The human endogenous retrovirus K (HERV-K) is the most recently acquired endogenous retrovirus in the human genome and is activated and expressed in many cancers and amyotrophic lateral sclerosis. We ...present the immature HERV-K capsid structure at 3.2 Å resolution determined from native virus-like particles using cryo-electron tomography and subtomogram averaging. The structure shows a hexamer unit oligomerized through a 6-helix bundle, which is stabilized by a small molecule analogous to IP6 in immature HIV-1 capsid. The HERV-K immature lattice is assembled via highly conserved dimer and trimer interfaces, as detailed through all-atom molecular dynamics simulations and supported by mutational studies. A large conformational change mediated by the linker between the N-terminal and the C-terminal domains of CA occurs during HERV-K maturation. Comparison between HERV-K and other retroviral immature capsid structures reveals a highly conserved mechanism for the assembly and maturation of retroviruses across genera and evolutionary time.
The N
-methyladenosine modification is one of the most abundant post-transcriptional modifications in ribonucleic acid (RNA) molecules. Using molecular dynamics simulations and alchemical free-energy ...calculations, we studied the structural and energetic implications of incorporating this modification in an adenine mononucleotide and an RNA hairpin structure. At the mononucleotide level, we found that the
configuration is more favorable than the
configuration by 2.05 ± 0.15 kcal/mol. The unfavorable effect of methylation was due to the steric overlap between the methyl group and a nitrogen atom in the purine ring. We then probed the effect of methylation in an RNA hairpin structure containing an AUCG tetraloop, which is recognized by a "reader" protein (YTHDC1) to promote transcriptional silencing of long noncoding RNAs. While methylation had no significant conformational effect on the hairpin stem, the methylated tetraloop showed enhanced conformational flexibility compared to the unmethylated tetraloop. The increased flexibility was associated with the outward flipping of two bases (A6 and U7) which formed stacking interactions with each other and with the C8 and G9 bases in the tetraloop, leading to a conformation similar to that in the RNA/reader protein complex. Therefore, methylation-induced conformational flexibility likely facilitates RNA recognition by the reader protein.
Interactions between RNA molecules and proteins are critical to many cellular processes and are implicated in various diseases. The RNA-peptide complexes are good model systems to probe the ...recognition mechanism of RNA by proteins. In this work, we report studies on the binding-unbinding process of a helical peptide from a viral RNA element using nonequilibrium molecular dynamics simulations. We explored the existence of various dissociation pathways with distinct free-energy profiles that reveal metastable states and distinct barriers to peptide dissociation. We also report the free-energy differences for each of the four pathways to be 96.47 ± 12.63, 96.1 ± 10.95, 91.83 ± 9.81, and 92 ± 11.32 kcal/mol. Based on the free-energy analysis, we further propose the preferred pathway and the mechanism of peptide dissociation. The preferred pathway is characterized by the formation of sequential hydrogen-bonding and salt-bridging interactions between several key arginine amino acids and the viral RNA nucleotides. Specifically, we identified one arginine amino acid (R8) of the peptide to play a significant role in the recognition mechanism of the peptide by the viral RNA molecule.
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an emerging class of small molecules that disrupt viral maturation by inducing the aberrant multimerization of IN. Here, we present cocrystal ...structures of HIV-1 IN with two potent ALLINIs, namely, BI-D and the drug candidate Pirmitegravir. The structures reveal atomistic details of the ALLINI-induced interface between the HIV-1 IN catalytic core and carboxyl-terminal domains (CCD and CTD). Projecting from their principal binding pocket on the IN CCD dimer, the compounds act as molecular glue by engaging a triad of invariant HIV-1 IN CTD residues, namely, Tyr226, Trp235, and Lys266, to nucleate the CTD-CCD interaction. The drug-induced interface involves the CTD SH3-like fold and extends to the beginning of the IN carboxyl-terminal tail region. We show that mutations of HIV-1 IN CTD residues that participate in the interface with the CCD greatly reduce the IN-aggregation properties of Pirmitegravir. Our results explain the mechanism of the ALLINI-induced condensation of HIV-1 IN and provide a reliable template for the rational development of this series of antiretrovirals through the optimization of their key contacts with the viral target.
Despite the remarkable success of combination antiretroviral therapy, HIV-1 remains among the major causes of human suffering and loss of life in poor and developing nations. To prevail in this drawn-out battle with the pandemic, it is essential to continue developing advanced antiviral agents to fight drug resistant HIV-1 variants. Allosteric integrase inhibitors (ALLINIs) are an emerging class of HIV-1 antagonists that are orthogonal to the current antiretroviral drugs. These small molecules act as highly specific molecular glue, which triggers the aggregation of HIV-1 integrase. In this work, we present high-resolution crystal structures that reveal the crucial interactions made by two potent ALLINIs, namely, BI-D and Pirmitegravir, with HIV-1 integrase. Our results explain the mechanism of drug action and will inform the development of this promising class of small molecules for future use in antiretroviral regimens.
Ribonucleic acids (RNAs) are conformationally flexible molecules that fold into three-dimensional structures and play an important role in different cellular processes as well as in the development ...of many diseases. RNA has therefore become an important target for developing novel therapeutic approaches. The biophysical processes underlying RNA function are often associated with rare structural transitions that play a key role in ligand recognition. In this work, we probe these rarely occurring transitions using nonequilibrium simulations by characterizing the dissociation of a ligand molecule from an HIV-1 viral RNA element. Specifically, we observed base-flipping rare events that are coupled with ligand binding/unbinding and also provided mechanistic details underlying these transitions.
Viruses remain a global threat to animals, plants, and humans. The type 1 human immunodeficiency virus (HIV-1) is a member of the retrovirus family and carries an RNA genome, which is reverse ...transcribed into viral DNA and further integrated into the host-cell DNA for viral replication and proliferation. The RNA structures from the HIV-1 genome provide valuable insights into the mechanisms underlying the viral replication cycle. Moreover, these structures serve as models for designing novel therapeutic approaches. Here, we review structural data on RNA from the HIV-1 genome as well as computational studies based on these structural data. The review is organized according to the type of structured RNA element which contributes to different steps in the viral replication cycle. This is followed by an overview of the HIV-1 transactivation response element (TAR) RNA as a model system for understanding dynamics and interactions in the viral RNA systems. The review concludes with a description of computational studies, highlighting the impact of biomolecular simulations in elucidating the mechanistic details of various steps in the HIV-1's replication cycle.
The Szeto-Schiller (SS) peptides are a subclass of cell-penetrating peptides that can specifically target mitochondria and mediate conditions caused by mitochondrial dysfunction. In this work, we ...constructed an iron-chelating SS peptide and studied its interaction with a mitochondrial-mimicking membrane using atomistic molecular dynamics (MD) simulations. We report that the peptide/membrane interaction is thermodynamically favorable, and the localization of the peptide to the membrane is driven by electrostatic interactions between the cationic residues and the anionic phospholipid headgroups. The insertion of the peptide into the membrane is driven by hydrophobic interactions between the aromatic side chains in the peptide and the lipid acyl tails. We also probed the translocation of the peptide across the membrane by applying nonequilibrium steered MD simulations and resolved the translocation pathway, free energy profile, and metastable states. We explored four distinct orientations of the peptide along the translocation pathway and found that one orientation was energetically more favorable than the other orientations. We tested a significantly slower pulling velocity on the most thermodynamically favorable system and compared metastable states during peptide translocation. We found that the peptide can optimize hydrophobic interactions with the membrane by having aromatic side chains interacting with the lipid acyl tails instead of forming π-π interactions with each other. The mechanistic insights emerging from our work will potentially facilitate improved peptide design with enhanced activity.
In my thesis work, I conducted molecular simulation studies to explore dynamics and interactions in nucleic acids. I began my work by applying conventional molecular dynamics (MD) simulations to ...study the local and global dynamics of the transactivation response (TAR) element from the type-1 human immunodeficiency virus (HIV-1) and the effect of binding of ligands on the dynamics of TAR RNA. I determined that the TAR RNA structure was stabilized on binding of ligands due to the decreased flexibility in helices that comprise TAR RNA. This rigidity of the TAR RNA structure was coupled with the decreased flipping of bulge nucleotides. I also observed that different initial conformations of TAR RNA converged to similar conformations in the course of MD simulations. Finally, I observed the formation of binding pockets in unliganded TAR structures that could accommodate ligands of various sizes. After comprehensively exploring the dynamics of TAR RNA with and without ligands, I conducted more specific studies on the interactions that were formed or broken during the (un)binding process of two ligands, a small molecule inhibitor and a helical peptide, from the viral RNA molecules using non-equilibrium simulations. Firstly, I observed that the dissociation of a small molecule is coupled with a base flipping event which I described using physical variables and thermodynamic properties. Secondly, I observed that the dissociation process of a helical peptide is facilitated by a network of hydrogen bonding and salt bridging interactions which are formed across four distinct dissociation pathways. I also resolved the free-energy profiles for each pathway which revealed metastable states and dissociation barriers. Based on the free-energy profiles, I proposed a preferred dissociation pathway and identified one arginine amino acid that plays an important role in the recognition of the peptide by the viral RNA. Next, I focused on studying a more complex reaction coordinate (RC) that could describe a base flipping mechanism in a double-stranded RNA (dsRNA) molecule using transition path sampling (TPS) methods. Additionally, I used the likelihood maximization method to determine a refined RC based on an ensemble of 1000 transition trajectories created by the path sampling algorithm. The refined RC consisted of two collective variables (CVs), a distance and a dihedral angle between the neighboring nucleotides and the flipping base. I also projected a free-energy profile along the refined RC which revealed three free-energy minima. I proposed that one of the free-energy minima represented a wobbled conformation of the flipping nucleobase. I also analyzed the reactive trajectories which showed that the base flipping is coupled with global conformational changes in a stem-loop of dsRNA. Outside of studies involving RNA, I conducted conventional MD simulations to study the dynamics of a porphyrin/DNA nanoassembly which revealed the overall left-handed orientation of the nanoassembly. I characterized the resulting porphyrin/DNA system using various physical variables. Overall, my thesis revealed the local and global dynamics of RNA as well as DNA systems, and perturbations to dynamics originating in binding of ligands of various sizes.
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