Histidine network regulates the structure-stability features of T7 endolysin native and partially folded conformations

Abstract

Conformational transitions displayed by proteins are a classical phenomenon associated with the structural intricacies that directly influence concomitant functionality. pH-dependent conformational switching is the combined outcome of pKa and pH that regulate the network of crucial interactions via side chain protonation-deprotonation, thus governing the structure-stability-activity of proteins. Histidine is a unique amino acid owing to its aromatic and ionizable imidazole side chain, which enables it to act as a hydrogen bond donor and acceptor under differential pH conditions/protonation events. The peptidoglycan degrading enzyme T7 endolysin possesses a conserved quad-His (H18, H48, H69, and H123) network at its hydrophobic core regulating the pH-induced conformational transition. However, the degree of involvement of individual His residue is currently unknown. In the current study, four T7L variants (H18K, H48K, H69K, and H123K) were designed, and the contribution of each site to T7L activity, conformational heterogeneity, reversibility, and stability of native/partially folded state has been assessed using a detailed biophysical, NMR, and cell-based analysis of amidase activity. The results delineated the prime responsibility of His48 followed by His69 and His18 in regulating the core structural stability, amidase activity, and pH-dependent conformational switching of T7L. The perturbations due to protonation of His network altered the associated polar and nonpolar interactions within the network and with the surrounding residues, thus essentially mediating the pH-dependent conformational transition of T7L. All the observed experimental evidence establishes the regulatory role of “histidine molecular switches” in the structure–activity paradigm of bacteriophage amidases.