Carlo Guardiani

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

Efficient Nonequilibrium Method for Binding Free Energy Calculations in Molecular Dynamics Simulations

We introduce an effective technique for the calculation of the binding free energy in drug-receptor systems using nonequilibrium molecular dynamics and application of the Jarzynski theorem. In essence, this novel methodology constitutes the nonequilibrium adaptation of an ancient free energy perturbation technique called Double Annihilation Method, invented more than 25 years ago [J. Chem. Phys. 1988, 89, 3742-3746] and upon which modern approaches of binding free energy computation in drug-receptor systems are heavily based. The proposed computational approach, termed Fast Switching Double Annihilation Methods (FS-DAM) in honor of its ancient ancestor, is applied to a prototypical example system with multiple binding sites, proving its computational potential and versatility in unraveling multiple site or allosteric binding processes.

The N-Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities.

The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms.

Conformational Landscape of N-Glycosylated Peptides Detecting Autoantibodies in Multiple Sclerosis, Revealed by Hamiltonian Replica Exchange

Synthetic N-glycosylated CSF114(Glc) and related peptides were proved to be able to recognize specific and high-affinity autoantibodies circulating in blood of relapsing-remitting multiple sclerosis (MS) patients and correlating with disease activity. The effect of these peptides has been linked to the β-turn structure around the minimal epitope Asn(Glc). In this work we performed Hamiltonian replica exchange molecular dynamics simulations on the central heptapeptide fragment of a CSF114(Glc)-derived peptide in water and in a water/hexafluoroacetone mixture, confirming a significant incidence of β-turn structures in both solvents. The structural similarity of the glycosylated and unglycosylated forms in all environments proves that the conformation of the heptapeptide is only marginally affected by the presence of the sugar. Moreover, the presence of a significant amount of bioactive hairpin-like conformations in the water environment suggests a possible use not only in the diagnosis but also in the treatment of MS.

Coarse Grained Modeling and Approaches to Protein Folding

The theoretical prediction of protein structures has become a field of increasing importance in both biology and physics. Reliable prediction methods in fact, would spare time consuming experimental X-ray and NMR techniques and they would represent a challenge for computational protein modeling as well. The well known limitations of all-atom models call for the development of coarse-grained protein descriptions including a minimal number of protein-like fea- tures, while being capable of mimicking the essence of protein folding mechanisms. In this paper we review the most im- portant classes of coarse-grained protein models in order of increasing complexity, starting from (over simplified) binary models, to models with one or two reaction centers per residue. We discuss how, despite their simplification, coarse- grained models constitute a viable approach to structure prediction and they shed light on many aspects of protein-folding problem.

A computational study of ion current modulation in hVDAC3 induced by disulfide bonds.

The human VDAC channel exists in three isoforms characterized by high sequence homology and structural similarity. Yet the function and mode of action of hVDAC3 are still elusive. The presence of six surface cysteines exposed to the oxidizing environment of the mitochondrial inter-membrane space suggests the possible establishment of intramolecular disulfide bonds. Two natural candidates for disulfide bridge formation are Cys2 and Cys8 that, located on the flexible N-terminal domain, can easily come in contact. A third potentially important residue is Cys122 that is close to Cys2 in the homology model of VDAC3. Here we analyzed the impact of SS bonds through molecular dynamics simulations of derivatives of hVDAC3 (dubbed SS-2-8, SS-2-122, SS-8-122) including a single disulfide bond. Simulations showed that in SS-8-122, the fragment 1-7 crosses the top part of the barrel partially occluding the pore and causing a 20% drop of conductance. In order to identify other potential channel-occluding disulfide bonds, we used a set of neural networks and structural bioinformatics algorithms, after filtering with the steric constraints imposed by the 3D-structure. We identified other three species, namely SS-8-65, SS-2-36 and SS-8-36. While the conductance of SS-8-65 and SS-2-36 is about 30% lower than that of the species without disulfide bonds, the conductance of SS-8-36 was 40-50% lower. The results show how VDAC3 is able to modulate its pore size and current by exploiting the mobility of the N-terminal and forming, upon external stimuli, disulfide bridges with cysteine residues located on the barrel and exposed to the inter-membrane space.

Sodium binding sites and permeation mechanism in the NaChBac channel : a molecular dynamics study

NaChBac was the first discovered bacterial sodium voltage-dependent channel, yet computational studies are still limited due to the lack of a crystal structure. In this work, a pore-only construct built using the NavMs template was investigated using unbiased molecular dynamics and metadynamics. The potential of mean force (PMF) from the unbiased run features four minima, three of which correspond to sites IN, CEN, and HFS discovered in NavAb. During the run, the selectivity filter (SF) is spontaneously occupied by two ions, and frequent access of a third one is often observed. In the innermost sites IN and CEN, Na+ is fully hydrated by six water molecules and occupies an on-axis position. In site HFS sodium interacts with a glutamate and a serine from the same subunit and is forced to adopt an off-axis placement. Metadynamics simulations biasing one and two ions show an energy barrier in the SF that prevents single-ion permeation. An analysis of the permeation mechanism was performed both computing minimum energy paths in the axial-axial PMF and through a combination of Markov state modeling and transition path theory. Both approaches reveal a knock-on mechanism involving at least two but possibly three ions. The currents predicted from the unbiased simulation using linear response theory are in excellent agreement with single-channel patch-clamp recordings.

Coarse-grained modeling of protein unspecifically bound to DNA

There is now a certain consensus that transcription factors (TFs) reach their target sites, where they regulate gene transcription, via a mechanism dubbed facilitated diffusion (FD). In FD, the TF cycles between events of 3D diffusion in solution (jumps), 1D diffusion along DNA (sliding), and small jumps (hopping), achieving association rates higher than for 3D diffusion alone. We investigate the FD phenomenology through molecular dynamics simulations in the framework of coarse-grained modeling. We show that, despite the crude approximations, the model generates, upon varying the equilibrium distance of the DNA-TF interaction, a phenomenology matching a number of experimental and numerical results obtained with more refined models. In particular, focusing on the kinematics of the process, we characterize the geometrical properties of TF trajectories during sliding. We find that sliding occurs via helical paths around the DNA helix, leading to a coupling of translation along the DNA axis with rotation around it. The 1D diffusion constant measured in simulations is found to be interwoven with the geometrical properties of sliding and we develop a simple argument that can be used to quantitatively reproduce the measured values.

A microscopic model of evolution of recombination

We study the evolution of recombination using a microscopic model developed within the frame of the theory of quantitative traits. Two components of fitness are considered: a static one that describes adaptation to environmental factors not related to the population itself, and a dynamic one that accounts for interactions between organisms, e.g. competition. We focus on the dynamics of colonization of an empty niche. As competition is a function of the population, selection pressure rapidly changes in time. The simulations show that both in the case of flat and steep static fitness landscapes, recombination provides a high velocity of movement in the phenotypic space thus allowing recombinants to colonize the highest fitness regions earlier than non-recombinants that are often driven to extinction. The stabilizing effects of competition and assortativity are also discussed. Finally, the analysis of phase diagrams shows that competition is the key factor for the evolution of recombination, while assortativity plays a significant role only in small populations.

Computational analysis of folding and mutation properties of C5 domain of myosin binding protein C

Thermal folding molecular dynamics simulations of the domain C5 of Myosin binding protein C were performed using a native‐centric model to study the role of three mutations related to Familial Hypertrophic Cardiomyopathy. Mutation of Asn755 causes the largest shift of the folding temperature, and the residue is located in the CFGA′ β‐sheet featuring the highest Φ‐values. The mutation thus appears to reduce the thermodynamic stability in agreement with experimental data. The mutations on Arg654 and Arg668, conversely, cause little change in the folding temperature and they reside in the low Φ‐value BDE β‐sheet, so that their pathological role cannot be related to impairment of the folding process but possibly to the binding with target molecules. As the typical signature of Domain C5 is the presence of a longer and destibilizing CD‐loop with respect to the other Ig‐like domains, we completed the work with a bioinformatic analysis of this loop showing a high density of negative charge and low hydrophobicity. This indicates the CD‐loop as a natively unfolded sequence with a likely coupling between folding and ligand binding. Proteins 2008.