β-glucosidase-catalyzed cellobiose conversion to glucose is the principal rate-limiting step in the deconstruction of lignocellulosic biomass to biofuel production as most β-glucosidases are feedback inhibited by glucose. Thus, deciphering the mechanism of glucose inhibition has been a prime focus for years. In this study, atomistic molecular dynamics simulations were performed to understand the effect of low (0.1 M) and high (0.8 M) glucose concentrations on the structure and dynamics of GH1 β-glucosidase (H0HC94) from Agrobacterium tumefaciens 5A. A protein structure network was constructed on each protein snapshot from the simulation, suggesting a glucose-induced conformational change of the enzyme in the high glucose concentration. Additionally, the conformational changes were characterized in terms of cliques and communities. The increasing number of cliques rigidified the residue fluctuations around the enzyme's tunnel in the high glucose concentration and hindered the glucose interaction at the active site. It was also supported by the low radial distribution of glucose and no glucose−enzyme hydrogen bonds at the active site tunnel. Moreover, the essential dynamic motions for catalysis were lost by the elevated number of glucose−enzyme interactions in the high glucose concentration. Furthermore, six secondary binding sites were predicted, which could induce uncompetitive inhibition of H0HC94. Overall, we propose a molecular basis of the H0HC94 inhibition, which will further help to design glucose-tolerant β-glucosidases for sustainable lignocellulosic biofuel production.

Understanding the behavior of ionic liquid tolerant hyperthermophilic endoglucanase Cel12A from Rhodothermus marinus in different concentrations of EmimAc.

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T-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 domain and the doubly phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly phosphorylated ITAM-ζ1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine residue of ITAM peptide to form an encounter complex leading to subsequent binding of second phosphotyrosine residue to the N-SH2 domain. We decipher a network of noncovalent interactions that allosterically couple the two SH2 domains during binding to doubly phosphorylated ITAMs. Mutation in the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in B-cells.