Calcium and domain interactions contribute to the thermostability of domains of the multimodular cellobiohydrolase, CbhA, a subunit of the Clostridium thermocellum cellulosome

Abstract

Each of three internal domains of multi-modular cellobiohydrolase CbhA from Clostridium thermocellum, X1(1), X1(2) (previously designated as fibronectin type 3-like modules, Fn3(1) and Fn3(2)) and family 3 carbohydrate-binding module (CBM3) binds 1 mol of Ca(2+). Structures and thermal stabilities of X1(1), X1(2), CBM3, X1(1)X1(2), and X1(1)X1(2)-CBM3 containing Ca(2+) (holo-proteins) and without Ca(2+) (apo-proteins) have been studied using CD spectroscopy. All domains are beta-proteins with irregular far-UV CD spectra due to the aromatic side chain contributions. The positive signal at 294 nm in the near-UV CD spectrum of X1(1) lacking a tryptophan residue might be attributed to the presence of aromatic clusters. Thermal denaturation of all proteins is reversible and results in the total loss of tertiary structure and preservation of significant amount of ordered secondary structure. Removal of Ca(2+) destabilizes polypeptides in a different way and to a different extent. It decreases the melting temperature ( T (m)) (by 20 degrees C) and co-operativity of thermal transition of X1(1), increases the number of transitions and lowers the co-operativity of unfolding of CBM3, and slightly decreases T (m)s (2.4-4.2 degrees C) of X1(2), X1(1)X1(2), and X1(1)X1(2)-CBM3. Transitions of X1(1)X1(2) and X1(1)X1(2)-CBM3 follow a two-state model regardless of the presence of Ca(2+). X1(1) is strongly stabilized in the apo-X1(1)X1(2) and apo-X1(1)X1(2)-CBM3 as they display T (m)s similar to those of individual and combined holo-modules. Observed CD spectra of X1(1)X1(2) and X1(1)X1(2)-CBM3 differ from those calculated as the simple weighted sum of individual modules. These differences are more prominent in spectra of apo-proteins. The results indicate the presence of inter-domain interactions in CbhA. Holo-modules, i.e. containing Ca(2+), behave essentially independently, but in the absence of Ca(2+) domain interactions are more important for the conformation of the polypeptides.