Association of epilepsy and neurological impairments with homozygous recessive missense mutations found in the genes responsible for ganglioside biosynthesis (ST3GAL5) and calcium voltage-gated channels (CACNA1H) - insights through molecular dynamic simulations.

With over 2.2 million cases, the incidence rate of epilepsy in Pakistan is far higher than the rest of the world due primarily to the frequent, traditionally imposed cousin marriages. In the present study, comprehensive whole exome sequencing (WES) analyses of a three-generation family with four affected members presenting 'unexplained' childhood absence epilepsy (CAE), seizures and dementia, was performed in a quest to identify heritable, epilepsy-causal gene variants to better aid in carrier screening and genetic counselling. The WES data was generated, analyzed, and validated through Sanger's sequencing, molecular dynamic simulation (MDS) analysis, and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) studies. Two homozygous recessive, missense mutations in ST3GAL5 (c.311A > G, p. His104Arg) and CACNA1H (c.6230G > A, p. Arg2077His) genes, earlier regarded as benign or of uncertain significance, have been identified as a potential etiology. Comparative MDS and free binding energy calculations revealed substantial structural perturbations in mutant forms of ST3GAL5 leading to decreased binding and reduced catalytic activity of the p.His104Arg and two other functional variants (p.Val74Glu and p.Arg288Ter) when compared with wild type. Our findings reinforce that WES analyses may uncover 'hidden', heritable variants and together with MDS and MM/GBSA may provide plausible clues to answer the unexplained causes of epilepsy for an effective management and better patient outcome. Further, revisit of epilepsy-associated mutational landscape in population context is imperative as the variants with 'benign' tags may turn out to be 'non-benign', when exist in combination with other benign.Communicated by Ramaswamy H. Sarma.

The Solvation of the E. coli CheY Phosphorylation Site Mapped by XFMS.

The Escherichia coli CheY protein belongs to a large bacterial response regulator superfamily. X-ray hydroxy radical foot-printing with mass spectroscopy (XFMS) has shown that allosteric activation of CheY by its motor target triggers a concerted internalization of aromatic sidechains. We reanalyzed the XFMS data to compare polar versus non-polar CheY residue positions. The polar residues around and including the 57D phosphorylated site had an elevated hydroxy radical reactivity. Bioinformatic measures revealed that a water-mediated hydrogen bond network connected this ring of residues with the central 57D. These residues solvated 57D to energetically stabilize the apo-CheY fold. The abundance of these reactive residues was reduced upon activation. This result was supported by the bioinformatics and consistent with the previously reported activation-induced increase in core hydrophobicity. It further illustrated XFMS detection of structural waters. Direct contacts between the ring residues and the phosphorylation site would stabilize the aspartyl phosphate. In addition, we report that the ring residue, 18R, is a constant central node in the 57D solvation network and that 18R non-polar substitutions determine CheY diversity as assessed by its evolutionary trace in bacteria with well-studied chemotaxis. These results showcase the importance of structured water dynamics for phosphorylation-mediated signal transduction.