Computational study of the mechanism of binding of antifungal icofungipen in the active site of eukaryotic isoleucyl tRNA synthetase from Candida albicans.

The eukaryotic fungal species Candida albicans is a critical infective pathogenic agent. The ß-amino acid, Icofungipen, is an effective inhibitor of Candida albicans. Icofungipen binds at the active site of the isoleucyl tRNA synthetase (IleRS) from Candida albicans (CaIleRS) and halts protein translation in fungus. In the present work, we have investigated the mechanism of binding of Icogungipen (abbreviated as IFP). Molecular dynamics (MD) simulations show that the carboxylic acid group of IFP in the CaIleRS: IFP complex is more oriented towards the Connective Polypeptide (CP) core loop compared to the carboxylic acid group of Ile in the CaIleRS: Ile complex. The Arg 410 of the CP core loop near the substrate is extended towards the IFP. Due to the difference in the conformation of residues of the CP core loop, the KMSKR loop is more proximal to the CP core loop in CaIleRS: IFP. The editing domain which is covalently linked with the CP core loop in the CaIleRS: IFP complex is also oriented in such a way that the active site cavity is narrow and longer. The metadynamics calculation shows that the IFP is trapped in a deeper potential well compared to Ile which is due to the effective closure of the gateway of the active site by KMSKR and CP core loop. The thin, long shape of the active site and the closed gate of the active site in CaIleRS: IFP complex is responsible for the effective capture of IFP relative to Ile in the active site.Communicated by Ramaswamy H. Sarma.

Computational Methods for Molecular Understanding of the Antibiotic-Aminoacyl tRNA Synthetase Interaction.

Developing an understanding of the interactions between an antibiotic and its binding site in a pathogen cell is the key to antibiotic design-an important cost-saving methodology compared to the costly and time-consuming random trial-and-error approach. The rapid development of antibiotic resistance provides an impetus for such studies. Recent years have witnessed the beginning of the use of combined computational techniques, including computer simulations and quantum mechanical computations, to understand how antibiotics bind at the active site of aminoacyl tRNA synthetases (aaRSs) from pathogens. Such computational protocols assist the knowledge-based design of antibiotics targeting aaRSs, which are their validated targets. After the ideas behind the protocols and their strategic planning are discussed, the protocols are described along with their major outcomes. This is followed by an integration of results from the different basic protocols. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Analysis of active-site residues from primary sequence of synthetase and transfer RNAs Basic Protocol 2: Molecular dynamics simulation-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex Basic Protocol 3: Quantum mechanical method-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex.

Dynamics of the Catalytic Active Site of Isoleucyl tRNA Synthetase from Staphylococcus aureus bound with Adenylate and Mupirocin.

The development of new antimicrobial drugs is critically needed due to the alarming increase in antibiotic resistance in bacterial pathogens. The active sites of different bacterial aminoacyl tRNA synthetases (aaRS) are validated targets of antibiotics. At present, the only aaRS inhibitor approved is mupirocin (MRC) which targets bacterial isoleucyl tRNA synthetase (IleRS). The present work is aimed at understanding the lacunae of knowledge concerning the active site conformational dynamics in IleRS in the presence of inhibitor mupirocin. With this end in view, we have carried out classical molecular dynamics simulation and metadynamics simulations of the open state of IleRS from Staphylococcus aureus (SaIleRS), the closed state tripartite complex bound with cognate adenylate (Ile-AMP) and tRNA, the closed state tripartite complex bound with noncognate MRC, and the closed state tripartite complex bound with tRNA and MRC with mutated SaIleRS (V588F). The present simulation established a dynamic picture of SaIleRS complexed with cognate and the noncognate substrates which are completely consistent with crystallographic and biochemical studies and explain the existing lacunae of knowledge. The active site is significantly more compact in the Ile-AMP bound complex compared to the open state due to the closure of the KMSKS and HMGH loops and clamping down of the tRNA acceptor end near the active site gate. The present result shows that the unusual open conformational state of the KMSKS loop observed in the cognate substrate-bound complex in the crystal is due to crystallographic constraints. Although the mupirocin tightly fits the catalytic active site in the MRC-bound complex, the nonanoic acid moiety is partly exposed to the water. The KMSKS loop is pushed open in the MRC-bound complex to accommodate the noncognate MRC. New tunnels open up, extending to the editing site in the complex. Out of its three broad segments, the C12 to C17 segment, the conjugated segment, and the nonanoic moiety, the conjugated part of MRC binds most effectively with the active site of the MRC-bound complex. The aromatic residues packing around the C12 to C17 segment of MRC stabilize the tRNA hairpin conformation in a similar way as observed in the TrpRS. The V588F mutation is weakening the interaction between this region of the active site and weakens the binding of MRC in the active site. This result explains why the V588F mutation is responsible for low-level mupirocin resistance. The free energy of unbinding the conjugated segment (and C12 to C17 segment, as well) largely contributes to the total free energy of unbinding the MRC. The active site organization of IleRS from eukaryotic Candida albicans is compared with the bacterial IleRS active site to understand the low binding affinity in eukaryotic IleRS. The present study could be a starting point of future studies related to the development of effective drug binding in the SaIleRS.

Inference of distant genetic relations in humans using "1000 genomes".

Nucleotide sequence differences on the whole-genome scale have been computed for 1,092 people from 14 populations publicly available by the 1000 Genomes Project. Total number of differences in genetic variants between 96,464 human pairs has been calculated. The distributions of these differences for individuals within European, Asian, or African origin were characterized by narrow unimodal peaks with mean values of 3.8, 3.5, and 5.1 million, respectively, and standard deviations of 0.1-0.03 million. The total numbers of genomic differences between pairs of all known relatives were found to be significantly lower than their respective population means and in reverse proportion to the distance of their consanguinity. By counting the total number of genomic differences it is possible to infer familial relations for people that share down to 6% of common loci identical-by-descent. Detection of familial relations can be radically improved when only very rare genetic variants are taken into account. Counting of total number of shared very rare single nucleotide polymorphisms (SNPs) from whole-genome sequences allows establishing distant familial relations for persons with eighth and ninth degrees of relationship. Using this analysis we predicted 271 distant familial pairwise relations among 1,092 individuals that have not been declared by 1000 Genomes Project. Particularly, among 89 British and 97 Chinese individuals we found three British-Chinese pairs with distant genetic relationships. Individuals from these pairs share identical-by-descent DNA fragments that represent 0.001%, 0.004%, and 0.01% of their genomes. With affordable whole-genome sequencing techniques, very rare SNPs should become important genetic markers for familial relationships and population stratification.