Energetics for displacing a single chain from the surface of microcrystalline cellulose into the active site of Acidothermus cellulolyticus Cel5A

C.E. Skopec1, M.E. Himmel2, J.F. Matthews1 and J.W. Brady1,3

1Department of Food Science, Stocking Hall, Cornell University, Ithaca, NY 14853 and 2National Renewable Energy Laboratory, National Bioenergy Center, 1617 Cole Boulevard, Golden, CO 80401-3393, USA

3 To whom correspondence should be addressed. e-mail: jwb7@cornell.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A series of molecular mechanics calculations were used to analyze the energetics for moving a single polysaccharide chain from the surface of microcrystalline cellulose into the binding cleft of the Cel5A cellulase from Acidothermus cellulolyticus. A build-up procedure was used to model the placement of a 12-residue oligosaccharide chain along the surface of the enzyme, using as a guide the four residues of the tetrasaccharide substrate co-crystallized with the protein in the crystallographic structure determination. The position of this 12-residue oligosaccharide was used to orient the enzyme properly above two different surfaces of cellulose 1ß, the (1,0,0) and the (1,1,0) faces of the crystal. Constrained molecular dynamics simulations were then used to pull a target chain directly below the enzyme up out of the crystal surface and into the binding groove. The energetics for this process were favorable for both faces, with the step face being more favorable than the planar face, implying that this surface could be hydrolyzed more readily.

Keywords: cellulase interactions/cellulase mechanisms/cellulose/conformational energy calculations


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The hydrolysis of cellulose, the major component of plant cell walls and the most abundant biological molecule in the biosphere, is a very slow process. The primary reason for its slow conversion to cellobiose and free glucose is that cellulose is completely insoluble under ambient environmental conditions. This insolubility has been the principal technical challenge in the cost-effective industrial production of glucose from cellulose for use in fermentations to produce alcohol (Wyman and Hinman, 1990Go). Certain organisms have evolved enzymes capable of hydrolyzing the glycosidic bonds in cellulose, but the turnover rates for these enzymes are low. It may be possible to overcome this problem by designing cellulases with improved activity through site-directed mutagenesis, but this approach will require an understanding of the mechanistic details of cellulose–cellulase interactions.

Cellulase enzymes can be classified into two types: exocellulases (including cellobiohydrolases) which attack the polymer chains only from their ends and endocellulases capable of breaking the chains at random internal positions. A great many cellulases have now been identified and characterized. Many of these enzymes consist of three domains: a globular catalytic domain (CD), a much smaller cellulose binding module (CBD) which is also globular and a non-globular linker peptide region connecting the first two domains. The growing number of identified cellulases have been classified into evolutionarily related families based on similarities in the primary sequences of their catalytic domains. Several cellulases have had the tertiary structures of their catalytic or binding domains determined by X-ray crystallography (Rouvinen et al., 1990Go; Davies et al., 1993Go; Spezio, 1993Go; Sakon et al., 1996Go; Tormo et al., 1996Go), which is of course essential for understanding the basis for their hydrolytic mechanism.

One particularly interesting cellulase is Cel5A from Acidothermus cellulolyticus. A.cellulolyticus is a thermotolerant, cellulolytic actinomycete bacterium (Baker et al., 1994Go). Isolated from an acidic hot spring in Yellowstone National Park, it secretes a number of highly thermostable, active enzymes which degrade cellulose. The Cel5A enzyme from this organism, initially referred to as E1, has been purified and the structure of its CD has been determined through X-ray crystallography (Sakon et al., 1996Go). Cel5A is a retaining endogluconase belonging to family 5 and clan GH-A. The Cel5A structure is designated as an ({alpha}/ß)8 barrel (Sakon et al., 1996Go). Family 5 is the largest cellulase family and all family 5 enzymes cleave the glycosidic bond with retention of configuration. Although the optimum temperature for Cel5A is 81°C, the enzyme has activity comparable to Trichoderma reesei endoglucanases at ambient temperatures (Ziegler et al., 2000Go). Cel5A has 521 amino acids, with 358 in the catalytic domain (Sakon et al., 1996Go). The catalytic domain alone functions synergistically with T.reesei Cel7A (CBH I).

Naturally occurring cellulose consists of both microcrystalline and amorphous regions. Cellulases from different sources have differing proportions of these amorphous and crystalline regions. In the enzymatic hydrolysis of cellulose, the rate of glucose turnover is affected by the degree of substrate crystallinity, the specific surface area, the degree of polymerization and the unit cell dimensions of the cellulose material (Lee et al., 1983Go; Mansfield et al., 1999Go). Several studies have investigated the effects of cellulose characteristics on the ability of cellulases to fragment crystalline cellulose into accessible, degradable strands (Chang et al., 1981Go; Lee et al., 1983Go; Rivers and Emert, 1988Go; Sinitsyn et al., 1991Go; Wilson, 1992Go; Irwin et al., 1993Go; Jackson et al., 1993Go; Kleman-Leyer et al., 1996Go; Mansfield et al., 1996Go; Lee et al., 2000Go; Schwarz, 2001Go).

Early workers on enzymatic degradation of cellulose thought that crystallinity was the major rate-limiting factor influencing the degree of hydrolysis (Mansfield et al., 1999Go). It was thought that only amorphous regions of the cellulose material are hydrolyzed. Studies of various organisms have found that they do contain cellulases capable of degrading crystalline cellulose or an insoluble artificially made cellulosic substrate such as filter-paper (Lee et al., 1983Go; Sinnott, 1990Go; Wilson, 1992Go; Irwin et al., 1993Go). For most cellobiohydrolases, the extent of hydrolysis is greater on crystalline cellulose when both the catalytic domain and the binding domain of the enzyme are present (Irwin et al., 1993Go; Teeri, 1997Go; Zhang et al., 1999Go; Lee et al., 2000Go). The cellulose binding domain is thought by some to be capable of disrupting the crystallinity and providing the catalytic domain with a suitable substrate.

In 1983, Lee et al. found that enzyme catalysis does not significantly affect the degree of polymerization (DP) of solid cellulose substrate. These authors proposed that cellulose chains are peeled off progressively from the fibrils by the cellulase enzymes since the DP remains constant during the course of hydrolysis (Lee et al., 1983Go). Kleman-Leyer et al. and Chang et al. have established that the extent of enzymatic hydrolysis is limited by the inherent degree of polymerization and also by the crystallinity and accessible surface area or pore size of the cellulose material, but again, found that the DP does not change as the reactions progress (Chang et al., 1981Go; Kleman-Leyer et al., 1996Go). This result may indicate that removing a chain of cellulose from the crystalline structure provides the means for hydrolysis of the entire chain.

In 1999, Lee et al. were able to show with atomic force microscopy that an endoglucanase from T. reesei caused peeling and smoothing of the crystalline cellulose fiber, which increased with the addition of an exocellulase (Lee et al., 2000Go). This finding gives rise to the idea that some cellulase mixtures are capable of degrading crystalline cellulose by removing whole chains from the surface and subsequently hydrolyzing the entire chain, leaving a new surface. This was not observed with cellulases lacking a binding domain. The Ac-Cel5A CD, however, is capable of hydrolyzing crystalline cellulose in the presence of exocellulases, without a binding domain (Baker et al., 1994Go) and yet is distinctly synergistic with exocellulases. These results support the concept of a ‘processive’ endoglucanase. Examination of the crystal structure of Cel5A shows the presence of a significant glycosyl-binding platform, extending along the surface of the protein to the –5 or –6 glycosyl sub-site positions. This extended sub-site-binding platform may mimic the function of the CB module (McCarter et al., 2002Go).

Whatever the mechanism or physical property causing the rate of glucose production to be slow, the disruption of crystalline cellulose requires the breaking of hydrogen bonds. When a chain of cellulose is separated from the crystal, hydrogen bonds between that chain and the rest of the crystal are lost and reorganization of the chain and the crystal must occur to minimize the change in energy. The energetics of removing a chain from an ordered layer in crystalline cellulose have not been previously examined. Nor have there been any studies of the effects of cellulase proteins, either CBDs or CDs, on the energetics of chain removal from a crystalline cellulose surface. However, if an enzyme such as Cel5A is able to hydrolyze microcrystalline cellulose, it would be necessary for the energy change for transferring a chain of cellulose from the crystal surface to the binding site to be either favorable or only slightly unfavorable. In either case, however, an energy barrier might exist along the reaction path for such a process which could substantially slow the overall hydrolysis rate. We report here a series of simulations which investigated the energy required to remove a chain of 12 glucose residues from a crystalline cellulose fragment and bind it into the active site of Ac-Cel5A. Ac-Cel5A makes a useful, simple model since only the catalytic domain is required for catalysis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The cellulose structure used for all the calculations reported here was cellulose Iß from the algae source Valonia, the crystal structure of which was proposed by Finkenstadt and Millane from X-ray fiber diffraction (Finekenstadt and Millane, 1998Go). A monoclinic crystal, this cellulose Iß structure has a {gamma} value of 96.3° with 2-fold screw symmetry and with lattice parameters a, b and c of 7.85, 8.27 and 10.38 Å, respectively (Finekenstadt and Millane, 1998Go). The unit cell consists of two cellobiose units. A four-layer, six-chain/layer, 12-residue/chain crystal was constructed with these lattice parameters using the crystal-building facility in the general molecular mechanics program CHARMM (Brooks et al., 1983Go). This crystal was long enough to accommodate the length and width of the Cel5A protein when it is placed on top of the crystal surface. The edge faces of the crystal fragment were the (1,0,0), (0,1,0,) and (0,0,1) planes. Figure 1 shows the unit cell directions and the exposed surfaces for this cellulose crystal. In the cellulose Iß crystal, there is extensive hydrogen bonding along the chains and between the chains of a layer. Two hydrogen bonds occur along the cellulose chains, from the O5 atom of one residue to the HO3 atom of the previous residue and from the HO3 atom of the same central residue to the O5 atom of the next residue in the chain. Three hydrogen bonds occur between chains: from the O3 atom of a residue in a central chain to the HO6 atom of an adjacent chain on one side and two to the adjacent chain on the other side, from HO6 to O3 and from O6 to HO2.



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Fig. 1. Three views of several crystal faces of the starting structure for the regular cellulose microcrystal constructed for these simulations. In the top view the (1,0,0) face is shown end-on, looking down the c-axis and the length of the regular ribbon-like chains, while the bottom view along the b-axis of this face is perpendicular to the length of the chains.

 
The model crystal was minimized using the Adopted Basis Newton–Rhapson (ABNR) algorithm for 50 steps without constraints, to relive any serious strains resulting from the setup procedure or inaccuracies in the crystal structure determination. Hydroxyl hydrogen rotation occurred during this minimization, but the ordered shape and primary alcohol positions remained constant. The bottom two layers of the crystal were fixed in this minimized position in subsequent calculations with the enzyme present.

The coordinates for the Cel5A CD were those determined by Sakon and co-workers and deposited in the Protein Data Bank under the code 1ECE-a (Sakon et al., 1996Go). The enzyme was crystallized with cellotetraose in the active site. This co-crystallization was achieved by growing the enzyme in excess cellobiose (0.3 M) to promote, or allow, the reverse process of cellobiose glycosidic condensation (cellobiose dehydration) for 1 month at room temperature (Sakon et al., 1996Go). For the present modeling purposes an oligomer of 12 glucose residues was constructed in the active site cleft based on the positions of the crystallographically determined coordinates for the four central residues of the chain. Although the active site binding cleft in Cel5A is much shallower than in other cellulases, superimposing an extended cellulose chain on to the coordinates of the four crystallographically-placed glucose residues requires a significant distortion of the polysaccharide chain conformation away from the flat ribbon found in the cellulose crystal, and the disruption of many of the interchain interactions found in the crystal.

The 12-glucose oligomer chain was built by first adding two glucose residues (one at either end of the chain) to the crystallographic tetramer, such that six residues filled the binding cleft. The residues were added using CHARMM and only the sugars were minimized with the ABNR algorithm for 100 steps while the protein was kept fixed. This was done to remove any steric clashes which may have resulted between the new residues and the protein. A Langevin molecular dynamics (MD) simulation (Brünger et al., 1984Go) of Cel5A with cellohexaose in the active site was then performed for 100 ps, with a 50 ps equilibration period during which only the sugars were allowed to move. The final structure was minimized with the ABNR algorithm for 100 steps before the next residues were added. This procedure ensured a stable structure and good placement of the substrate residues before the chain was extended. Again two residues were added, followed by the same minimization and dynamics regimen and this procedure was repeated three times. The result was a 12-residue chain in the active site. Although the resulting placement of the chain is not guaranteed to be correct, it provides a guide for the orientation of the enzyme over the cellulose surface in such a way as to minimize the distortions of the surface cellulose chains required to pull one chain up from the surface and into the binding site.

The model Cel5A with the 12-oligomer chain in the active site was placed on two faces of the model cellulose crystal using the molecular graphics program Quanta® (MSI, 1994Go). The two faces chosen were the (1,0,0) face and the (1,1,0) face of cellulose Iß. The enzyme was oriented such that the ends of the substrate entering and exiting the active site binding cleft ran parallel to the chains in the top face of the crystal surfaces. The ends of the bound chain were lined up against a chain within the crystal surface. This was done so that the enzyme was placed as closely as possible in the proper position with respect to the crystal surface. Thus the enzyme was situated above the crystal surfaces with the active site of Cel5A over a specific chain. This chain in the crystal was designated as the chain of interest and the one that which would be moved from the crystal surface and into the binding cleft. Once the enzyme was properly oriented, the 12-oligomer bound chain was then deleted from the enzyme.

The original coordinates of the cellotetraose that was crystallized in the active site of Cel5A were next added back to the complex of the enzyme on the crystal as virtual atoms to serve as a guide for positioning the surface chain in the active site. The hydrogen atoms of this virtual cellotetraose were deleted and the heavy atoms were changed to represent atoms of zero radius and zero charge. These original substrate atoms were subsequently referred to as ‘dummy atoms’. The dummy atoms represented specific positions in the active site onto which the atoms in the chain of interest could be mapped. The distance between the real atoms in the surface chain and the target dummy atoms in the active site was then decreased over a series of dynamics simulations such that the chain moved from the crystal surface and into the enzyme binding cleft. The whole complex now involved the enzyme situated over a chain on the surface of the cellulose crystal. Figure 2 illustrates the starting structure for moving the chain from the crystal into the active site of Cel5A from the (1,0,0) face and Figure 3 illustrates the starting structure for moving the chain from the crystal into the active site of Cel5A from the (1,1,0) face. As can be seen from the position of the ‘dummy’ tetrasaccharide atoms, the distance that the surface chain must be moved is not insubstantial.



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Fig. 2. The starting structure for moving the chain from the (1,0,0) crystal surface into the active site of Cel5A. (A) View from the (0,0,1) face; (B) view from the (0,1,0) face. The protein is shown as a ribbon structure for the backbone only. The yellow atoms represent the experimental coordinates for the cellotetraose that was co-crystallized in the active site.

 


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Fig. 3. The starting structure for moving the chain from the (1,1,0) crystal surface into the active site of Cel5A. (A) View from the (0,0,1) face; (B) view from the (–1,1,0) face. The protein is shown as a ribbon structure for the backbone only. The yellow atoms represent the experimental coordinates for the cellotetraose that was co-crystallized in the active site.

 
For both faces, the starting structure was equilibrated for 100 ps, using a Langevin integration scheme (Brünger et al., 1984Go) at 300 K. Non-bonded interactions were truncated at 13.0 Å on a neutral group-by-group basis after being made to go smoothly to zero between 10.0 and 12.0 Å using ST2-type switching functions (Stillinger and Rahman, 1974Go; Brooks et al., 1983Go). Chemical bond lengths involving hydrogen atoms were kept rigid using the constraint algorithm SHAKE (van Gunsteren and Berendsen, 1977Go). Owing to the inherent asymmetry of the system, no attempts were made to extend the surface through the application of crystal periodicity.

The targeted cellulose chain was moved into the binding groove of the cellulase as a function of distance using a series of constrained Langevin MD simulations. The centers of geometry between the dummy atoms in the active site and the atoms in the chain to be pulled up were equilibrated at a specific starting distance. This distance was constrained using an umbrella potential with a force constant of 40.0 kcal/mol·Å2. For the (1,1,0) surface, the chain was held at a distance of 5.0 Å for a 100 ps simulation and for the (1,0,0) surface the chain was held at 4.5 Å for a 100 ps simulation. The final coordinates for the simulations were minimized to an energy tolerance of 0.02 kcal/mol using the Conjugate Gradient algorithm. The constraint distance between the chain atoms and the dummy atoms was then decreased by 0.5 Å over a subsequent 100 ps simulation. This simulation was started from the last step of the previous calculation. This process was repeated until the distance was equal to zero between the centers of geometry of the chain and the dummy atoms in the active site. The energy of each structure was plotted as a function of distance for both faces to obtain an energy trend for moving the chain from the surface and into the active site of Cel5A.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cel5A docked on the (1,0,0) face of cellulose Iß

The final structure of the chain in the active site is shown in Figure 4. The chain was moved into the binding cleft over the course of 0.9 ns of constrained MD simulation. The distance between the centers of geometries of the chain and the dummy atoms decreased by a total of 4.5 Å over the entire series of simulations. The energy of each minimized structure along the trajectory was plotted against each constraint distance as the chain was moved in the direction of the dummy atoms situated in the binding cleft of Cel5A. The energy profile that resulted, shown in Figure 5, illustrates that the removal of a chain of cellulose from the crystal structure and its subsequent interaction with the enzyme is a favorable process. The reported energies are relative to that of the starting structure, which was the equilibrated structure of Cel5A on the (1,0,0) surface with the chain of interest flat in the crystal. The very large magnitudes of the energy changes suggest that this procedure, which ignored solvent water molecules, among other approximations, was not quantitatively realistic, but the overall trend is as might be expected.



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Fig. 4. Final structure of Cel5A on the (1,0,0) face of the cellulose Iß crystal with the chain of interest in the binding cleft colored yellow, the conserved residues in the active site colored green, the backbone colored red and the crystal colored blue. (A) View from the reducing end of the (0,0,1) face; (B) view of the (0,1,0) face.

 


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Fig. 5. Energy profile of the total system for pulling a chain of cellulose from the regular (1,0,0) crystal surface and into the active site of Cel5A, relative to the equilibrated, minimized structure of Cel5A on the (1,0,0) face of the cellulose Iß crystal (squares); energy profile for just the interaction energy of the chain with Cel5A as the chain of cellulose is moved from the regular (1,0,0) crystal face and into the active site of Cel5A, relative to the minimized, equilibrated structure of Cel5A on the (1,0,0) surface with the chain of interest flat in the crystal (triangles); and the interaction energy of the chain of interest with just the crystal (diamonds).

 
Although this process was ‘downhill’ in energy, with a total difference of 80 kcal/mol between the maximum and minimum energy geometries, there appeared to be an increase in the energy again as the separation distance approached zero. This increase in energy was 33.1 kcal/mol. Almost all of this increase came from the protein and chain interaction energy, which increased by 30.4 kcal/mol at the 0.0 Å distance. The overall direction in the interaction energy is downhill as would be expected. As the chain was moved from the crystal and into the binding cleft, there was an increase in the number of interactions that occurred between Cel5A and the chain. The energy trend for just the interaction energy of the chain of interest with Cel5A is also shown in Figure 5. This figure exhibits the same trend over the final simulation of the trajectory, as the chain moved into the binding cleft from 0.5 Å away, indicating that a change in the conformation of the protein or the chain occurred during this simulation which made the interaction energy of the structure unfavorable.

Comparing the structure that was obtained after constraining the distance at 0.5 Å to that which was constrained at 0.0 Å, there is a significant change in the position of His116, a conserved residue in the active site of Cel5A. Figure 6 shows the {chi} angle of His116 during the 100 ps of the 0.0 distance simulation. The consequence of the conformational transition seen in Figure 6 is shown in Figure 7. This depicts a change in a favorable hydrogen bond between His116 H{epsilon}2 and O2 of the residue in the –1 position. These atoms are colored yellow. The second residue is Trp319, which aligns with His116 for a hydrophobic interaction. The alignment of this stacking is more parallel in the 0.5 Å structure. This hydrophobic interaction occurs in the original crystal structure and His116 makes a hydrogen bond with the O3 atom of the –1 residue. As the chain was pulled closer to the active site, less room was available to the residues within the binding cleft. His116 had to tilt to accommodate the incoming chain. Similarly, there was also a major change in the {phi} and {psi} angles of the +1 and +2 residues in the chain at the 0.0 Å distance. The {phi} and {psi} trends for these residues are shown in Figure 8.



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Fig. 6. {chi} angle of residue His116 during the 100 ps simulation at the 0.0 Å constraint distance (see text).

 


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Fig. 7. The altered position of His116 in the 0.0 Å constraint distance structure (B) as compared to the 0.5 Å distance structure which is lower in energy (A). The favorable N{epsilon}2 (yellow) hydrogen bond to the O2 (yellow) of the –1 residue in the chain is removed during the 0.0 Å distance structure. The second red residue is Trp319, which makes a hydrophobic interaction with His116.

 


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Fig. 8. The {phi} and {psi} angle values for the glycosidic linkage of the sugar residues in the +1 and +2 positions showing a change in conformation when the distance between the center of geometries is decreased from 0.5 to 0.0 Å.

 
The large change in the {phi} and {psi} angles contributed to the increase in energy that occurred as the chain moved from the 0.5 Å distance and into the active site. This conformational change alters the hydrogen bonds of the residues in the +1 and +2 positions compared with that in the structure at the 0.5 Å distance, such that two hydrogen bonds are lost. One of these is between the HO2 of the –1 residue and OH of Tyr245 and the other is between the O3 of the +1 residue to the H{epsilon}1 of the N{epsilon}1 in Trp213.

During the process of the chain leaving the crystal, reorganization of the hydrogen bonding within the crystal also occurred and new interactions between the chain and enzyme were formed. Not surprisingly, examination of the hydrogen bonding shows that an increase in the number of interactions between the chain and the enzyme occurred during the course of the simulation. Consequently, the number of hydrogen bonds decreased between the chain and the crystal. Table I gives the number of hydrogen bonds that occurred after minimization of the final structure at each distance.


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Table I. Hydrogen bond numbers at each distance along the trajectory for the (1,0,0) face calculation showing similar counts for the total number of interactions, but a difference in the number of hydrogen bonds between segments
 
The low-energy structures for just the protein and the chain, that energy which does not include the rest of the crystal, occur at the 1.0 and 0.5 Å distances. This is where the largest number of hydrogen bonds occurred between Cel5A and the chain. Three hydrogen bonds were lost between the chain of interest and Cel5A when the chain was moved 0.5 Å from the active site to the 0.0 Å distance. As was stated, two of the hydrogen bonds were lost owing to the conformational change in the {phi} and {psi} angles and the third hydrogen bond was lost due to the change in the {chi} angle of His116 discussed above.

As the chain is removed from the crystal, the crystal undergoes a transformation from a perfectly ordered structure to a structure more indicative of amorphous regions in cellulose. The crystal fragment which was used in this calculation was very small and the contribution of the electrostatic interaction to the total energy due to the alignment of the glucose chain dipoles is large. This dipole energy contribution cannot be separated from the energy contribution of hydrogen bonds in the electrostatic energy term. The energy change of just the crystal as the chain is moved from the surface and into the active site of Cel5A is therefore hard to specify accurately. The interaction energy of the crystal with the chain increased as the chain was removed from the cellulose surface. This energy trend is also shown in Figure 5. As the chain atoms moved further from the crystal atoms, a decrease in the number of favorable interactions occurred and the interaction energy increased.

The r.m.s. differences between the final structure obtained at the 0.0 Å separation distance and the original crystal structure are given in Table II. There is a significant r.m.s. difference for the cellotetraose, but not a significant change in the protein. The conformational difference between the original cellotetraose structure and the residues of the chain that were moved into the active site are depicted in Figure 9. The original crystallographic position of the four-residue chain is shown in yellow and the chain moved from the cellulose crystal is indicated in blue. There is a large deviation in the –1 residue of chain. Figure 10 shows the relationship between the polysaccharide chain atoms in the final structure and those of the five conserved residues in the active site. The final distance from the Glu162 residue to the glycosidic oxygen is 4.53 Å. The conserved residues are labeled and colored red in Figure 10 and the residues in the cellulose chain are numbered and colored blue.


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Table II. R.m.s. deviation between the final structure at the 0.0 Å distance and the original (1,0,0) face crystal structure
 


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Fig. 9. The original substrate as crystallized by Sakon et al. (1996) and the final chain structure that was obtained at the 0.0 Å distance for the (1,0,0) surface calculation. The original substrate is yellow and the final structure is blue. The (1->4) glycosidic bond goes left to right.

 


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Fig. 10. The final active site structure showing the five conserved residues involved in binding of the substrate and the activity of Cel5A. The distance from the OH of Glu162 to the glycosidic oxygen (yellow) is 4.53 Å.

 
The r.m.s. deviation of the five conserved residues in the active site which were in close contact with the substrate in the crystal structure from those same residues in the final structure at the 0.0 Å constraint distance was 1.52 Å. The docking procedure used here did not alter the conformation of the protein, including the residues in the active site, to a great extent. The energy for the original cellotetraose substrate was greater by 21.5 kcal/mol than that of the four residues mapped onto the dummy atoms from the chain of interest. However, this procedure did a poor job of mapping the chain from the crystal on to the active site dummy atoms for this face. The glycosidic oxygen did not come within nucleophilic attack distance of Glu282 and hydrogen bonds found in the original structure were not present in the final structure at 0.0 Å. In the crystal structure model, His116 hydrogen bonds to the O3 group of the –1 residue and Asn161 hydrogen bonds to the O2 group of the –1 residue. However, both of these amino acids were too far from the sugar in the –1 position in the minimized coordinates of the 0.0 Å constraint distance structure. It is thought that in the first steps of the hydrolysis reaction residue Glu162 hydrogen bonds to the glycosidic oxygen of the scissile bond. In the final minimized structure at 0.0 Å distance, these two atoms are too far apart for a hydrogen bond to occur. Also, this calculation was done without any sort of periodic boundary conditions. The edge effects influenced the movement of the end residues in the crystal. This can be seen in Figure 4. An end residue of the chain of interest made a hydrogen bond with Gln89 of Cel5A. This may be an important interaction in the movement of the chain; however, Cel5A situated on an extended crystal surface or one with periodic boundaries would need to be modeled to confirm this.

Other residues were found to hydrogen bond to the chain of interest at the 0.0 Å distance. These residues were Asn80, Gln89, Arg117, His164, Asp324 and Ser325. These residues may be important in assisting the movement of the chain from the crystal surface and up into the binding cleft. Mutagenesis studies would help to determine the extent to which these interactions are compulsory for hydrolysis.

Cel5A docked on the (1,1,0) face of cellulose Iß

The final structure of the chain in the active site of Cel5A, after being moved from within the (1,1,0) surface of the crystal, is shown in Figure 11. With the (1,1,0) surface, the enzyme is located on the face where the chains are tilted at a 45° angle, such that both the tops and sides of the chains constitute the accessible surface of the top layer. This orientation makes this face more hydrophilic then the (1,0,0) face and more capable of making hydrogen bonds with the enzyme.



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Fig. 11. Final structure of Cel5A on the (1,1,0) face of the cellulose Iß crystal with the chain of interest in the binding cleft colored yellow, the conserved residues in the active site green, the backbone of Cel5A red and the crystal blue. (A) View from the reducing end of the (0,0,1) face; (B) view of the (–1,1,0) face.

 
The energy trend for moving the cellulose chain of 12 residues from the (1,1,0) face of the regular crystalline cellulose complex into the active site of Cel5A is shown in Figure 12. The process was overall a ‘downhill’ one, with an energy barrier at the 1.5 Å distance mark. This barrier had a relative magnitude of 28.5 kcal/mol. The overall process lowered the energy of the system by 107.7 kcal/mol, relative to the starting structure, which was the minimized, equilibrated structure of Cel5A on the (1,1,0) step face with the chain of interest flat in the crystal. Again, the very large magnitude of the overall energy change probably is not quantitatively accurate owing to the many approximations.



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Fig. 12. Total energy trend (circles) for moving a chain from the (1,1,0) face of the regular crystalline cellulose fragment and into the active site of Cel5A, relative to the starting structure; interaction energy for just the chain of interest with the rest of the crystal relative to the starting structure for the (1,1,0) face (triangles); and the energy trend for just the Cel5A as a function of distance that the chain of interest is from the active site, relative to the starting structure (squares).

 
The interaction energy of the moving chain with just the rest of the crystal is also shown in Figure 12. Not surprisingly, this interaction energy consistently increases, but the largest increase occurred at the same distance that the barrier was seen in the total energy profile. When considering just the energy of the crystal and the removed chain, the energy barrier was 34.6 kcal/mol. As the chain was moving from the crystal and into the active site of Cel5A, the hydrogen bonds that occurred along and between the chains had to break. The {phi} and {psi} angles of the chain can change to preserve these hydrogen bonds for a certain distance, but at some point the chain is distorted too much for these bonds to be maintained. After the chain was moved 3.0 Å, the interaction energy increased greatly, indicating that at this point most of the chain no longer was in close contact with crystal.

The hydrogen bond numbers for all distances of the chain from the Cel5A on the (1,1,0) surface are given in Table III. At the 1.5 Å distance the number of hydrogen bonds between the chain and the crystal decreased as expected based on the energy profile. It is at this point that the residues of the chain of interest are too far from the crystal to maintain five hydrogen bonds that previously existed. At the same time the chain is still too far from Cel5A for the chain residues to hydrogen bond with the residues of Cel5A within the binding cleft. For example, at the 2.0 Å distance, the –1 residue has its hydroxymethyl group 3.12 Å (oxygen–oxygen distance) from the hydroxymethyl group of the residue in the chain below. In the 1.5 Å structure, this distance increased to 4.92 Å and neither residue made a new hydrogen bond. The greatest number of hydrogen bonds between the chain and Cel5A occurred at the 0.0 Å distance, which is the lowest energy structure. Both the protein energy and the crystal energy decrease at the 0.0 Å distance. This may indicate a reordering of the hydrogen bonds to maximize inter-segment interactions within the crystal and inter-segment interactions within the protein in addition to protein chain interactions. The energy trend for just the protein is also depicted in Figure 12, showing the decrease in energy which occurred as the chain moved the last 0.5 Å.


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Table III. Hydrogen bond numbers at each distance along the trajectory for the (1,1,0) face showing similar counts for the total number of interactions, but a difference in the number of hydrogen bonds between segments
 
The r.m.s. differences for the final structure of Cel5A on the (1,1,0) face are given in Table IV. The r.m.s. deviation for the substrate only for the (1,1,0) surface calculation was much lower than that which was calculated for the (1,0,0) surface. The procedure worked better for mapping the chain of interest on to the dummy atoms in this case. The final structure for the four residues of interest in the chain is superimposed on to the original cellotetraose structure in Figure 13, showing good overlay of the two substrates. The final active site structure is shown in Figure 14. The distance between the catalytic hydroxyl of Glu162 and the glycosidic oxygen atom of the scissile bond is 2.94 Å. This residue is positioned nicely for acid–base deglycosylation of the glycosyl-enzyme intermediate.


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Table IV. R.m.s. deviation between the final structure at the 0.0 Å distance and the original crystal structure for the (1,1,0) face calculation
 


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Fig. 13. The final position of the chain that was moved into the active site from the (1,1,0) face, superimposed on the experimental tetrasaccharide substrate coordinates from the crystal structure.

 


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Fig. 14. The final position of the chain that was moved into the active site superimposed on the experimental tetrasaccharide substrate coordinates from the crystal structure.

 
Other residues made hydrogen bonds along the chain of interest as it was moved from the surface into the active site. These residues were His39, Trp42, Asn80, Arg117, Gln123, Gln204, Tyr245, Gln247 and Asp324. Of these residues, Asn80, Arg117 and Asp324 were also found to make close contact with the chain in the (1,0,0) surface calculation.

The energetic analyses reported here were calculated without considering any contributions from water molecules and in particular those which might be located between the protein and the cellulose surface. Since this enzyme is a glycosidase, at least one such water molecule must be located in this region, close to the active site, in order to effect the hydrolysis, and other molecules may be trapped or incorporated in this interfacial region as well. Because of the great strength of water hydrogen bonds, the contribution of such water molecules could be important.

A full analysis of the contribution from water is beyond the scope of this study, but Figure 15 demonstrates that water molecules are probably present. During the course of the simulations, voids large enough to accommodate a water molecule existed, either transiently or persistently, between the protein and the surface. To explore this possibility, the final frame of the MD simulations was energy-minimized and then superimposed on a previously equilibrated box of water molecules, with only those water molecules which were further than 2.4 Å but closer than 3.5 Å from any protein or cellulose heavy atom being retained. The cubic water box was then rotated about the principal axes and the procedure repeated to reduce the chance of overlooking a water-sized void owing to the random placement of water molecules in the box. Several voids were found, as can be seen from Figure 15. As might be expected, pulling the chain up out of the surface created a void in the crystal surface layer large enough to accommodate several water molecules, although it is not clear that in an actual disruption process any would have been close enough to enter this hole or that the chain would have allowed their penetration even if they were adjacent. As can be seen from the figure, several other molecules could also be placed in the interfacial region, including several more along the binding groove, close to the active site, and at least one can be placed in a void off the center line of the binding groove. Although the average energy contribution of these various water molecules would be difficult to determine, the total interaction energy of just the latter molecule with the rest of the system in this single artificial configuration was found to be –15.3 kcal/mol.



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Fig. 15. A stereo figure (Varshney et al., 1994Go; Humphrey et al., 1996Go) illustrating the positions of voids large enough to contain a water molecule in the final structure from the calculation for the (1,0,0) face. Water molecules, shown in purple and white, were placed in voids in the interfacial region between the protein and the crystal surface as described in the text. The top layer of the cellulose crystal is shown in gray. The view is looking up from within the cellulose surface toward the ‘underside’ of the protein and the interfacial region.

 
Conclusions

The process of moving a chain of cellulose from an ordered cellulose crystal structure into the active site of Cel5A was found to be overall an energetically favorable process for both of the crystal faces of cellulose Iß studied. The increase in energy that occurred at the end of the process along the trajectory for the (1,0,0) face resulted from a combination of the increase in the energies of both the protein and the chain. The increase was caused by a change in the {phi} and {psi} angles of the +1 and +2 residues in the chain and a rotation about the {chi} angle of residue His116. For this crystal face the procedure employed here mapped the chain on to the cellotetraose substrate poorly, but did not significantly affect the active site residues which make contact to the chain in the crystal structure. The chain orientation in the (1,0,0) surface requires a twist to occur in order to avoid steric clashes in the active site. In the structure calculated for the complex, His116 made a favorable interaction with the chain, but owing to the distance constraints, this residue had to alter its position to accommodate the chain. The chain moved towards a conformation lower in energy than that of the crystal structure, but was unable to make close contact with Glu162 or Glu282. It would be interesting to characterize the ability of Cel5A to hydrolyze cellulose experimentally through a mutation of His116 to a smaller, less bulky residue. The hydrophobic interaction with Trp139 would also need to be considered. Other residues which hydrogen bond to the chain may play a role in crystalline cellulose disruption. Mutagenesis studies could also help identify if any of these are essential for hydrolysis.

The barrier which occurred along the trajectory for the (1,1,0) face was caused by an increase in both the chain and crystal energies due to a loss of hydrogen bonds between the moving chain and the rest of the crystal. The final conformation of the chain from the (1,1,0) surface mapped on to the crystallographic coordinates of the tetrasaccharide substrate fairly well. In this structure the glycosidic oxygen of the scissile bond was in close contact with Glu162 and the lowest energy conformation for the entire complex occurred at the 0.0 Å constraint or overlap distance. Again, residues along the binding cleft which were in close contact with the chain of interest may be important for the disruption of the crystalline surface. Early contact with the chain by these residues would help minimize the effects of the loss of interactions of the chain with the crystal. Mutagenesis studies on these residues may help to understand their role in cellulose breakdown.

The results of these simulations appear reasonable in their qualitative trends, since a decrease in energy for pulling a chain into the cellulase binding groove would be expected if the enzyme is indeed able to disrupt crystalline cellulose and it seems reasonable that it would be easier to extract one of the more exposed chains from the (1,1,0) step face than from the flat (1,0,0) face. The magnitudes of the energy changes seem large, however, and probably are not quantitatively accurate. There are many approximations involved in these simulations which could affect the magnitudes of the total calculated energies. It is, of course, difficult to estimate the effects of any inaccuracies in the force field, but the use of long-range cutoffs for the non-bonded energy terms could have the effect of reducing the total binding energy of the target chain to the rest of the crystal. The finite and relatively small size of the crystal potentially could also have been significant. In particular, the short length of the crystal would decrease the energy needed to slide the target chain out of register since the total number of rings binding to the neighboring chains and resisting the disruption was small. In addition, no solvent was present in these simulations. The background field of the solvent could easily affect the disruption energy. Also, although most of the solvent must be excluded from the interface region when the flat face of the enzyme binds to the cellulose crystal surface, effectively dehydrating both, presumably at least one water molecule must be incorporated into the region of the catalytic site to accomplish the hydrolysis. Since no such water was included in the present simulations, it is impossible to estimate how its absence affected the calculated energetics. Nevertheless, the present simulations provide at least a qualitative model of the disruption process. Furthermore, the observation that the bulk of the favorable energy change upon binding comes from protein–carbohydrate interactions suggests that point mutations in specific residues could affect the energetics significantly and that potentially mutagenesis could produce an enzyme with enhanced activity.


    Acknowledgements
 
The authors thank Joshua Sakon for helpful discussions and advance access to the crystal coordinates for E1. The authors also thank David Wilson for useful discussions. This work was supported by subcontract XCO-8-17101-01 from the National Renewable Energy Laboratory.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received January 21, 2003; revised October 6, 2003; accepted October 9, 2003





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