Cumulative phylogenetic, sequence and structural analysis of Insulin superfamily proteins provide unique structure-function insights.

The insulin superfamily proteins (ISPs), in particular, insulin, IGFs and relaxin proteins are key modulators of animal physiology. They are known to have evolved from the same ancestral gene and have diverged into proteins with varied sequences and distinct functions, but maintain a similar structural architecture stabilized by highly conserved disulphide bridges. The recent surge of sequence data and the structures of these proteins prompted a need for a comprehensive analysis, which connects the evolution of these sequences (427 sequences) in the light of available functional and structural information including representative complex structures of ISPs with their cognate receptors. This study reveals (a) unusually high sequence conservation of IGFs (>90 % conservation in 184 sequences) and provides a possible structure-based rationale for such high sequence conservation; (b) provides an updated definition of the receptor-binding signature motif of the functionally diverse relaxin family members (c) provides a probable non-canonical C-peptide cleavage site in a few insulin sequences. The high conservation of IGFs appears to represent a classic case of resistance to sequence diversity exerted by physiologically important interactions with multiple partners. We also propose a probable mechanism for C-peptide cleavage in a few distinct insulin sequences and redefine the receptor-binding signature motif of the relaxin family. Lastly, we provide a basis for minimally modified insulin mutants with potential therapeutic application, inspired by concomitant changes observed in other insulin superfamily protein members supported by molecular dynamics simulation.

Alternate conformations found in protein structures implies biological functions: A case study using cyclophilin A.

Protein dynamics linked to numerous biomolecular functions, such as ligand binding, allosteric regulation, and catalysis, must be better understood at the atomic level. Reactive atoms of key residues drive a repertoire of biomolecular functions by flipping between alternate conformations or conformational substates, seldom found in protein structures. Probing such sparsely sampled alternate conformations would provide mechanistic insight into many biological functions. We are therefore interested in evaluating the instance of amino acids adopted alternate conformations, either in backbone or side-chain atoms or in both. Accordingly, over 70000 protein structures appear to contain alternate conformations only 'A' and 'B' for any atom, particularly the instance of amino acids that adopted alternate conformations are more for Arg, Cys, Met, and Ser than others. The resulting protein structure analysis depicts that amino acids with alternate conformations are mainly found in the helical and ß-regions and are often seen in high-resolution X-ray crystal structures. Furthermore, a case study on human cyclophilin A (CypA) was performed to explain the pre-existing intrinsic dynamics of catalytically critical residues from the CypA and how such intrinsic dynamics perturbed upon Ser99Thr mutation using molecular dynamics simulations on the ns-µs timescale. Simulation results demonstrated that the Ser99Thr mutation had impaired the alternate conformations or the catalytically productive micro-environment of Phe113, mimicking the experimentally observed perturbation captured by X-ray crystallography. In brief, a deeper comprehension of alternate conformations adopted by the amino acids may shed light on the interplay between protein structure, dynamics, and function.

Functional characterization of a hypothetical protein (TTHA1873) from Thermus thermophilus.

Thermus thermophilus is an extremely thermophilic organism that thrives at a temperature of 65°C. T. thermophilus genome has ~2218 genes, out of which 66% (1482 genes) have been annotated, and the remaining 34% (736 genes) are assigned as hypothetical proteins. In this work, biochemical and biophysical experiments were performed to characterize the hypothetical protein TTHA1873 from T. thermophilus. The hypothetical protein TTHA1873 acts as a nuclease, which indiscreetly cuts methylated and non-methylated DNA in divalent metal ions and relaxes the plasmid DNA in the presence of ATP. The chelation of metal ions with EDTA inhibits its activity. These results suggest that the hypothetical protein TTHA1873 would be a CRISPR-associated protein with non-specific DNase activity and ATP-dependent DNA-relaxing activity.

In vitro and in silico perspectives to explain anticancer activity of a novel syringic acid analog ((4-(1H-1, 3-benzodiazol-2-yl)-2, 6-dimethoxy phenol)) through apoptosis activation and NFkB inhibition in K562 leukemia cells.

Syringic acid (SA) is an active carcinogenesis inhibitor; however, the low bioavailability and unstable functional groups hinder its activity. Here, a chemically synthesized novel SA analog (SA10) is evaluated for its anticancer activity using in-vitro and in-silico studies. K562 cell line study revealed that SA10 had shown a higher rate of inhibition (IC50 = 50.40 µg/mL) than its parental compound, SA (IC50 = 96.92 µg/mL), at 50 µM concentration. The inhibition ratio was also been evaluated by checking the expression level of NFkB and Bcl-2 and showing that SA10 has two-fold increase in the inhibitory mechanism than SA. This result demonstrates that SA10 acts as an NFkB inhibitor and an apoptosis inducer. Further, molecular docking and simulation have been performed to get insights into the possible inhibitory mechanism of SA and SA10 on NFkB at the atomistic level. The molecular docking results exemplify that both SA and SA10 bind to the active site of NFkB, thereby interfering with the association between DNA and NFkB. SA10 exhibits a more robust binding affinity than SA and is firmly docked well into the interior of the NFkB, as confirmed by MM-PBSA calculations. In a nutshell, the Benzimidazole scaffold containing SA10 has shown more NFkB inhibitory activity in K562 cells than SA, which could be helpful as an ideal therapeutic NFkB inhibitor for treating cancers.

Mutation-Dependent Refolding of Prion Protein Unveils Amyloidogenic-Related Structural Ramifications: Insights from Molecular Dynamics Simulations.

The main focus of prion structural biology studies is to understand the molecular basis of prion diseases caused by misfolding, and aggregation of the cellular prion protein PrPC remains elusive. Several genetic mutations are linked with human prion diseases and driven by the conformational conversion of PrPC to the toxic PrPSc. The main goal of this study is to gain a better insight into the molecular effect of disease-associated V210I mutation on this process by molecular dynamics simulations. This inherited mutation elicited copious structural changes in the ß1-α1-ß2 subdomain, including an unfolding of a helix α1 and the elongation of the ß-sheet. These unusual structural changes likely appeared to detach the ß1-α1-ß2 subdomain from the α2-α3 core, an early misfolding event necessary for the conformational conversion of PrPC to PrPSc. Ultimately, the unfolded α1 and its prior ß1-α1 loop further engaged with unrestrained conformational dynamics and were widely considered as amyloidogenic-inducing traits. Furthermore, the resulting folding intermediate possesses a highly unstable ß1-α1-ß2 subdomain, thereby enhancing the aggregation of misfolded PrPC through intermolecular interactions between frequently refolding regions. Briefly, these remarkable changes as seen in the mutant ß1-α1-ß2 subdomain are consistent with previous experimental results and thus provide a molecular basis of PrPC misfolding associated with the conformational conversion of PrPC to PrPSc.

Deciphering the Molecular Effects of Mutations on ATRX Cause ATRX Syndrome: A Molecular Dynamics Study.

α-thalassemia mental retardation X-linked (ATRX) syndrome is caused by the dysfunction of ATRFfigX protein. The present study explored the structural consequences influenced by two observed mutations V194I and C220R on ADD domain of ATRX protein by applying all atom molecular dynamics (MD) simulation. MD result showed that both the mutants exhibited wide variations in their backbone dynamics, as a result, mutant V210I showed complete distortion on α3 and the mutant C220R displayed a biased disruption on α2-3. The interference in the local folding of α-helices in both the mutants resulted by the loss of hydrogen bonds mediated by the backbone atoms. Principle component analysis (PCA) elucidated that both the mutants endured a diverse conformational dynamics, consequently adopted thermodynamically different conformational state. Besides, binding residues in both the mutants showed more structural disorder, thereby unable to recognize the hallmark modification, K9me3 (tri-methylated lysine at position 9) of histone H3 peptide and it was not conducive for the wild type ADD domain like functionality. Altogether, our findings provide knowledge to understand the structural and functional relationship of disease-associated mutations, V194I and C220R on ADD domain as well as gain further insights into the molecular pathogenesis of ATRX syndrome. J. Cell. Biochem. 118: 3318-3327, 2017. © 2017 Wiley Periodicals, Inc.

Unraveling the molecular effects of mutation L270P on Wiskkot-Aldrich syndrome protein: insights from molecular dynamics approach.

Missense mutation L270P disrupts the auto-inhibited state of "Wiskkot-Aldrich syndrome protein" (WASP), thereby constitutively activating the mutant structure, a key event for pathogenesis of X-linked neutropenia (XLN). In this study, we comprehensively deciphered the molecular feature of activated mutant structure by all atom molecular dynamics (MD) approach. MD analysis revealed that mutant structure exposed a wide variation in the spatial environment of atoms, resulting in enhanced residue flexibility. The increased flexibility of residues favored to decrease the number of intra-molecular hydrogen bonding interactions in mutant structure. The reduction of hydrogen bonds in the mutant structure resulted to disrupt the local folding of secondary structural elements that eventually affect the proper folding of mutants. The unfolded state of mutant structure established more number of inter-molecular hydrogen bonding interaction at interface level due to the conformational variability, thus mediated high binding affinity with its interacting partner, Cdc42.