TABLE 1.
Effect of C. thermocellum cohesin mutations on type I cohesin-dockerin binding affinity
Mutations in both the hydrophobic and hydrophilic interface patches (Figs. 2 and 3) can significantly reduce binding. Predicted ΔΔG values were calculated based on the Protein Data Bank structure 1OHZ (12) using the Rosetta interface protocol (33). Predicted hot spots (mutations to alanine with ΔΔG >1.0 kcal/mol) within the interface patches were experimentally validated by iELISA. Fig. 4 summarizes the predicted effect on binding of mutations as calculated by additional computational protocols.
| Mutation | Calculated ΔΔG Rosetta 2.3a | Experimental ΔΔG | Experimental IC50b |
|---|---|---|---|
| REU | kcal/mol | nm | |
| WT | 0 | 1.1b | |
| Hydrophobic patch | |||
| L83A | 2.2 | 3.0 | 123c |
| L83S | 2.4 | 3.4 | 257c,d |
| Polar patch | |||
| N37A | 2.2 | 0 | 1 |
| N37D | 2.1 | 2.5 | 70 |
| N37L | 1.9 | >4e | >103 |
| D39A | 3.0 | >4 | >103c |
| D39N | 0.3 | >4 | >103f |
| E131A | 1.3 | 1.8 | 20 |
| N37A/E120A | 2.2 | 1.2 | 7c |
| N37A/D39A | 5.2 | >4 | >103 |
| N37A/E131A | 3.5 | 1.0 | 6 |
| N37A/D39A/E131A | 6.4 | >4 | >103 |
| Additional positions | |||
| V41A | 0.0 | ||
| V41Y | 0.3 | 0.7 | 4f |
| D70A | 0.1 | ||
| D70S | 0.2 | −0.1 | 1f |
| Y74A | 1.8 | −0.1g | |
| V81S | 0.1 | 1.9 | 30f |
| A85L | 0.5 | −0.1 | 1f |
| E86A | 1.6 | ||
| E86S | 1.6 | 0.4 g | |
| E120A | 0.0 | ||
| D39S/Y74A | 4.4 | 1.1g | |
| D39S/Y74A/E86S | 6 | 2.8g | |
| D39N/D70S/V81S | 0.5 | >3 | >200f |
a In Rosetta energy units (REU); optimized to correlate with kcal/mol.
b IC50 values in this study are normalized relative to the binding of wild-type C. thermocellum cohesin to C. thermocellum dockerin (i.e. 1.1, first row).
c Reported in Ref. 26.
d The standard error of the mean of logarithmic values of IC50 exceeds the half-log value for this measure.
e Very weak binding could not be fitted and was therefore assigned ΔΔG values of >4 kcal/mol corresponding to the largest measured concentration.
f Measured also previously by enzyme-linked interaction assay (29).
g Measured by isothermal titration calorimetry (65).