NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

Abstract

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.

Catalysis by a rigid enzyme

“…Thus, these observations suggest that the conversion of (ES)1 into (ES)2 could be related to the rate-limiting step in catalysis. A similar slow transition process was reported for a different type of glycosidase, chitosanase, when titrated with chitosan hexaose (24). In that study, the slow exchange was attributed to a conformational change of the protein induced by ligand 25 binding.…”

NMR and computational methods for molecular resolution of allosteric pathways in enzyme complexes

“…One example, Chemical Shift Covariance Analysis (CHESCA) first reported by Melacini and coworkers (Selvaratnam et al 2011), analyzes the chemical shifts of multiple allosteric states of a protein to identify pairwise correlations in covariant residues that compose an allosteric network. Alternatively, protocols to fit NMR lineshapes in a series of 2D spectra collected as allosteric effectors are titrated into protein samples have been described to extract both thermodynamic (K and ΔG) and kinetic information (k ex , k T → R , and k R → T ) (Kovrigin 2012;Shinya et al 2017;Waudby et al 2016). The sensitivity of these methods to communication between active and allosteric sites is limited by the assumption that extrinsic factors such as buffer conditions, temperature, and pH have little effect on the allosteric ensemble, as well as by the fact that the chemical shift itself contains contributions from ligand binding, structural reshuffling at protein interfaces, and small pK a shifts as hydrogen bond networks are altered.…”

Dynamics of Ligand Binding to a Rigid Glycosidase**

“…Asimilar slow process was reported for ad ifferent type of glycosidase,c hitosanase, when titrated with chitosan hexaose. [16] In that study,the slow exchange was attributed to ac onformational change of the protein induced by ligand binding.However,for BCX-E78Q the similarity of the PCS of resonances in (ES) 1 and (ES) 2 indicates that the conversion from (ES) 1 to (ES) 2 does not lead to am ajor structural rearrangement of the enzyme ( Figure 2C). This observation poses the question what the slow transition step in BCX-E78Q Michaelis complex represents.…”