The main goal of our laboratory is to understand the physical and chemical properties of complex biological systems, in particular their function emerging from structure. To address these questions, we use and develop a broad spectrum of computational tools fully integrated with experimental data. Multiscale models and dynamic integrative modeling are used to investigate the assembly and function of molecular assemblies mimicking conditions of the cellular environment.

Current Research Highlights

July 2018: Mapping the sensing spots of aerolysin for single oligonucleotides analysis.

Nanopore sensing is a powerful single-molecule method for DNA and protein sequencing. Recent studies have demonstrated that aerolysin exhibits a high sensitivity for single-molecule detection. However, the lack of the atomic resolution structure of aerolysin pore has hindered the understanding of its sensing capabilities. Herein, we integrate nanopore experimental results and molecular simulations based on a recent pore structural model to precisely map the sensing spots of this toxin for ssDNA translocation. Rationally probing ssDNA length and composition upon pore translocation provides new important insights for molecular determinants of the aerolysin nanopore. Computational and experimental results reveal two critical sensing spots (R220, K238) generating two constriction points along the pore lumen. Taking advantage of the sensing spots, all four nucleobases, cytosine methylation and oxidation of guanine can be clearly identified in a mixture sample. The results provide evidence for the potential of aerolysin as a nanosensor for DNA sequencing.

December 2017: Conserved Lipid and Small-Molecule Modulation of COQ8.

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by CoQ-like precursors and by binding to membranes containing cardiolipin, a prevalent mitochondrial lipid enriched at the IMM where COQ8 resides. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, our work advances our understanding of the core COQ8 biochemical function across evolution (ATPase activity), reveals how the positioning of COQ8 on the IMM is key to its activation, and provides effective new tools for the further investigation of the role of COQ8 in CoQ biosynthesis.

October 2017: Recruitment of the amyloid precursor protein by γ-secretase at the synaptic plasma membrane.

Γ-secretase is a membrane-embedded protease that cleaves single transmembrane helical domains of various integral membrane proteins. The amyloid precursor protein (APP) is an important substrate due to its pathological relevance to Alzheimer’s disease. The mechanism of the cleavage of APP by γ-secretase that leads to accumulation of Alzheimer’s disease causing amyloid-β (Aβ) is still unknown. Coarse-grained molecular dynamics simulations in this study reveal initial lipids raft formation near the catalytic site of γ-secretase as well as changes in dynamic behavior of γ-secretase once interacting with APP. The results suggest a precursor of the APP binding mode and hint at conformational changes of γ-secretase in the nicastrin (NCT) domain upon APP binding.

May 2017: Signal Sensing and Transduction by Histidine Kinases as Unveiled through Studies on a Temperature Sensor.

Histidine kinases (HK) are the sensory proteins of two-component systems, responsible for a large fraction of bacterial responses to stimuli and environmental changes. Prototypical HKs are membrane-bound proteins that phosphorylate cognate response regulator proteins in the cytoplasm upon signal detection in the membrane or periplasm. HKs stand as potential drug targets but also constitute fascinating systems for studying proteins at work, specifically regarding the chemistry and mechanics of signal detection, transduction through the membrane, and regulation of catalytic outputs. In this Account, we focus on Bacillus subtilis DesK, a membrane-bound HK part of a two-component system that maintains appropriate membrane fluidity at low growth temperatures. Unlike most HKs, DesK has no extracytoplasmic signal-sensing domains; instead, sensing is carried out by 10 transmembrane helices (coming from two protomers) arranged in an unknown structure. The fifth transmembrane helix from each protomer connects, without any of the intermediate domains found in other HKs, into the dimerization and histidine phosphotransfer (DHp) domain located in the cytoplasm, which is followed by the ATP-binding domains (ABD). Throughout the years, genetic, biochemical, structural, and computational studies on wild-type, mutant, and truncated versions of DesK allowed us to dissect several aspects of DesK’s functioning, pushing forward a more general understanding of its own structure/function relationships as well as those of other HKs. We have shown that the sensing mechanism is rooted in temperature-dependent membrane properties, most likely a combination of thickness, fluidity, and water permeability, and we have proposed possible mechanisms by which DesK senses these properties and transduces the signals. X-ray structures and computational models have revealed structural features of TM and cytoplasmic regions in DesK’s kinase- and phosphatase-competent states. Biochemical and genetic experiments and molecular simulations further showed that reversible formation of a two-helix coiled coil in the fifth TM segment and the N-terminus of the cytoplasmic domain is essential for the sensing and signal transduction mechanisms. Together with other structural and functional works, the emerging picture suggests that diverse HKs possess distinct sensing and transduction mechanisms but share as rather general features (i) a symmetric phosphatase state and an asymmetric kinase state and (ii) similar functional outputs on the conserved DHp and ABD domains, achieved through different mechanisms that depend on the nature of the initial signal. We here advance (iii) an important role for TM prolines in transducing the initial signals to the cytoplasmic coiled coils, based on simulations of DesK’s TM helices and our previous work on a related HK, PhoQ. Lastly, evidence for DesK, PhoQ, BvgS, and DctB HKs shows that (iv) overall catalytic output is tuned by a delicate balance between hydration potentials, coiled coil stability, and exposure of hydrophobic surface patches at their cytoplasmic coiled coils and at the N-terminal and C-terminal sides of their TM helices. This balance is so delicate that small perturbations, either physiological signals or induced by mutations, lead to large remodeling of the underlying conformational landscape achieving clear-cut changes in catalytic output, mirroring the required response speed of these systems for proper biological function.

April 2017: Protein post-translational modifications: In silico prediction tools and molecular modeling.
Post-translational modifications (PTMs) occur in almost all proteins and play an important role in numerous biological processes by significantly affecting proteins structure and dynamics. Several computational approaches have been developed to study PTMs (e.g., phosphorylation, sumoylation or palmitoylation) showing the importance of these techniques in predicting modified sites that can be further investigated with experimental approaches. In this review, we summarize some of the available online platforms and their contribution in the study of PTMs. Moreover, we discuss the emerging capabilities of molecular modeling and simulation that are able to complement these bioinformatics methods, providing deeper molecular insights into the biological function of post-translational modified proteins.

March 2017: Disentangling constraints using viability evolution principles in integrative modeling of macromolecular assemblies.

Predicting the structure of large molecular assemblies remains a challenging task in structural biology when using integrative modeling approaches. One of the main issues stems from the treatment of heterogeneous experimental data used to predict the architecture of native complexes. We propose a new method, applied here for the first time to a set of symmetrical complexes, based on evolutionary computation that treats every available experimental input independently, bypassing the need to balance weight components assigned to aggregated fitness functions during optimization.

October 2016: Olfactory receptor pseudo-pseudogenes.

Pseudogenes are generally considered to be non-functional DNA sequences that arise through nonsense or frame-shift mutations of protein-coding genes. Although certain pseudogene-derived RNAs have regulatory roles, and some pseudogene fragments are translated, no clear functions for pseudogene-derived proteins are known. Olfactory receptor families contain many pseudogenes, which reflect low selection pressures on loci no longer relevant to the fitness of a species. Here we report the characterization of a pseudogene in the chemosensory variant ionotropic glutamate receptor repertoire of Drosophila sechellia, an insect endemic to the Seychelles that feeds almost exclusively on the ripe fruit of Morinda citrifolia. This locus, D. sechellia Ir75a, bears a premature termination codon (PTC) that appears to be fixed in the population. However, D. sechellia Ir75a encodes a functional receptor, owing to efficient translational read-through of the PTC. Read-through is detected only in neurons and is independent of the type of termination codon, but depends on the sequence downstream of the PTC. Furthermore, although the intact Drosophila melanogaster Ir75a orthologue detects acetic acid—a chemical cue important for locating fermenting food found only at trace levels in Morinda fruit—D. sechellia Ir75a has evolved distinct odour-tuning properties through amino-acid changes in its ligand-binding domain. We identify functional PTC-containing loci within different olfactory receptor repertoires and species, suggesting that such ‘pseudo-pseudogenes’ could represent a widespread phenomenon.

August 2016 : Molecular Effects of Concentrated Solutes on Protein Hydration, Dynamics, and Electrostatics

Most studies of protein structure and function are performed in dilute conditions, but proteins typically experience high solute concentrations in their physiological scenarios and biotechnological applications. High solute concentrations have well-known effects on coarse protein traits like stability, diffusion, and shape, but likely also perturb other traits through finer effects pertinent at the residue and atomic levels. Here, NMR and molecular dynamics investigations on ubiquitin disclose variable interactions with concentrated solutes that lead to localized perturbations of the protein’s surface, hydration, electrostatics, and dynamics, all dependent on solute size and chemical properties. Most strikingly, small polar uncharged molecules are sticky on the protein surface, whereas charged small molecules are not, but the latter still perturb the internal protein electrostatics as they diffuse nearby. Meanwhile, interactions with macromolecular crowders are favored mainly through hydrophobic, but not through polar, surface patches. All the tested small solutes strongly slow down water exchange at the protein surface, whereas macromolecular crowders do not exert such strong perturbation. Finally, molecular dynamics simulations predict that unspecific interactions slow down microsecond- to millisecond-timescale protein dynamics despite having only mild effects on pico- to nanosecond fluctuations as corroborated by NMR. We discuss our results in the light of recent advances in understanding proteins inside living cells, focusing on the physical chemistry of quinary structure and cellular organization, and we reinforce the idea that proteins should be studied in native-like media to achieve a faithful description of their function.