Enzyme–Enzyme Docking and Interactional Energy
HADDOCK [41] was employed to investigate the EEIs of TeCel7A with TrCel6A, EG, BG and TeCel7A itself, respectively. Table 1 shows the docking results for these assemblies, wherein TeCel7A–EG processes the best Z–score. Binding affinity (ΔG) and the dissociation constant (Kd) were then measured by PRODIGY [42]. TeCel7A–TrCel6A features the strongest affinity of − 17.2 kcal/mol. These results suggest that the two enzymes can interact powerfully, mainly owing to their flexible SELs (see Fig. 1). However, TeCel7A is not apt to bind with itself in view of the lowest HADDOCK score, Z–Score and binding affinity of TeCel7A–TeCel7A assembly, compared with the other enzyme–enzyme complexes.
Table 1
Results of HADDOCK for assemblies of TeCel7A with TrCel6A, EG, BG and TeCel7A itself.
| TeCel7A–TrCel6A | TeCel7A–EG | TeCel7A–BG | TeCel7A–TeCel7A |
HADDOCK score | –57.3 ± 11.5 | –92.9 ± 3.8 | –59.3 ± 7.8 | –40.7 ± 3.9 |
Z–Score | –2.1 | –2.7 | –1.9 | –0.8 |
ΔG (kacl/mol) | –17.2 | –12.7 | –15.3 | –10.5 |
On the basis of protein–protein docking, multi–microsecond MD simulations were then employed to decipher their underlying mechanism in detail. The four initial structures of TeCel7A, TrCel6A, EG, BG and the four protein-protein complexes of TeCel7A–TrCel6A, TeCel7A–EG, TeCel7A–BG and TeCel7A–TeCel7A were sufficiently relaxed in an explicit water environment and each followed by a 500 ns MD simulation, except for TeCel7A–TrCel6A, which followed by 1.5 µs. The evolution of the center-of-mass (COM) distance between the two proteins in each complex structure (Fig. S1) suggests that the well-equilibrated states have been reached. As reported in Table 2, the binding free energies calculated by the MM-PBSA method indicate that the two exo–lytic cellulases possess the strongest binding free energy among the four enzyme-enzyme assemblies. In view of results of docking and MD simulations, we therefore suggest that TrCel6A is the most promising one to influence the hydrolytic function of TeCel7A.
Table 2
Binding free energiesa for enzyme–enzyme complexes.
System | ΔG | ΔEMMb | ΔGPB | ΔGSA |
TeCel7A-TrCel6A | -23.4 | -1232.2 | -3207.8 | -2.1 |
TeCel7A-EG | -7.8 | -519.6 | -996.1 | -48.4 |
TeCel7A-BG | -16.2 | -158.0 | -3076.4 | -14.8 |
TeCel7A-TeCel7A | -14.6 | -284.1 | -2562.7 | -69.1 |
a All quantities are in kcal mol− 1. b ΔEMM energy includes the intermolecular noncovalent interactions and the change of the conformational energies. |
Binding Modes and Interaction Interface of TeCel7A–TrCel6A
As depicted in Fig. 2a, the optimal docking pose for TeCel7A–TrCel6A indicates that the two exo–lytic cellulases bind with each other with their SELs. Furthermore, the other enzymes were found to combine to the region around SELs of TeCel7A as well (Fig. S2). Eight hydrophobic residues in TeCel7A (Ala375, Ala376, Met378, Leu379, Ala389, Ile396, Ala397 and Val407) were found to locate at the interactional interfaces of the complexes (Fig. 2b). It is well known that the unproductive adsorption caused by the binding of hydrophobic sites between cellulases and lignin is one of the main limiting factors in the enzymatic hydrolysis [23, 43]. EEIs can properly prevent cellulases from the adsorption of lignin residues, thereby contributing to increasing the efficiency of enzymatic hydrolysis.
Influence of TrCel6A on Flexibility and Catalytic Region of TeCel7A
As depicted in Fig. 3a, the interactional interface of TeCel7A–TrCel6A is changed after 1.5 µs MD simulation, like that of other enzyme-enzyme complexes (Fig. S3). The phenomena indicate that this nonspecific association between enzymes cannot always sustain. Since the enzymes should work on their exclusive substrates by traditional synergetic forms, we, therefore, suggest that the separation of the enzyme-enzyme complexes after EEIs is beneficial to hydrolysis cycle. Figure 3b shows the root mean square fluctuation (RMSF) values of the residues in TeCel7A with and without combining TrCel6A. It is apparent that flexibility of the loops A1, B1, B2 and B3 is improved significantly by EEIs. In the enzymatic hydrolysis, improved flexibility of the SELs will accelerate dissociation − the rate–limiting step of GH7 CBHI degrading cellulose alone, increasing the yield of cellobiose.
It is acknowledged that the initial binding of TeCel7A to cellulose is certainly influenced by the opening of SELs [44, 45]. As shown in Fig. 4a, the cross–sectional area (CSA) formed by the center of mass (COM) distances of loops B2, B3 and A3 were measured. Figure 4b indicates that the CSA of TeCel7A is expanded notably by association with TrCel6A. Before the degradation of lignocellulose by TeCel7A, the EEIs between the two exo–lytic cellulases in hydrolytic system will help the initial association of TeCel7A with cellulose. Andersen et al. have reported that the highest synergy was observed at the beginning of hydrolysis [46], consistent with our theoretical analysis.
Dethreading Process
Vermaas et al. have reported that dethreading is the predominant mechanism for dissociation of GH7 CBHI from cellulose [30]. We, therefore, speculate that highly extent of synergy between the two exo–lytic cellulases occurs in dethreading process − the longest time-consuming action of GH7 CBHI degrading cellulose. As shown in Fig. 5a, loops A1, B1 and B2 absorb on the surface of cellulose before dissociation. The notably improved flexibility of these three loops after EEIs (see Fig. 3b) can enhance motility of CBHI, helping the enzyme leave the surface of substrate. The results of Vermaas et al. further indicate that − 1 to − 3 transition is the rate–limiting step for dethreading, especially − 1 to − 2 [30]. As illustrated in Fig. 5b, many hydrogen bonds are formed between − 1 glucosyl ring and the enzyme, influencing the rate of dethreading significantly. Acceleration of dethreading was deemed to depend on modulating interactions around the − 1 state [30]. Loop B3, closest to the − 1 site is, therefore, the most promising SEL to influence dethreading. In view of the improved flexibility of loop B3 (see Fig. 3b) after EEIs, it will interact with − 1 glucosyl ring frequently, facilitating motion of the substrate. We, therefore, suggest that the key to the synergy of exo–lytic cellulases is to improve the flexibility of loop B3, thus accelerating dethreading.
Existence of EEIs and the Role of Carbohydrate Binding Module (CBM)
To verify the existence of combination between exo–lytic cellulases experimentally, MST experiment were performed, characterizing the binding of TeCel7A to TrCel6A. The relevant result indicates that the interactions of TeCel7A with TrCel6A exist, but weak (Kd = 140 ± 60 µM, see Fig. 6). In view of the result of Fig. 3a and the corresponding theoretical analysis, the weak combination between exo–lytic cellulases is conjectured to be reasonable. The degree of cooperation between them is, therefore, suggested to depend on the frequency of EEIs. In the study of Badino et al., the synergetic extent of the two exo–lytic cellulases achieved the highest level when both the two enzymes possess CBMs [21]. In addition, their synergy was decreased step by step if one or both of them do not have CBMs [21]. We conjecture that CBMs can enable the two exo–lytic cellulases to locate at the surface of cellulose to interact adequately, enhancing the degree of EEIs. Overall, the synergy between the exo–lytic cellulases can arise from increased flexibility of the key loops in TeCel7A by EEIs, degree of which is improved by their CBMs.