Mechanism of darunavir binding to monomeric HIV-1 protease: a step forward in the rational design of dimerization inhibitors.

HIV protease (HIVPR) is a key target in AIDS therapeutics. All ten FDA-approved drugs that compete with substrates in binding to this dimeric enzyme's active site have become ineffective due to the emergence of drug resistant mutants. Blocking the dimerization interface of HIVPR is thus being explored as an alternate strategy. The latest drug, darunavir (DRV), which exhibited a high genetic barrier to viral resistance, is said to have a dual mode of action - (i) binding to the dimeric active site, and (ii) preventing the dimerization by binding to the HIVPR monomer. Despite several reports on DRV complexation with dimeric HIVPR, the mode and mechanism of the binding of DRV to the HIVPR monomer are poorly understood. In this study, we utilized all-atomic MD simulations and umbrella sampling techniques to identify the best possible binding mode of DRV to the monomeric HIVPR and its mechanism of association. The results suggest that DRV binds between the active site and the flap of the monomer, and the flap plays a crucial role in directing the drug to bind and driving the other protein domains to undergo induced fit changes for stronger complexation. The obtained binding mode of DRV was validated by comparing with various mutational data from clinical isolates to reported in vitro mutations. The identified binding pose was also able to successfully reproduce the experimental Ki value in the picomolar range. The residue-level information extracted from this study could accelerate the structure-based drug designing approaches targeting HIVPR dimerization.

Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage.

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 (eSpCas9). Hence, this may also be true in GeoCas9.

Identifying crucial E-protein residues responsible for unusual stability of Zika virus envelope.

An outbreak of Zika virus (ZIKV) infections in 2015-16 that caused microcephaly and other congenital abnormalities in newborns prompted intense research across the globe. These studies have suggested that ZIKV can survive high temperatures and harsh physiological conditions, unlike the other flaviviruses such as dengue virus (DENV). In contrast, recent cryo-electron microscopy studies have shown very similar architecture of the ZIKV and DENV envelopes that constitute the primary level of viral protection. Encouraged by these findings, here we attempt to identify the crucial protein residues that make the ZIKV envelope so robust by employing coarse-grained and all-atomic molecular dynamics simulations and computational mutagenesis studies. In accordance with more recent cryo-electron microscopy findings, our simulation results exhibited stable ZIKV envelope protein shell both at 29oC and 40°C, whereas the DENV2 shell loosened up significantly at 40°C. Subsequently, we simulated a series of ZIKV variants to identify the specific domain and residues involved in maintaining the structural integrity of the viral protein shell at high temperatures. Our results suggest that the DIII domain-more specifically, the CD- and FG-loop residues of the ZIKV protein shell-play a crucial role in making the virus envelope thermostable by inducing strong raft-raft interactions. These findings can accelerate the rational design of ZIKV therapeutics.

Electrostatics Plays a Crucial Role in HIV-1 Protease Substrate Binding, Drugs Fail to Take Advantage.

HIV-1 protease (HIVPR) is an important drug target for combating AIDS. This enzyme is an aspartyl protease that is functionally active in its dimeric form. Nuclear magnetic resonance reports have convincingly shown that a pseudosymmetry exists at the HIVPR active site, where only one of the two aspartates remains protonated over the pH range of 2.5-7.0. To date, all HIVPR-targeted drug design strategies focused on maximizing the size-shape complementarity and van der Waals interactions of the small molecule drugs with the deprotonated, symmetric active site envelope of crystallized HIVPR. However, these strategies were ineffective with the emergence of drug resistant protease variants, primarily due to the steric clashes at the active site. In this study, we traced a specificity in the substrate binding motif that emerges primarily from the asymmetrical electrostatic potential present in the protease active site due to the uneven protonation. Our detailed results from atomistic molecular dynamics simulations show that while such a specific mode of substrate binding involves significant electrostatic interactions, none of the existing drugs or inhibitors could utilize this electrostatic hot spot. As the electrostatic is long-range interaction, it can provide sufficient binding strength without the necessity of increasing the bulkiness of the inhibitors. We propose that introducing the electrostatic component along with optimal fitting at the binding pocket could pave the way for promising designs that might be more effective against both wild type and HIVPR resistant variants.

Fine-tuning pH sensor H98 by remote essential residues in the hydrogen-bond network of mTASK-3.

TASK-3 generates a background K+ conductance which when inhibited by acidification depolarizes membrane potential and increases cell excitability. These channels sense pH by protonation of histidine residue H98, but recent evidence revealed that several other amino acid residues also contribute to TASK-3 pH sensitivity, suggesting that the pH sensitivity is determined by an intermolecular network. Here we use electrophysiology and molecular modeling to characterize the nature and requisite role(s) of multiple amino acids in pH sensing by TASK-3. Our results suggest that the pH sensor H98 and consequently pH sensitivity is influenced by remote amino acids that function as a hydrogen-bonding network to modulate ionic conductivity. Among the residues in the network, E30 and K79 are the most important for passing external signals near residue S31 to H98. The hydrogen-bond network plays a key role in selectivity or pH sensing in mTASK-3, and E30 and S31 in the network can modulate the conductive properties (E30) or reverse the pH sensitivity and selectivity of the channel (S31). Molecular dynamics simulations and pK1/2 calculation revealed that double mutants involving H98 + S31 primarily regulate the structure stability of the pore selectivity filter and pore loop regions, further strengthen the stability of the cradle suspension system, and alter the ionization state of E30 and K79, thereby preventing pore conformational change that normally occurs in response to varying extracellular pH. These results demonstrate that crucial residues in the hydrogen-bond network can remotely tune the pH sensing of mTASK-3 and may be a potential allosteric regulatory site for therapeutic molecule development.

Machines on Genes through the Computational Microscope.

Macromolecular machines acting on genes are at the core of life's fundamental processes, including DNA replication and repair, gene transcription and regulation, chromatin packaging, RNA splicing, and genome editing. Here, we report the increasing role of computational biophysics in characterizing the mechanisms of "machines on genes", focusing on innovative applications of computational methods and their integration with structural and biophysical experiments. We showcase how state-of-the-art computational methods, including classical and ab initio molecular dynamics to enhanced sampling techniques, and coarse-grained approaches are used for understanding and exploring gene machines for real-world applications. As this review unfolds, advanced computational methods describe the biophysical function that is unseen through experimental techniques, accomplishing the power of the "computational microscope", an expression coined by Klaus Schulten to highlight the extraordinary capability of computer simulations. Pushing the frontiers of computational biophysics toward a pragmatic representation of large multimegadalton biomolecular complexes is instrumental in bridging the gap between experimentally obtained macroscopic observables and the molecular principles playing at the microscopic level. This understanding will help harness molecular machines for medical, pharmaceutical, and biotechnological purposes.

Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids.

Synergistic-aggregation and cross-seeding by two different proteins/peptides in the amyloid aggregation are well evident in various neurological disorders including Alzheimer's disease. Here, we show co-storage of human Prolactin (PRL), which is associated with lactation in mammals, and neuropeptide galanin (GAL) as functional amyloids in secretory granules (SGs) of the female rat. Using a wide variety of biophysical studies, we show that irrespective of the difference in sequence and structure, both hormones facilitate their synergic aggregation to amyloid fibrils. Although each hormone possesses homotypic seeding ability, a unidirectional cross-seeding of GAL aggregation by PRL seeds and the inability of cross seeding by mixed fibrils suggest tight regulation of functional amyloid formation by these hormones for their efficient storage in SGs. Further, the faster release of functional hormones from mixed fibrils compared to the corresponding individual amyloid, suggests a novel mechanism of heterologous amyloid formation in functional amyloids of SGs in the pituitary.

The formation of plaques of proteins called 'amyloids' in the brain is one of the hallmark characteristics of both Alzheimer's and Parkinson's disease, but amyloids can form in many tissues and organs, often disrupting normal activity. A lot of the research into amyloids has focused on their role in disease, but it turns out that amyloids can also appear in healthy tissues. For example, some protein hormones form amyloids that act as storage depots, helping cells to release the hormone when it is needed. Normally, amyloids are made mostly of a single type of protein or protein fragment associated with a particular disease like Alzheimer's. Often, this type of amyloid promotes plaque formation in other proteins, which aggravates other diseases (for example, the amyloids that form in Alzheimer's can lead to Parkinson's disease or type II diabetes getting worse).The plaques start growing from small amyloid fragments called seeds. In mixed amyloids ­ amyloids made of two types of proteins ­ seeds made of one protein can trigger the formation of amyloids of the other protein. This raises the question, is this true for hormones? The body often releases more than one hormone at a time from the same tissue; for example, the pituitary gland releases prolactin and galanin simultaneously. However, these hormones have completely different structures, so whether they can form a mixed amyloid is unclear. To answer this question, Chatterjee et al. first determined that, within the pituitary gland of female rats, prolactin and galanin could be found together in the same cells, forming mixed amyloids. To understand out how this happens, Chatterjee et al. tried seeding new amyloids using either prolactin or galanin. This revealed that only prolactin seeds were able to trigger the formation of galanin amyloids. Chatterjee et al. also found that the mixed amyloids could release the hormones faster than amyloids made from either protein alone. Together, these results suggest that the collaboration between these two proteins may help maintain hormone balance in the body. Problems with hormone storage and release lead to various human diseases, including prolactinoma. Understanding amyloid storage depots could reveal new ways to control hormone levels. Further research could also help to explain more about well-studied diseases linked to amyloids, like Alzheimer's.

Hydrogen bonding catalysis by water in epoxide ring opening reaction.

Water can act as catalyst is perhaps the most intriguing property reported of this molecule in the last decade. However, despite being an integral part of many enzyme structures, the role of water in catalyzing enzymatic reactions remains sparsely studied. In a recent study, we have shown that the epoxide ring opening in aspartate proteases follows a two-step process involving water. In this work, we attempt to unravel the electronic basis of the co-catalytic role of water in the epoxide ring opening reaction by employing high-level quantum mechanical calculations at M06-2X/6-31+G(d,p) level of accuracy. Our computed electron density and its reduced gradient show that water anchor the reactant molecules through strong H-bond bridges. In addition, the strong ionizing power of water allows better charge delocalization to stabilize the transition states and oxyanion intermediate. Electrostatic analyses suggest greater charge transfer from the aspartates to the epoxide in the transition state, which is found to be exergonic in nature rendering a low-barrier reaction compared to a control system where water was omitted in the reaction field. This elucidated mechanism at electronic level could promote further research to search for the co-catalytic role of water in other enzymes.

Molecular Basis of Differential Stability and Temperature Sensitivity of ZIKA versus Dengue Virus Protein Shells.

Rapid spread of ZIKA virus (ZIKV) and its association with severe birth defects have raised worldwide concern. Recent studies have shown that ZIKV retains its infectivity and remains structurally stable at temperatures up to 40 °C, unlike dengue and other flaviviruses. In spite of recent cryo-EM structures that showed similar architecture of ZIKA and dengue virus (DENV) E protein shells, little is known that makes ZIKV so temperature insensitive. Here, we attempt to unravel the molecular basis of greater thermal stability of ZIKV over DENV2 by executing atomistic molecular dynamics (MD) simulations on the viral E protein shells at 37 °C. Our results suggest that ZIKA E protein shell retains its structural integrity through stronger inter-raft communications facilitated by a series of electrostatic and H-bonding interactions among multiple inter-raft residues. In comparison, the DENV2 E protein shell surface was loosly packed that exhibited holes at all 3-fold vertices, in close agreement with another EM structure solved at 37 °C. The residue-level information obtained from our study could pave way for designing small molecule inhibitors and specific antibodies to inhibit ZIKV E protein assembly and membrane fusion.

Mechanistic Study on the Antibacterial Activity of Self-Assembled Poly(aryl ether)-Based Amphiphilic Dendrimers.

The increased threat of bacterial resistance against conventional antibiotics has warranted the need for development of membrane targeting antibacterial agents. Several self-assembled cationic amphiphiles with different supramolecular structures have been reported in recent years for potent antibacterial activity with increased specificity. In this study, we describe the self-assembly and antibacterial activity of four lower generation poly(aryl ether)-based amphiphilic dendrimers (AD-1, AD-2, AD-3, and AD-4) containing terminal amine (PAMAM-based), ester, and hydrazide functional groups with varied hydrophobicity. Among the four dendrimers under study, the amine-terminated dendrimer (AD-1) displayed potent antibacterial activity. The ratio of surface cationic charge to hydrophobicity had a significant effect on the antibacterial activity, where AD-3 dendrimer with increased surface cationic charges exhibited a higher minimum inhibitory concentration (MIC) than AD-1. AD-2 (ester terminated) and AD-4 (hydrazide terminated) dendrimers did not show any bactericidal activity. The amphiphilic dendrimer-bacteria interactions, further validated by binding studies, also showed significant changes in bacterial morphology, effective membrane permeation, and depolarization by AD-1 in comparison with AD-3. Molecular dynamics simulations of AD-1 and AD-3 on bacterial membrane patches further corroborated the experimental findings. The structural conformation of AD-1 dendrimer facilitated increased membrane interaction compared to AD-3 dendrimer. AD-1 also displayed selectivity to bacterial membranes over fibroblast cells (4× MIC), corroborating the significance of an optimal hydrophobicity for potent antibacterial activity with no cytotoxicity. The self-assembled (poly(aryl ether)-PAMAM-based) dendrimer (AD-1) also exhibited potent antibacterial activity in comparison with conventional higher generation dendrimers, establishing the implication of self-assembly for bactericidal activity. Moreover, the detailed mechanistic study reveals that optimal tuning of the hydrophobicity of amphiphilic dendrimers plays a crucial role in membrane disruption of bacteria. We believe that this study will provide valuable insights into the design strategies of amphiphilic dendrimers as antibacterial agents for efficient membrane disruption.