Deciphering Isoniazid Drug Resistance Mechanisms on Dimeric Mycobacterium tuberculosis KatG via Post-molecular Dynamics Analyses Including Combined Dynamic Residue Network Metrics

Abstract

Resistance mutations in Mycobacterium tuberculosis ( Mtb ) catalase peroxidase protein (KatG), an essential enzyme in isoniazid (INH) activation, reduce the sensitivity of Mtb to first-line drugs, hence presenting challenges in tuberculosis (TB) management. Thus, understanding the mutational imposed resistance mechanisms remains of utmost importance in the quest to reduce the TB burden. Herein, effects of 11 high confidence mutations in the KatG structure and residue network communication patterns were determined using extensive computational approaches. Combined traditional post-molecular dynamics analysis and comparative essential dynamics revealed that the mutant proteins have significant loop flexibility around the heme binding pocket and enhanced asymmetric protomer behavior with respect to wild-type (WT) protein. Heme contact analysis between WT and mutant proteins identified a reduction to no contact between heme and residue His270, a covalent bond vital for the heme-enabled KatG catalytic activity. Betweenness centrality calculations showed large hub ensembles with new hubs especially around the binding cavity and expanded to the dimerization domain via interface in the mutant systems, providing possible compensatory allosteric communication paths for the active site as a result of the mutations which may destabilize the heme binding pocket and the loops in its vicinity. Additionally, an interesting observation came from Eigencentrality hubs, most of which are located in the C-terminal domain, indicating relevance of the domain in the protease functionality. Overall, our results provide insight toward the mechanisms involved in KatG-INH resistance in addition to identifying key regions in the enzyme functionality, which can be used for future drug design.

Evolutionary progression of collective mutations in Omicron sub-lineages towards efficient RBD-hACE2: Allosteric communications between and within viral and human proteins

“…Remarkably, almost all the residues at positions 326, 355-357, 375, 378 and 379 that lost EC hub status experienced higher residue fluctuation in the Omicron sub-lineages compared to the WT (Table S4). This relationship between EC and residue flexibility has previously been shown in [75] .…”

“…Residues Ala403, Ile436, Leu444, Thr449, Cys498, Asn501 and His505, which are documented epitopes for neutralizing antibody binding [57] , [131] , [132] , had hub status in the WT which was lost in majority of the Omicron sub-lineages (BA.1, BA3_10, BA3_15 and BA.4). Recently, we showed a relationship between a dynamically stable C-terminal domain of the KatG protein and a high concentration of EC hubs in the domain, implying that highly influential residues are associated with stable regions [75] . Likewise, here, the Omicron sub-lineages experienced higher RBD residue fluctuation compared to the WT which could explain the scarcity of EC hubs.…”

“…Here, like in our previous studies [75] , [77] , [110] we employed five DRN analysis metrics; averaged betweenness centrality (BC) , averaged closeness centrality (CC) , averaged degree centrality ( DC) , averaged eigenvector centrality (EC) and averaged katz centrality ( KC) to explore the communication network differences between the WT and the Omicron sub-lineage RBD-hACE2 protein complexes . The centrality hubs for each metric were identified using a global cutoff of top 5% for the RBD and 4% for the hACE2 proteins.…”

“…Previously, we defined “centrality hubs” as any node that forms part of the set of highest centrality nodes for any given averaged centrality metric [76] , [78] . Here, the metric specific DRN centrality hubs were identified using the previously applied analysis algorithm [75] , [77] , [78] . The algorithm vectorises metric specific results of all the systems in descending order before ranking them and obtaining the hub-threshold value based on a set percentage cut off.…”

“…Trajectory analysis, comparative essential dynamics (ED) calculations and RBD-hACE2 interface residue interaction frequency analysis revealed: 1) a flexible RBM that resulted in conformational variability of the Omicron sub-lineages compared to the WT; 2) highly flexible antigenic hotspots in the RBD which could hinder neutralizing antibody binding; 3) increased residue interactions and interaction frequency between the viral and human proteins in the Omicron variants compared to the WT; 4) significant allosteric effects of mutations in some sub-lineages on ACE2. Furthermore, dynamic residue network (DRN) analysis using our recently developed algorithm [75] , [76] , [77] , [78] identified, for the first time, the high centrality communication paths bridging the RBD to the hACE2 core, and the allosteric changes in residue network patterns in both the RBD and hACE2 resulting from collective Omicron sub-lineage mutations.…”