Research Highlights

• 2018

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.

• 2017

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.

 • 2016

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.

August 2016: Effect of the Synaptic Plasma Membrane on the Stability of the Amyloid Precursor Protein Homodimer.

The proteolytic cleavage of the transmembrane (TM) domain of the Amyloid Precursor Protein (APP) releases amyloid-β (Αβ) peptides, which accumulation in the brain tissue is an early indicator of Alzheimer’s disease. We used multiscale molecular dynamics simulations to investigate the stability of APP-TM dimer in realistic models of the synaptic plasma membrane (SPM). Between the two possible dimerization motifs proposed by NMR and EPR, namely G709XXXA713 and G700XXXG704XXXG708, our study revealed that the dimer promoted by the G709XXXA713 motif is not stable in the SPM due to the competition with highly unsaturated lipids that constitute the SPM. Under the same conditions, the dimer promoted by the G700XXXG704XXXG708 motif is instead the most stable species and likely the most biologically relevant. Regardless of the dimerization state, both these motifs can be involved in the recruitment of cholesterol molecules.

August 2016: Unorthodox Kinase Activity of the ancient UbiB family member COQ8A.

The ancient UbiB protein kinase-like (PKL) family is widespread, comprising one- quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. It has five human members (ADCK1–5), which are largely uncharacterized. We demonstrate that COQ8A (ADCK3) possesses conserved unorthodox PKL functionalities essential for maintaining a CoQ biosynthesis complex. Loss of COQ8A disrupts this complex, causing CoQ deficiency and cerebellar ataxia. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Structural investigation of the new nucleotide-bound structure of COQ8A reveals that the movement of two loops in the signature KxGQ domain of COQ8A opens two hydrophobic pockets on the protein, which are not present in the apo COQ8A structure. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.​

June 2016: Detection and sequence/structure mapping of biophysical constraints to protein variation in saturated mutational libraries and protein sequence alignments with a dedicated server.

Protein variability can now be studied by measuring high-resolution tolerance-to-substitution maps and fitness landscapes in saturated mutational libraries. But these rich and expensive datasets are typically interpreted coarsely, restricting detailed analyses to positions of extremely high or low variability or dubbed important beforehand based on existing knowledge about active sites, interaction surfaces, (de)stabilizing mutations, etc.
Our new webserver PsychoProt (freely available without registration at or at helps to detect, quantify, and sequence/structure map the biophysical and biochemical traits that shape amino acid preferences throughout a protein as determined by deep-sequencing of saturated mutational libraries or from large alignments of naturally occurring variants.

May 2016: Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins. 

Paramyxovirus fusion proteins (F), critical for viral entry and infection, initially fold into a metastable prefusion state and, upon triggering, refold irreversibly to a stable postfusion state to physically mediate membrane fusion. The large-scale conformational changes that occur in the F-refolding pathway are understood, but a detailed structural understanding of F-protein metastability remains elusive. Here, stabilizing and destabilizing mutations of the parainfluenza virus 5 fusion protein were examined to reveal that the immobilization of the N-terminal helix stabilizes paramyxovirus prefusion F proteins. The N-terminal helix, the interaction of which with domain II appears to be a critical early step in the F-protein refolding pathway, presents a novel alternative target for structure-based antiviral therapeutics.

April 2016: Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process.

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

February 2016: Pore-forming toxins: ancient, but never really out of fashion.

Pore-forming toxins (PFTs) are virulence factors produced by many pathogenic bacteria and have long fascinated structural biologists, microbiologists and immunologists. Interestingly, pore-forming proteins with remarkably similar structures to PFTs are found in vertebrates and constitute part of their immune system. Recently, structural studies of several PFTs have provided important mechanistic insights into the metamorphosis of PFTs from soluble inactive monomers to cytolytic transmembrane assemblies. In this Review, we discuss the diverse pore architectures and membrane insertion mechanisms that have been revealed by these studies, and we consider how these features contribute to binding specificity for different membrane targets. Finally, we explore the potential of these structural insights to enable the development of novel therapeutic strategies that would prevent both the establishment of bacterial resistance and an excessive immune response.

• 2015

December 2015: LipidBuilder: A Framework To Build Realistic Models for Biological Membranes.

The physical and chemical characterization of biological membranes is of fundamental importance for understanding the functional role of lipid bilayers in shaping cells and organelles, steering vesicle trafficking and promoting membrane-protein signaling. Molecular dynamics simulations stand as a powerful tool to probe the properties of membranes at atomistic level. However, the biological membrane is highly complex, and closely mimicking its physiological constitution in silico is not a straightforward task. Here, we present LipidBuilder, a framework for creating and storing models of biologically relevant phospholipid species with acyl tails of heterogeneous composition. LipidBuilder also enables the assembly of these database-stored lipids into realistic bilayers featuring asymmetric distribution on layer leaflets and concentration of given membrane constituents as defined, for example, by lipidomics experiments. The ability of LipidBuilder to assemble robust membrane models was validated by simulating membranes of homogeneous lipid composition for which experimental data are available. Furthermore, taking advantage of the extensive lipid headgroup repertoire, we assembled models of membranes of heterogeneous nature as naturally found in viral (phage PRD1), bacterial (Salmonella enterica, Laurinavicius, S.; Kakela, R.; Somerharju, P.; Bamford, D. H.; Virology 2004, 322, 328336) and plant (Chlorella kessleri, Rezanka, T.; Podojil, M.; J. Chromatogr. 1989, 463, 397408) organisms. These realistic membrane models were built using a near-exact lipid composition revealed from analytical chemistry experiments. We suggest LipidBuilder as a useful tool to model biological membranes of near-biological complexity, and as a robust complement to the current efforts to characterize the biophysical properties of biological membrane using molecular simulation.

October 2015: A coiled coil switch mediates cold sensing by the thermosensory protein DesK.

The thermosensor histidine kinase DesK from Bacillus subtilis senses changes in membrane fluidity initiating an adaptive response. Structural changes in DesK have been implicated in transmembrane signaling, but direct evidence is still lacking. On the basis of structure-guided mutagenesis, we now propose a mechanism of DesK-mediated signal sensing and transduction. The data indicate that stabilization/destabilization of a 2-helix coiled coil, which connects the transmembrane sensory domain of DesK to its cytosolic catalytic region, is crucial to control its signaling state. Computational modeling and simulations reveal couplings between protein, water and membrane mechanics. We propose that membrane thickening is the main driving force for signal sensing and that it acts by inducing helix stretching and rotation prompting an asymmetric kinase-competent state. Overall, the known structural changes of the sensor kinase, as well as further dynamic rearrangements that we now predict, consistently link structure determinants to activity modulation.

May 2015: Assessing the potential of atomistic molecular dynamics simulations to probe reversible protein-protein recognition and binding.

Protein-protein recognition and binding are governed by diffusion, noncovalent forces and conformational flexibility, entangled in a way that only molecular dynamics simulations can dissect at high resolution. Here we exploited ubiquitin’s noncovalent dimerization equilibrium to assess the potential of atomistic simulations to reproduce reversible protein-protein binding, by running submicrosecond simulations of systems with multiple copies of the protein at millimolar concentrations. The simulations essentially fail because they lead to aggregates, yet they reproduce some specificity in the binding interfaces as observed in known covalent and noncovalent ubiquitin dimers. Following similar observations in literature we hint at electrostatics and water descriptions as the main liable force field elements, and propose that their optimization should consider observables relevant to multi-protein systems and unfolded proteins. Within limitations, analysis of binding events suggests salient features of protein-protein recognition and binding, to be retested with improved force fields. Among them, that specific configurations of relative direction and orientation seem to trigger fast binding of two molecules, even over 50 Å distances; that conformational selection can take place within surface-to-surface distances of 10 to 40 Å i.e. well before actual intermolecular contact; and that establishment of contacts between molecules further locks their conformations and relative orientations.

May 2015: Understanding and Engineering Thermostability in DNA Ligase from Thermococcus sp. 1519.

The physical chemical principles underlying enzymatic thermostability are keys to understand the way evolution has shaped proteins to adapt to a broad range of temperatures. Understanding the molecular determinants at the basis of protein thermostability is also an important factor for engineering more thermoresistant enzymes to be used in the industrial setting, such as, for instance, DNA ligases, which are important for DNA replication and repair and have been long used in molecular biology and biotechnology. Here, we first address the origin of thermostability in the thermophilic DNA ligase from archaeon Thermococcus sp. 1519 and identify thermosensitive regions using molecular modeling and simulations. In addition, we predict mutations that can enhance thermostability of the enzyme through bioinformatics analyses. We show that thermosensitive regions of this enzyme are stabilized at higher temperatures by optimization of charged groups on the surface, and we predict that thermostability can be further increased by further optimization of the network among these charged groups. Engineering this DNA ligase by introducing selected mutations (i.e., A287K, G304D, S364I, and A387K) eventually produced a significant and additive increase in the half-life of the enzyme when compared to that of the wild type.

Full-size image (105 K)March 2015: The importance of dynamics in integrative modeling of supramolecular assemblies.

Revealing the atomistic architecture of supramolecular complexes is a fundamental step toward a deeper understanding of cellular functioning. To date, this formidable task is facilitated by an emerging array of integrative modeling approaches that combine experimental data from different sources. One major challenge these methods have to face is the treatment of the dynamic rearrangements of the individual subunits upon assembly. While this flexibility can be sampled at different levels, integrating native dynamic determinants with available experimental inputs can provide an effective way to reveal the molecular recognition mechanisms at the basis of supramolecular assembly.

March 2015: A model for dsDNA binding by the WHD domains of the Hop2-Mnd1 protein complex.

In meiotic DNA recombination, the Hop2-Mnd1 complex promotes Dmc1-mediated single-stranded DNA (ssDNA) invasion into homologous chromosomes to form a synaptic complex by a yet-unclear mechanism. We used available structural information to deduce how the WHD pair of Hop2-Mnd1 might bind dsDNA. The WHDs of both Hop2 and Mnd1 are structurally most similar to the WHD of transcription regulator TtgV among the known structures of the WHDs in complex with DNA according to the program Dali. Structural superposition of the TtgV:dsDNA complex onto both WHDs of Hop2 and Mnd1 indicated that binding of the juxtaposed WHDs to a continuous DNA is likely to require severe distortion of the DNA. To investigate further, we modeled dsDNA bound to the WHDs based on the TtgV:dsDNA structure, and after geometry optimization, could confirm indeed that dsDNA is highly perturbed in the model. Based on this initial model, we performed MD simulations to further test the stabilityof the complex and the structural changes produced upon dsDNA binding. MD simulations revealed a distortion inthe base pairing in between the WHDs.

February 2015: How structural and physicochemical determinants shape sequence constraints in a functional enzyme.

The need for interfacing structural biology and biophysics to molecular evolution is being increasingly recognized. One part of the big problem is to understand how physics and chemistry shape the sequence space available to functional proteins, while satisfying the needs of biology. Here we present a quantitative, structure-based analysis of a high-resolution map describing the tolerance to all substitutions in all positions of a functional enzyme, namely a TEM lactamase previously studied through deep sequencing of mutants growing in competition experiments with selection against ampicillin. Substitutions are rarely observed within 7 Å of the active site, a stringency that is relaxed slowly and extends up to 15-20 Å, with buried residues being especially sensitive. Substitution patterns in over one third of the residues can be quantitatively modeled by monotonic dependencies on amino acid descriptors and predictions of changes in folding stability. Amino acid volume and steric hindrance shape constraints on the protein core; hydrophobicity and solubility shape constraints on hydrophobic clusters underneath the surface, and on salt bridges and polar networks at the protein surface together with charge and hydrogen bonding capacity. Amino acid solubility, flexibility and conformational descriptors also provide additional constraints at many locations. These findings provide fundamental insights into the chemistry underlying protein evolution and design, by quantitating links between sequence and different protein traits, illuminating subtle and unexpected sequence-trait relationships and pinpointing what traits are sacrificed upon gain-of-function mutation.

 • 2014

Full-size image (41 K) August 2014: A dimerization interface mediated by functionally critical residues creates interfacial disulfide bonds and copper sites in CueP.

CueP confers bacterial copper resistance in the periplasm, particularly under anaerobic conditions, through an unknown mechanism. The only available structure and limited solution data suggest that CueP forms noncovalent dimers in solution, whereas sequence conservation suggests important roles for three cysteines and two histidines as copper ligands. Here we report evidence of a dimerization equilibrium mediated by a newly identified interface of functional relevance, which occludes internal copper sites and disulfide bonds but allows for intra- and interchain disulfide bonding, an extensive disulfide relay, and interfacial copper sites. Our results suggest a role for CueP linking redox-state sensing and copper detoxification.

Abstract Image April 2014: Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics.

We present here in a thorough description of the effects of high glucose concentrations on the diffusion, hydration and internal dynamics of ubiquitin, as predicted from extensive molecular dynamics simulations on several systems described at fully atomistic level. We observe that the protein acts as a seed that speeds up the natural propensity of glucose to cluster at high concentration; the sugar molecules thus aggregate around the protein trapping it inside a dynamic cage. This process extensively dehydrates the protein surface, restricts the motions of the remaining water molecules, and drags the large-scale, collective motions of protein atoms slowing down the rate of exploration of the conformational space despite only a slight dampening of fast, local dynamics. We discuss how these effects could be relevant to the function of sugars as preservation agents in biological materials, and how crowding by small sticky molecules could modulate proteins across different reaction coordinates inside the cellular cytosol.

January 2014: Molecular dynamics simulations of apocupredoxins: insights into the formation and stabilization of copper sites under entatic control.

Cupredoxins perform copper-mediated long-range electron transfer (ET) in biological systems. Their copper-binding sites have evolved to force copper ions into ET-competent systems with decreased reorganization energy, increased reduction potential, and a distinct electronic structure compared with those of non-ET-competent copper complexes. The entatic or rack-induced state hypothesis explains these special properties in terms of the strain that the protein matrix exerts on the metal ions. This idea is supported by X-ray structures of apocupredoxins displaying “closed” arrangements of the copper ligands like those observed in the holoproteins; however, it implies completely buried copper-binding atoms, conflicting with the notion that they must be exposed for copper loading. On the other hand, a recent work based on NMR showed that the copper-binding regions of apocupredoxins are flexible in solution. We have explored five cupredoxins in their “closed” apo forms through molecular dynamics simulations. We observed that prearranged ligand conformations are not stable as the X-ray data suggest, although they do form part of the dynamic landscape of the apoproteins. This translates into variable flexibility of the copper-binding regions within a rigid fold, accompanied by fluctuations of the hydrogen bonds around the copper ligands. Major conformations with solvent-exposed copper-binding atoms could allow initial binding of the copper ions. An eventual subsequent incursion to the closed state would result in binding of the remaining ligands, trapping the closed conformation thanks to the additional binding energy and the fastening of noncovalent interactions that make up the rack.

 • 2013

August 2013: All-atom simulations of crowding effects on ubiquitin dynamics.

It is well-known that crowded environments affect the stability of proteins, with strong biological and biotechnological implications; however, beyond this, crowding is also expected to affect the dynamic properties of proteins, an idea that is hard to probe experimentally. Here we report on a simulation study aimed at evaluating the effects of crowding on internal protein dynamics, based on fully all-atom descriptions of the protein, the solvent and the crowder. Our model system consists of ubiquitin, a protein whose dynamic features are closely related to its ability to bind to multiple partners, in a 325 g L⁻¹ solution of glucose in water, a condition widely employed in in vitro studies of crowding effects. We observe a slight reduction in loop flexibility accompanied by a dramatic restriction of the conformational space explored in the timescale of the simulations (∼0.5 µs), indicating that crowding slows down collective motions and the rate of exploration of the conformational space. This effect is attributed to the extensive and long-lasting interactions observed between protein residues and glucose molecules throughout the entire protein surface. Potential implications of the observed effects are discussed.

August 2013: Swirling into the membrane. Near-atomistic models of the prepore and membrane-inserted pore conformations derived from a combination of crystallography, cryo-EM, single-particle analysis, molecular simulation and modeling reveal a swirling mechanism of membrane insertion and pore formation by aerolysin. Our integrative effort to unveil the mechanism of pore formation in bacteria done in direct collaboration with the van der Goot’s lab has just appeared in Nature Chemical Biology. See also press release at EPFL News Mediacom.

July 2013: Injectisome flexibility in between membranes.  Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells. We present the first structural analysis of the Yersinia injectisome. Unexpectedly, the crystal structure of the inner membrane component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented this elasticity. We modeled the ring-shaped YscDJ ring at the inner membrane and found that together with the secretin YscC at the outer membrane were stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We thus suggest that elasticity is critical for these two-membrane spanning protein complexes to cope with variations in the intermembrane distance. Kudryashev et al. eLife 2013;2:e00792. DOI: 10.7554/eLife.00792.

July 2013: Simplified models for molecular simulation of proteins. We present a new generation of coarse-grained (CG) potentials that account for a simplified electrostatic description of soluble proteins. The treatment of permanent electrostatic dipoles of the backbone and polar side-chains allows to simulate multiprotein complexes maintaining their molecular interfaces. An efficient heuristic algorithm based on particle swarm optimization (as implemented in our parallel optimization workbench – power) is used for the derivation of CG parameters via a force-matching procedure. The ability of this protocol to deal with high dimensional search spaces hints to the generation of a fully transferable CG force field. ASAP article in J. Chem. Theory Comput. 2013.

 July 2013Swarm intelligence hits dynamic modeling. We present a swarm intelligence-based method that, by accounting for steric interactions and a limited amount of experimental spatial restraints, predicts the arrangement of proteins in symmetric assemblies. Importantly, the native flexibility of each protein subunit is taken into account as extracted from molecular dynamics simulations. We show that this is a key ingredient for the prediction of biologically functional assemblies when, upon oligomerization, subunits explore activated states undergoing significant conformational changes. Featuted article in Structure, 2013, 21(7), 1097.

This novel dynamic modeling approach is implemented in our parallel optmization workbench (power).

January 2013The transmembrane core of a two-component system.The PhoQP two-component system is a signaling complex essential for bacterial virulence and cationic antimicrobial peptide resistance. PhoQ is the histidine kinase chemoreceptor of this tandem machine and assembles in a homodimer conformation spanning the bacterial inner membrane. We present an atomistic model of the key transmembrane (TM) domain assembled by using molecular simulations, guided by experimental cross-linking data. A concerted displacement of the TM helices at the  periplasmic side is found to modulate a rotation at the cytoplasmic end, supporting the transduction of the chemical signal through a combination of   scissoring and rotational movement of the TM helices. Published in PLoS Comput Biol 9(1): e1002878.

 January 2013: Metals on the move. Type II topoisomerase (topoII) is a metalloenzyme targeted by clinical antibiotics and anticancer agents. We integrate existing structural data with molecular simulation and propose a model for the yet uncharacterized structure of the reactant state of topoII. This model describes a canonical two-metal-ion mechanism and suggests how the metals could rearrange at the catalytic pocket during enzymatic turnover, explaining also experimental evidence for topoII inhibition. Cover article in J. Chem. Theory Comput., 2013, 9 (2), 857.

 • 2012

December 2012: Present in bacterial and mitochondrial membranes,cardiolipins have a unique dimeric structure, and under physiological conditions can be unprotonated or singly protonated. We report exhaustive   models of cardiolipins consistent with commonly used force fields. The proposed models will contribute to the study of the assembly of more realistic bacterial and mitochondrial membranes and the investigation of the role of cardiolipins for the biophysical and biochemical properties of membranes and membrane-embedded proteins. Published in J. Chem. Theory Comput., 2013, 9 (1), 670.

 • 2011

July 2011: A dual chaperone for aerolysin. The C-terminal peptide (CTP) of the pore-forming toxing aerolysin not only prevents premature formation of the oligomeric pore-forming structure, but is required for the initial folding of the protein, despite its C-terminal location. This is the first example of an intramolecular chaperone having the dual function. Furthermore, we pinpointed in silico and validated experimentally single-point mutations leading to CTP-aerolysin destabilization and explored the implication of these findings for the heptamerization process. The work, conducted in collaboration with the van der Goot’ lab, was published on PLoS Pathogens.
• 2010

July 2010: One ruler is enough. The needle length of the Yersinia injectisome is determined by Yop secretion protein P (YscP), an early substrate of the injectisome itself. There is a linear correlation between the length of YscP and the length of the needle, suggesting that YscP acts as a molecular ruler. However, it is not known whether one single molecule of YscP suffices to control the length of one needle or whether several molecules of YscP are exported in alternation with the needle subunit YscF until the needle length matches the ruler length, which would stop needle growth. To address this question, different strains expressing simultaneously a short and a long version of YscP were engineered (yscP388 and yscP686). The experimentally obtained needle length distribution was compared with the distributions predicted by stochastic modeling of the various possible scenarios. The experimental data are compatible with the single ruler model and not with the scenarios involving more than one ruler per needle. The work conducted in collaboration with Prof. Cornelis’ Lab in Basel has just appeared in the early publications of the Proceeding of the National Academy of Sciences USA.


January 2010: Backbone dipoles stabilizes CG proteins.  We introduce a nonradial potential term for coarse-grained (CG) molecular simulations of proteins. This term mimics the backbone dipole−dipole interactions and accounts for the needed directionality to form stable folded secondary structure elements. We show that α-helical and β-sheet peptide chains are correctly described in dynamics without the need of introducing any a priori bias potentials or ad hoc parametrizations, which limit broader applicability of CG simulations for proteins. Moreover, our model is able to catch the formation of supersecondary structural motifs, like transitions from long single α-helices to helix−coil−helix or β-hairpin assemblies. This novel scheme requires the structural information of Cα beads only; it does not introduce any additional degrees of freedom to the system and has a general formulation, which allows it to be used in synergy with various CG protocols, leading to an improved description of the structural and dynamic properties of protein assemblies and networks. More details can be found in the full article appeared this month in the Journal of Chemical Theory and Computation, 2010, 6: 315

• 2009

m2web January 2009. The M2 protein from the influenza A virus is commonly described as a pH-activated proton channel. It conducts in fact protons to the viral interior permitting the uncoating of the viral RNA and fusion of the viral envelope with the endosomal bilayer. His37 is crucial for proton conduction lining the transmembrane region of the pore. Molecular simulations based on the first X-ray structure of the channel suggest that the protein prefers a conformation in which the channel is open to the environment on the outside of the virus but closed to the interior. Diffusion of protons into the channel and protonation of His37 stabilizes an oppositely gated conformation. Thus, protons might be conducted through a transporter-like mechanism, in which the protein alternates between two different conformations, and His37 is protonated/deprotonated during each turnover. The transporter-like mechanism is consistent with the known properties of the M2 bundle, including its relatively low rate of proton flux and its strong rectifying behavior (Proc Natl Acad Sci USA).

• 2008

December 2008. The computational study done in collaboration with the Cornelis’ Lab in Basel on the length of the Yersinia injectisome needle has just appeared in Molecular Microbiology. The analysis of the correlation between the size of YscP protein and the needle length in Yersinia enterocolitica reinforces the hypothesis that YscP acts as a molecular ruler, and hints that the secondary structure of YscP might influence needle length. The calculated lengths, when the YscP helical content is preserved, correlates strikingly with the measured needle length with a constant difference of ∼29 nm, which corresponds approximately to the size of the basal body. Thus, our results support the ruler model and show that the functional ruler has a helical structure. (“The helical content of the YscP molecular ruler determines the length of the Yersinia injectisome” in Molecular Microbiology).


August 2008. Our new scheme for multiscale molecular simulations has appeared this month on the Journal of Chemical Theory and Computation, as cover article. Multiscale approaches can be used to study intermolecular interactions in proteins. Interface regions are treated atomistically, while coarse-grained models describe the remaining part of the system. We show that the electrostatic potential of the coarse-grained region can be reconstructed by a multipolar expansion, based on the topology of the backbone centroids only. (“Topologically based multipolar reconstruction of electrostatic interactions in multiscale simulations of proteins”, J. Chem. Theory Comp. 4(8), 1378, 200810.1021/ct800122x).

Scheme of a RNase H

July 2008. Our research on the catalytic mechanism of ribonuclease H has just appeared on the Journal of the American Chemical Society. RNase H hydrolyzes the phosphodiester linkages of the RNA strand in RNA-DNA hybrids, and represents a very promising target for anti-HIV drug design. We find that the two metal ions act cooperatively, facilitating nucleophile formation and stabilizing both the transition state and the leaving group. The twoMg ions also support the formation of a meta-stable phosphorane intermediate along the reaction (“Phosphodiester cleavage in ribonuclease H occurs via an associative two-metal-aided catalytic mechanism”, J. Am. Chem. Soc. 200810.1021/ja8005786) highlighted on the third issue of JACS Select dedicated to “Molecular Modeling of Complex Chemical Systems“.