Anton Project Summaries


Identification of conformational transitions in the outward-facing structure of the sodium-coupled leucine transporter, LeuTAa. (TRAJECTORIES COMING SOON)

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}.

PI: Bahar, Ivet

Understanding the mechanics of energy conversion in Na​+ dependent co­transporters

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.

PI: Grabe, Michael

Metabolite permeation and voltage-­gating of the mitochondrial channel VDAC

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.

PI: Grabe, Michael

Evolutionary Pathways of Engineered Sitagliptinases through Microsecond Molecular Dynamics (TRAJECTORIES COMING SOON)

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)

PI: Houk, Kendall

Detailed Characterization of the Equilibrium Fluctuations of the Engrailed Homeodomain

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.

PI: Langmead, Christopher James

Nanoscale structure in sphingolipid mixtures

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.

PI: Lyman, Eduard

Unraveling anomalous subdiffusion in heterogeneous membranes

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.

PI: Lyman, Edward

Microsecond scale simulations to characterize skeletal muscle Ca2+-binding protein troponin C

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.

PI: McCammon, Andrew

Microsecond scale simulations to characterize skeletal muscle Ca2+-binding protein parvalbumin

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.

PI: McCammon, Andrew

Transient Formation of Water-Conducting States in Membrane Transporter vSGLT

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.

PI: Tajkhorshid, Emad