An envelope glycoprotein, gp41, is crucial for the entry of human immunodeficiency virus (HIV) into the host cell. The 20−23 N-terminal amino acid sequence of gp41 plays an important role in promoting fusion between viral and host cells. Interestingly, the structure and function of the fusion peptide are extremely sensitive to the characteristics of the lipid environment. In this present work, we have extensively utilized steady-state and time-resolved fluorescence spectroscopy in tandem with molecular dynamics simulation to elucidate peptide binding and peptide-induced perturbation to the membrane. We have used two depth-dependent fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its trimethylammonium derivative (TMA-DPH), to monitor the effect of peptide binding along the bilayer normal and have reconciled the experimental observation with the insights from the simulated molecular events. We have further monitored the effect of membrane cholesterol on peptide-induced membrane perturbation. The molecular dynamics simulation data show that the peptide alters the membrane properties in the vicinity of the peptide and it penetrates to a larger extent into the bilayer when the membrane contains cholesterol. Our results clearly elucidate that cholesterol alters the membrane physical properties in favor of membrane fusion and interaction pattern of the fusion peptide with the membrane in a concentration-dependent fashion. The role of cholesterol is specifically important as the host eukaryotic cells contain a decent amount of cholesterol that might be critical for the entry of HIV into the host cells.

Plants need to maintain a low Na+/K+ ratio for their survival and growth when there is high sodium concentration in soil. Under these circumstances, the high affinity K+ transporter (HKT) and its homologs are known to perform a critical role with HKT1;5 as a major player in maintaining Na+ concentration. Preferential expression of HKT1;5 in roots compared to shoots was observed in rice and rice-like genotypes from real time PCR, microarray, and RNAseq experiments and data. Its expression trend was generally higher under increasing salt stress in sensitive IR29, tolerant Pokkali, both glycophytes; as well as the distant wild rice halophyte, Porteresia coarctata, indicative of its importance during salt stress. These results were supported by a low Na+/K+ ratio in Pokkali, but a much lower one in P. coarctata. HKT1;5 has functional variability among salt sensitive and tolerant varieties and multiple sequence alignment of sequences of HKT1;5 from Oryza species and P. coarctata showed 4 major amino acid substitutions (140 P/A/T/I, 184 H/R, D332H, V395L), with similarity amongst the tolerant genotypes and the halophyte but in variance with sensitive ones. The best predicted 3D structure of HKT1;5 was generated using Ktrab potassium transporter as template. Among the four substitutions, conserved presence of aspartate (332) and valine (395) in opposite faces of the membrane along the Na+/K+ channel was observed only for the tolerant and halophytic genotypes. A model based on above, as well as molecular dynamics simulation study showed that valine is unable to generate strong hydrophobic network with its surroundings in comparison to leucine due to reduced side chain length. The resultant alteration in pore rigidity increases the likelihood of Na+ transport from xylem sap to parenchyma and further to soil. The model also proposes that the presence of aspartate at the 332 position possibly leads to frequent polar interactions with the extracellular loop polar residues which may shift the loop away from the opening of the constriction at the pore and therefore permit easy efflux of the Na+. These two substitutions of the HKT1;5 transporter probably help tolerant varieties maintain better Na+/K+ ratio for survival under salt stress.

No abstract

Activation of the pro-apoptotic BAX protein, a BCL-2 family member, is known to trigger apoptosis by forming pores in the mitochondrial outer membrane (MOM). While in the cytosol, release of its transmembrane C-terminal helix (called α9 helix) from a well-characterized binding pocket (BC groove) and subsequent permeabilization of the MOM are understood to be the initiating events of the activation. Concerning what initiates BAX activation, so far one plausible suggestion has been that the transient attachment of BH3-only peptide at a distal site from the BC groove triggers the activation process. Yet how this pivotal step displaces α9 from the BC groove has remained poorly understood. Using a combination of standard molecular dynamics and enhanced sampling methods, the energy landscape of BIM (BH3-only peptide) induced BAX activation has been computed, and the molecular origin of those events is hereby reported in atomistic detail. The simulated transition pathway of α9 release reveals that BIM subdues the energetic cost of the process by reducing the activation energy barrier to some extent but mostly by minimizing the free energy difference between the active (α9-released) and inactive (α9-bound) states. Interestingly, the flexibility of the α9 helix itself plays a decisive role in this mechanism. The impact of BIM encounter at the distal site is found to propagate to the α9 (BC groove bound) mostly through conserved pathways of residue level interactions. Overall, the thermodynamic basis of the "hit-and-run" mechanism for activation of the BCL-2 family is presented reconciling the available biochemical observations.

Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (e SpCas9). Hence, this may also be true in GeoCas9.