydrophobic coupling interactions and thus the CBM is tightly bound to the surface, while able to retain two-dimensional translational freedom.

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This critical property of translational freedom may be inhibited when the hydroxyl groups on a reducing end of a broken cellulose chain, which would now be puckered away from the surface, interact with the amino acids on the 1–5 loop of the CBM, or when the CBM distorts in response to the reducing end of the cellulose chain, enabling a new hydrophobic interaction with the cellulose surface using the fourth tyrosine residue. In this way, this CBM type may ‘recognize’ and respond productively to the reducing end of a cellulose chain. In a recent CBM/cellulose computational docking study reported by, the CBM was placed in a particular orientation relative to cellulose based on the premise that the CBM ‘dives under’ the top cellulose layer.

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This work illustrated possible sites for interaction, primarily H bonds, on the topside of the CBM with the displaced cellodextrin. In contrast, our present study permits the CBM to ‘ride’ near the broken cellodextrin lying in the cellulose surface and find, using MD, low-energy conformations. It is possible that both the surface decrystallization model proposed here and the cellulose reducing end entry model proposed by Mulakala and Reilly are consistent with biological function.

Acknowledgments The authors thank Drs Mark Miller and Fran Berman for their support regarding the computational resources of the San Diego Supercomputer Center during the course of this work. Dr Charles Brooks and Dr Philip Mason also provided valuable comments and guidance to the authors.

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This research was supported in part by the National Science Foundation through the San Diego Supercomputer Center under grant SCI-0438741. DOE Office of the Biomass Program also provided funding for this work.