Clarisse G. Ricci

Molecular mechanism of peroxisome proliferator-activated receptor α activation by WY14643 : a new mode of ligand recognition and receptor stabilization

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.

CHARMM Force Field Parameterization of Peroxisome Proliferator-Activated Receptor γ Ligands

The peroxisome proliferator-activated receptor γ (PPARγ) ligands are important therapeutic drugs for the treatment of type 2 diabetes, obesity and cardiovascular diseases. In particular, partial agonists and non-agonists are interesting targets to reduce glucose levels, presenting few side effects in comparison to full agonists. In this work, we present a set of CHARMM-based parameters of a molecular mechanics force field for two PPARγ ligands, GQ16 and SR1664. GQ16 belongs to the thiazolidinedione class of drugs and it is a PPARγ partial agonist that has been shown to promote the “browning” of white adipose tissue. SR1664 is the precursor of the PPARγ non-agonist class of ligands that activates PPARγ in a non-classical manner. Here, we use quantum chemical calculations consistent with the CHARMM protocol to obtain bonded and non-bonded parameters, including partial atomic charges and effective torsion potentials for both molecules. The newly parameterized models were evaluated by examining the behavior of GQ16 and SR1664 free in water and bound to the ligand binding pocket of PPARγ using molecular dynamics simulations. The potential parameters derived here are readily transferable to a variety of pharmaceutical compounds and similar PPARγ ligands.

Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex

Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors.

Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9

CRISPR-Cas9 is a genome editing technology with major impact in life sciences. In this system, the endonuclease Cas9 generates double strand breaks in DNA upon RNA-guided recognition of a complementary DNA sequence, which strictly requires the presence of a protospacer adjacent motif (PAM) next to the target site. Although PAM recognition is essential for cleavage, it is unknown whether and how PAM binding activates Cas9 for DNA cleavage at spatially distant sites. Here, we find evidence of a PAM-induced allosteric mechanism revealed by microsecond molecular dynamics simulations. PAM acts as an allosteric effector and triggers the interdependent conformational dynamics of the Cas9 catalytic domains (HNH and RuvC), responsible for concerted cleavage of the two DNA strands. Targeting such an allosteric mechanism should enable control of CRISPR-Cas9 functionality.

“Martinizing” the Variational Implicit Solvent Method (VISM): Solvation Free Energy for Coarse-Grained Proteins

Solvation is a fundamental driving force in many biological processes including biomolecular recognition and self-assembly, not to mention protein folding, dynamics, and function. The variational implicit solvent method (VISM) is a theoretical tool currently developed and optimized to estimate solvation free energies for systems of very complex topology, such as biomolecules. VISM’s theoretical framework makes it unique because it couples hydrophobic, van der Waals, and electrostatic interactions as a functional of the solvation interface. By minimizing this functional, VISM produces the solvation interface as an output of the theory. In this work, we push VISM to larger scale applications by combining it with coarse-grained solute Hamiltonians adapted from the MARTINI framework, a well-established mesoscale force field for modeling large-scale biomolecule assemblies. We show how MARTINI-VISM (MVISM) compares with atomistic VISM (AVISM) for a small set of proteins differing in size, shape, and charge distribution. We also demonstrate MVISM’s suitability to study the solvation properties of an interesting encounter complex, barnase–barstar. The promising results suggest that coarse-graining the protein with the MARTINI force field is indeed a valuable step to broaden VISM’s and MARTINI’s applications in the near future.

DNA and IκBα Both Induce Long-Range Conformational Changes in NFκB

We recently discovered that IκBα enhances the rate of release of nuclear factor kappa B (NFκB) from DNA target sites in a process we have termed molecular stripping. Coarse-grained molecular dynamics simulations of the stripping pathway revealed two mechanisms for the enhanced release rate: the negatively charged PEST region of IκBα electrostatically repels the DNA, and the binding of IκBα appears to twist the NFκB heterodimer so that the DNA can no longer bind. Here, we report amide hydrogen/deuterium exchange data that reveal long-range allosteric changes in the NFκB (RelA-p50) heterodimer induced by DNA or IκBα binding. The data suggest that the two Ig-like subdomains of each Rel-homology region, which are connected by a flexible linker in the heterodimer, communicate in such a way that when DNA binds to the N-terminal DNA-binding domains, the nuclear localization signal becomes more highly exchanging. Conversely, when IκBα binds to the dimerization domains, amide exchange throughout the DNA-binding domains is decreased as if the entire domain is becoming globally stabilized. The results help understand how the subtle mechanism of molecular stripping actually occurs.

Dynamic Structure and Inhibition of a Malaria Drug Target: Geranylgeranyl Diphosphate Synthase

We report a molecular dynamics investigation of the structure, function, and inhibition of geranylgeranyl diphosphate synthase (GGPPS), a potential drug target, from the malaria parasite Plasmodium vivax. We discovered several GGPPS inhibitors, benzoic acids, and determined their structures crystallographically. We then used molecular dynamics simulations to investigate the dynamics of three such inhibitors and two bisphosphonate inhibitors, zoledronate and a lipophilic analogue of zoledronate, as well as the enzymes product, GGPP. We were able to identify the main motions that govern substrate binding and product release as well as the molecular features required for GGPPS inhibition by both classes of inhibitor. The results are of broad general interest because they represent the first detailed investigation of the mechanism of action, and inhibition, of an important antimalarial drug target, geranylgeranyl diphosphate synthase, and may help guide the development of other, novel inhibitors as new drug leads.

Molecular mechanism of off-target effects in CRISPR-Cas9

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations – using Gaussian accelerated MD (GaMD) – are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to “lock” the HNH domain, thereby identifying the ability to “lock” HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to “trap” somehow the HNH domain in an inactive “conformational checkpoint” state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

RelA-Containing NFκB Dimers Have Strikingly Different DNA-Binding Cavities in the Absence of DNA

The main nuclear factor kappa B transcription factor family members RelA-p50 heterodimer and RelA homodimer have different biological functions and show different transcriptional activation profiles. To investigate whether the two family members adopt a similar conformation in their free states, we performed hydrogen-deuterium exchange mass spectrometry, all-atom molecular dynamics simulations, and stopped-flow binding kinetics experiments. Surprisingly, the N-terminal DNA-binding domains adopt an open conformation in RelA-p50 but a closed conformation in RelA homodimer. Both hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations indicate the formation of an interface between the N-terminal DNA-binding domains only in the RelA homodimer. Such an interface would be expected to impede DNA binding, and stopped-flow binding kinetics show that association of DNA is slower for the homodimer as compared to the heterodimer. Our results show that the DNA-binding cavity in the RelA-p50 heterodimer is open for DNA binding, whereas in the RelA homodimer, it is occluded.

Tailoring the Variational Implicit Solvent Method for New Challenges: Biomolecular Recognition and Assembly

Predicting solvation free energies and describing the complex water behavior that plays an important role in essentially all biological processes is a major challenge from the computational standpoint. While an atomistic, explicit description of the solvent can turn out to be too expensive in large biomolecular systems, most implicit solvent methods fail to capture “dewetting” effects and heterogeneous hydration by relying on a pre-established (i.e., guessed) solvation interface. Here we focus on the Variational Implicit Solvent Method, an implicit solvent method that adds water “plasticity” back to the picture by formulating the solvation free energy as a functional of all possible solvation interfaces. We survey VISMs applications to the problem of molecular recognition and report some of the most recent efforts to tailor VISM for more challenging scenarios, with the ultimate goal of including thermal fluctuations into the framework. The advances reported herein pave the way to make VISM a uniquely successful approach to characterize complex solvation properties in the recognition and binding of large-scale biomolecular complexes.