Unraveling anomalous subdiffusion in heterogeneous membranesPI: Lyman, Edward
Description: Simulations of mixtures of DPPC:DOPC + chol in a ratio of 1:1 +20 mol %. Four different types of trajectories are available, listed in the order they appear below: (1) Lo/Ld coexistence at T < Tm, where Tm is the miscibility transition temperature. (2) The homogeneous phase at T > Tm. (3) The Lo phase at the same T as (1). (4) The Ld phase at the same T as (1). Compositions of the two phases were taken from Veatch et al Biophys J 86:2910(2004).
Keywords: Cholesterol, liquid ordered, miscibility phase transition.
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Microsecond scale simulations to characterize skeletal muscle Ca2+-binding protein troponin CPI: McCammon, Andrew
Description: Troponin (Tn) plays an important role in calcium signaling events in both cardiac and skeletal muscle contraction. Troponin is a hetero-trimeric complex consisting of troponin C (TnC, calcium binding subunit), troponin I (TnI, inhibitory subunit) and troponin T (TnT, tropomyosin binding subunit). Troponin C is a calcium-sensitive protein that initiates muscle contraction. In the calcium-free state troponin inhibits contraction. Calcium binding to TnC initiates a chain of conformational changes that release troponin I’s contractile inhibition on the thin filament and subsequently allow for force generation to occur. Similarly, dissociation of calcium from troponin C is associated with muscle relaxation. Interesting differences between the cardiac and skeletal isoforms of the N-terminal domain of TnC exist. While both molecules consist of two EF-hands and thus two potential Ca2+ binding sites, the cardiac isoform only binds one calcium ion. In cardiac TnC site I is completely defunct for calcium binding which is due to several amino acid substitutions with respect to site I in skeletal TnC. Site II, the low-affinity, Ca2+-specific Ca2+-binding site is generally considered the only site directly involved in calcium regulation of cardiac muscle contraction. Ca2+-binding to site II of cardiac TnC does not induce an opening transition akin to skeletal TnC but leaves the structure more or less unperturbed in the closed conformation. It was the aim of this proposal to elucidate the impact of the different calcium binding patterns in skeletal and cardiac TnC on the dynamics of the molecule. Particular focus was centered on the opening and closing of the hydrophobic patch that binds the TnI switch peptide. |
Microsecond scale simulations to characterize skeletal muscle Ca2+-binding protein parvalbuminPI: McCammon, Andrew
Description: Contractile function is strongly dependent on the availability of freely diffusing Ca2+ during systole. Altering the available free Ca2+ for binding myofilament proteins opens the door for treating a variety of contractile diseases including those affecting cardiac, skeletal or smooth-muscle tissue, for which the sensitivity to calcium is abnormal. Recent studies suggest that the contractile response to cytosolic Ca2+ can be modulated directly by engineering variants of the myofilament protein, troponin, or indirectly by modifying secondary proteins that impact that availability of free calcium. In particular, transfection of cardiac cells with parvalbumin, a potent Ca2+ binding protein expressed in skeletal muscle and neurological cells, was shown to delay the decay in the calcium transient during relaxation and partially restore contractile function in a mouse model of heart failure. Parvalbumin, PV, a member of the EF-hand family, consists of two isoforms. In mammals, the α-isoform localizes to skeletal muscle tissue, whereas the β isoform is predominantly found in the brain. PV has been the subject of intense experimental characterization, of which several studies have examined the molecular basis for the attenuated Ca2+ affinity in β-PV relative to α-PV. The altered Ca2+ binding conformation is believed to arise due to variations in the stability of the two isoforms, and in part due to the β-PV Ca2+-free (apo) state presenting greater thermal stability than the α-PV. Based on our previous computational studies of TnC, we found that altering the packing of helix bundles comprising the EF-hands led to significant changes in Ca2+ affinity. In a similar note, we anticipate that bundle residue mutations could modulate the energetics of helix packing and in turn, alter Ca2+ binding. Hence, we proposed using molecular dynamics simulations to estimate the stability of EF-hand bundle packing, as well as quantify the correlation between stable packing in the apo and holo states and experimentally-determined Ca2+ affinities for the wild-type β-parvalbumin structure. Given spectroscopic and computational evidence that the timescale of TnC conformational changes are at least nanosecond-long, our study was critically-dependent on simulations spanning microseconds in length. |
Understanding the mechanics of energy conversion in Na+ dependent cotransportersPI: Grabe, Michael
Description: Membrane transport proteins that utilize a 5helix inverted repeat motif have recently emerged as one of the largest structural classes of secondary active transporters. These membrane proteins are responsible for transporting small molecules such as amino acids and sugars across membranes. They use electrochemical gradients to concentrate these substrates via an alternating access mechanism originally outlined in the 1960s. This project aimed to understand several key aspects of this mechanism at the molecular level through simulations of the sodiumgalactose cotransporter vSGLT. |
Detailed Characterization of the Equilibrium Fluctuations of the Engrailed HomeodomainPI: Langmead, Christopher James
Description: Three simulations (at 300, 330, and 350K) of approx. 50 microseconds each, of the equilibrium fluctuations of the D. melanogaster engrailed homeodomain in explicit solvent. |
Evolutionary Pathways of Engineered Sitagliptinases through Microsecond Molecular Dynamics (TRAJECTORIES COMING SOON)PI: Houk, Kendall
Description: We extended our studies on the origins of enzyme evolution to a transaminase for the commercial synthesis of the diabetes drug sitagliptin (Januvia®), Merck’s largest selling drug. Intriguingly, no differences in the active site configuration of natural and evolved enzymes (either active or inactive) have been found either in the solid state or throughout 200 ns MD simulations in water. Initial explorations suggest an important role of protein-protein interactions in these catalytic complexes.
Our goal is to understand how directed evolution leads to this highly active catalyst through long timescale MD simulations with Anton. The information obtained throughout this study will be incorporated in our inside-out enzyme design protocol.
Due to the nature of this project, in which we need to analyze different mutants of the same protein, which were generated with homology modeling methods from an unpublished proprietary x-ray structure, all trajectories were obtained exactly under the same simulation conditions. (TRAJECTORIES COMING SOON) |
Metabolite permeation and voltage-gating of the mitochondrial channel VDACPI: Grabe, Michael
Description: Eukaryotic cells require efficient exchange of metabolites, such as ATP and ADP, between the mitochondria and the rest of the cell. This exchange is mediated by the most abundant protein in the outer mitochondrial membrane, the Voltage Dependent Anion Channel (VDAC), which governs the flux of anions, cations, and metabolites between the cytoplasm and the intermembrane space of the mitochondria. In addition to its role in bioenergetics, VDAC modulates the organelle’s permeability implicating VDAC in the metabolic stress of cardiovascular disease, cancer and mitochondrialdependent apoptotic cell death. Despite these vital roles, mitochondrial permeability and its regulation remain poorly understood.
This project sought to determine whether ATP can permeate the crystalographic structure of mVDAC1, as well as to generally elucidate how the presence of ATP within the lumen of the channel effects the conduction properties of monovalent ions. |
Transient Formation of Water-Conducting States in Membrane Transporter vSGLTPI: Tajkhorshid, Emad
Description: We performed a large set of extended equilibrium molecular dynamics simulations on several classes of membrane transporters, in different conformational states, to test the phenomenon in diverse transporter classes and to investigate the underlying molecular mechanism of water transport through membrane transporters. This 1 microsecond simulation for vSGLT is one of the simulations in our project. The simulations reveal spontaneous formation of transient water-conducting (channel-like) states allowing passive water diffusion through the lumen of the transporters. These channel-like states are permeable to water but occluded to substrate, thereby not hindering the uphill transport of the primary substrate, i.e., the alternating access model remains applicable to the substrate. The rise of such water-conducting states during the large-scale structural transitions of the transporter protein is indicative of imperfections in the coordinated closing and opening motions of the cytoplasmic and extracellular gates. We propose that the observed water-conducting states likely represent a universal phenomenon in membrane transporters, which offers an expanded understanding of alternating access mechanism. |
Nanoscale structure in sphingolipid mixturesPI: Lyman, Eduard
Description: Simulations of ternary mixtures of Palmitoyl sphingomyelin in order to assess nanoscale structure in Lo phases and the nature of boundaries between boundaries between phases. |
Non-canonical voltage-sensor pore coupling in the hyperpolarized cyclic nucleotide gated channelPI: Chanda, Baron
Description: HCN1 is a member of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels family. It regulates rhythmic activity in excitable cells by promoting inward cation current in response to membrane hyperpolarization. Activation by hyperpolarization is a hallmark of HCN channels; while other voltage-gated ion channels open upon membrane depolarization, members of the HCN family require an opposite polarity to conduct ions, suggesting a unique voltage-sensing mechanism. In this work, we aimed to uncover this mechanism using a cryo-EM structure of the HCN1 channel and molecular dynamics simulations. Since this structure corresponds to the resting state, we applied a strong hyperpolarizing electric field to promote its activation. The same strategy has previously been shown to provide remarkable insight into the voltage-sensing mechanism of another voltage-gated ion channel, the Kv1.2/2.1 chimera [Jensen et al., 2012]. To further speed up HCN1 activation, we mutated two selected residues of the voltage sensor domain into more hydrophilic ones; this trick was inspired by the experimental studies of Lacroix and Bezanilla [Lacroix & Bezanilla, 2012] and aimed to decrease the friction between the helices of the voltage sensor domain during activation.
Molecular dynamics simulations of the HCN1 mutant under a hyperpolarizing electric field indeed revealed a novel voltage-sensing mechanism. Some of its features, such as the displacement of the S4 helix and the rearrangement of the salt-bridges inside the voltage sensor domain, were similar to those reported for other voltage-gated ion channels. However, the S4 displacement was at least two times smaller compared to the Kv1.2/2.1 chimera; an additional gating charge was transferred through the rearrangement of a local electric field inside the voltage sensor domain; and finally, the S4 helix was broken in two parts in the activated state, one of which interacted with the S1-S3 helices and the other oriented parallel to the membrane surface. Remarkably, overall six voltage sensor domains in two independent trajectories followed the same activation mechanism and converged to a similar conformation. The results of this work have inspired further experiments to test crucial properties of the newly revealed activation mechanism and the activated state of HCN1.
References: Jensen, M. Ø. et al. Mechanism of Voltage Gating in Potassium Channels.
Science 336, 229 (2012).
Lacroix, J. J. & Bezanilla, F. Tuning the voltage-sensor motion with a single
residue. Biophys. J. 103, L23-25 (2012). |
Mechanism of Phosphate Release in Myosin VI.PI: Mugnai, Mauro
Description: Myosin motors step along the filamentous actin. These enzymes power their movement by hydrolyzing ATP into ADP and phosphate (Pi). The products of the reaction are released, a new ATP binds and the cycle continues.
The nature of the nucleotide bound to the myosin motor is associated with precise structural conformations of the enzyme, which have been identified via experimental studies. However, the paths connecting these metastable structures are of outstanding importance in order to decipher how myosin works. With the simulations performed on Anton 2, we investigated the mechanism of phosphate release from the nucleotide binding site, which is a key step of the myosin cycle because is associated with the conformational transition that exerts force on the actin filament.
The simulations are started using a structure generated by X-ray crystallography and corresponding to the phosphate-release (PiR) state. The post-hydrolysis phosphate anion is in the vicinity of ADP (also negatively charged) and a magnesium cation (Mg2+), so it is reasonable to expect that the strong electrostatic interaction between Mg2+ and Pi contribute to holding the phosphate in the vicinity of its post-ATP-hydrolysis location. It is therefore interesting to focus on the solvation shell of the cation.
We ran a series of simulations from the PiR conformation, and we noticed that although the conformation of the nucleotide binding site changes during the simulation, the phosphate remains in the vicinity of the cation. In contrast, if at the beginning of the simulation the phosphate is rotated, this perturbation enabled the release of Pi in different trajectories. Strikingly, we observed that the escape of Pi was always preceded by the hydration of Mg2+ by 4 water molecules, upwards from the 2 water molecules seen in crystal structure. We therefore surmised that the hydration of Mg2+ by 4 water molecules triggers the release of Pi.
By analyzing the different trajectories, we investigated the pathway of phosphate release, showing that multiple escape routes are possible, although most of the time the phosphate populates the release channel predicted on the basis of experimental evidence
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Identification of conformational transitions in the outward-facing structure of the sodium-coupled leucine transporter, LeuTAa. (TRAJECTORIES COMING SOON)PI: Bahar, Ivet
Description: The bacterial sodium-coupled leucine/alanine transporter LeuT is broadly used as a model system for studying the transport mechanism of neurotransmitters because of its structural and functional homology to mammalian transporters such as serotonin, dopamine, or norepinephrine transporters, and because of the resolution of its structure in different states. Although the binding sites (S1 for substrate, and Na1 and Na2 for two co-transported sodium ions) have been resolved, we still lack a mechanistic understanding of coupled Na+- and substrate-binding events. We present here results from extensive (>20 μs) unbiased molecular dynamics simulations generated using the latest computing technology. Simulations show that sodium binds initially the Na1 site, but not Na2, and, consistently, sodium unbinding/escape to the extracellular (EC) region first takes place at Na2, succeeded by Na1. Na2 diffusion back to the EC medium requires prior dissociation of substrate from S1. Significantly, Na+ binding (and unbinding) consistently involves a transient binding to a newly discovered site, Na1″, near S1, as an intermediate state. A robust sequence of substrate uptake events coupled to sodium bindings and translocations between those sites assisted by hydration emerges from the simulations: (i) bindings of a first Na+ to Na1″, translocation to Na1, a second Na+ to vacated Na1″ and then to Na2, and substrate to S1; (ii) rotation of Phe253 aromatic group to seclude the substrate from the EC region; and (iii) concerted tilting of TM1b and TM6a toward TM3 and TM8 to close the EC vestibule. {TRAJECTORIES COMING SOON}.
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Binding Mechanism of the Matrix Domain of HIV-1 Gag on Lipid MembranesPI: Voth, Gregory
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Breakage of the Oligomeric CaMKII Hub by the Regulatory Segment of the KinasePI: Kuriyan, John
Description: Built and simulated two systems, one of a dodecameric hub with each subunit connected via a disordered linker to the regulatory segment (13 µs), and one of a dodecameric hub without the linkers and regulatory segments (6 µs). Observed that two out of the twelve regulatory segments spontaneously dock onto the hub assembly, on diametrically opposite sides of the hub. Docking is accommodated by a considerable distortion of the hub assembly, from a ring-like shape to an oval shape. At the interfaces where docking occurred, the subunits on either side moved away from each other, causing the interfaces to open. The remaining interfaces in the hub closed. Simulations of the hub without the linkers and regulatory segments indicated that the hub itself is intrinsically highly dynamic, with sub-units undergoing considerable movement with respect to each other. Docking at the interfaces by regulatory regions reduces this movement, essentially locking the subunits of the oligomer into rigid conformations with respect to each other. We suggest that docking by a third regulatory segment, at one of the closed interfaces, would destabilize the hub, leading its disassembly. |
Lipophilic Modulation of Cardiac Channel Activity as an Anti- Arrhythmic TherapyPI: Larsson, Peter
Description: The cardiac action potential is primarily generated by sodium and calcium channels, which depolarize the membrane potential, and by potassium channels that repolarize the membrane potential and terminate the action potential. The major cardiac potassium currents contributing to action potential stability are IKr (enabled by hERG1 channel) and IKs (KCNQ1 channel) that contribute to the action potential termination. Over 300 different inherited mutations have been found in IKs channels that cause cardiac arrhythmias in patients, while drug-induced arrhythmias related to IKr currents are impeding and very common side-effects across all classes of drugs commonly known as Long QT syndrome (LQTS). We built on the available Cryo-EM structures of these channels to (i) develop a comprehensive experimental/computational pipeline for structure-function relations and structure-functional properties of cardiac ion channels, and (ii) explore the specific hypothesis that binding of membrane-localized agents to highly-specific and unique binding pockets in hERG1 or KCNQ1 channels can potentially be explored as an anti-arrhythmic therapy for LQTS and prevention of sudden cardiac deaths. For example, relatively minor perturbation of the PUFA lipid tail converts the lipid from a non-specific interacting partner to potential IKs activator. A special emphasis will be placed on the methodological aspects of the Cryo-EM structures refinement with MD ensembles-based techniques and specific application to voltage-gated K+ channels.
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