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Computational Study of Anthracycline Interactions with Membrane-Embedded P-Glycoprotein
Sunday, February 28, 2016 cellular functions that include signal transduction, material transport, and energy conversion. Performing these functions requires the proteins to integrate within the membrane in the correct topology, or the overall orientation of the protein relative to the membrane. The integration of most proteins proceeds via the Sec translocon, a conserved protein-conducting channel that allows a nascent protein chain to access the membrane while being fed into the channel during translation. Previous studies have established that the Sec-facilitated integration of single-spanning proteins depends on factors including nascent chain hydrophobicity and charge; however, the extent to which these properties influence the integration and topology of multispanning proteins is less clear. Here, we use coarse-grained simulations to investigate the Sec-facilitated membrane integration of multispanning proteins on realistic biological timescales. We employ a simulation model that enables access to a timescale of minutes while retaining sufficient chemical accuracy to capture the forces that drive membrane integration. This study focuses on understanding two experimental observations: first, the finding that the protein EmrE exhibits two possible topologies in a 1:1 stoichiometry; and second, the finding that some marginally hydrophobic domains efficiently integrate into the membrane as part of a multispanning protein, despite inefficiently integrating as a single-spanning protein. This work provides mechanistic explanations for these observations, leading to insight into the sequence-level determinants of multispanning membrane protein integration and topology (1). (1) R.C. Van Lehn, B. Zhang, and T. F. Miller III, ‘‘Regulation of multispanning membrane protein topology by post-translational annealing,’’ eLife, in press. 299-Pos Board B79 Heterogeneity of the Hydrophobic Core of a Membrane Protein Complex Satarupa Bhaduri1, Stanislav D. Zakharov1, S Saif Hasan1, Valentyn Stadnytskyi1, Lukasz Bujnowicz2, Marcin Sarewicz3, Artur Osyczka3. 1 Biological Sciences, Purdue University, West Lafayette, IN, USA, 2 Department of Molecular BiophysicsFaculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland, 3Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland. The cytochrome bc1 complex utilizes two pairs of b-hemes in a mostly symmetric dimer to accomplish trans-membrane electron transfer and generation of a trans-membrane proton electrochemical potential gradient. Analysis of the structure of the complex in the purple photosynthetic bacterium, Rhodobacter capsulatus, allowed determination of the identity of the strongly interacting heme pair. Determination of the preferred heme sites of electron residence utilized Soret band excitonic circular dichroism (CD) spectra to assay heme heme interactions. Simultaneous kinetics of heme reduction and generation of the CD spectra imply that the thermodynamically favorable pair-wise heme occupancy utilizes the intra-monomer b-heme pair that exhibits different heme redox potentials on the electrochemically positive and negative sides of the cytochrome complex. This contradicts the expectation, based on the assumption of a dielectrically homogeneous protein environment, that the two higher potential hemes on the electronegative cytoplasmic side of the complex would be preferentially reduced. Relative to the protein medium in the structure that separates the two higher potential hemes on the electronegative side of the complex, a relatively large dielectric constant must exist between the intra-monomer b-hemes on the two sides of the complex, thus allowing increased polarizabilty and a smaller electrostatic repulsion between the reduced hemes. Thus, the distribution of dielectric constants in the core of the intra-membrane cytochrome complex, which mediate inter-heme electrostatic interactions, and which are a measure of local hydrophobicity, is heterogeneous. 300-Pos Board B80 Probing Conformational Equilibria in Flexible Recognition by Molecular Dynamics and EPR Jennifer M. Hays1, Marissa Kieber2, Tsega Solomon2, Linda Columbus2, Peter M. Kasson3. 1 Biomedical Engineering, University of Virginia, Charlottesville, VA, USA, 2 Chemistry, University of Virginia, Charlottesville, VA, USA, 3Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA. We have developed a combined experimental and computational approach for understanding the conformational equilibria of opacity associated adhesin (Opa) protein binding to human carcinoembryonic cell adhesin molecule 1 (CEACAM). This binding is a flexible recognition process: although the Opa-CEACAM binding event is high-affinity and shows some specificity, the unliganded state of Opa is extremely flexible, and Opa is highly tolerant of
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mutations that still permit CEACAM recognition. To elucidate the nature of this binding process, we combine electron paramagnetic resonance experiments and molecular dynamics simulation as follows. Double electron electron resonance (DEER) distance measurements between Opa and CEACAM residues are used to inform molecular dynamics simulations of the Opa-CEACAM complex. We incorporate DEER measurements by adding spring potentials to the system Hamiltonian. In order to thoroughly sample the system’s free energy landscape, we use a MARTINI coarse-grained model, cluster the resultant trajectories, and begin additional atomistic simulations from cluster centers. The benefits of this approach are twofold. First, we can direct DEER experiments by identifying under-determined regions of phase space. We identify these regions via a maximum-information-minimum-redundancy approach: the mutual information is computed between each pair Opa-CEACAM residue pair and the system’s configuration, and then highly informative pairs are measured using DEER. Second, once the model is validated by DEER, we can use the same clustering methods to identify which conformational states and specific interactions contribute to Opa-CEACAM binding. 301-Pos Board B81 Computational Study of Anthracycline Interactions with MembraneEmbedded P-Glycoprotein Eric K. Wong, J. Alfredo Freites, Douglas J. Tobias. University of California, Irvine, Irvine, CA, USA. P-glycoprotein (P-gp), an ATP-dependent efflux pump, is responsible for multidrug resistance when overexpressed in cancer cells due to its broad substrate specificity. A clear understanding of the P-gp binding pathway and specificity is necessary for the design of effective anti-tumor agents. Daunorubicin and idarubicin are chemotherapeutic agents susceptible to P-gp-mediated efflux. Although both drugs are structurally similar, idarubicin is four times more lipophilic and is resistant to P-gp-mediated efflux. A combination of microsecond timescale atomistic molecular dynamics (MD) simulations and adaptive biasing force simulations were used to sample conformations of the protein and drugs in a lipid bilayer. Our simulations show that a lipid-solvated cavity in P-gp more accurately reproduces the inward-facing conformation found in the crystal structures. More importantly the substrate portal, the gate between the cavity and the membrane inner leaflet, remains open to drug entry when it has been previously observed to close in simulation. Unrestrained MD simulations of daunorubicin embedded in a lipid bilayer show drug diffusion from bulk lipid to this substrate portal. Potential of mean force (PMF) calculations of the drugs along the membrane normal show minima in the hydrophobic core of the membrane. Though the drug centers-of-mass are at similar membrane depths, the methoxy functional group in daunorubicin results in the drug orienting close to the head group region of the membrane. Idarubicin, which lacks the methoxy group, orients its hydrophobic ring system towards the membrane center. Continuing studies are aimed at determining the consequences of drug orientation in the membrane as they enter the protein cavity through the substrate portal as well as characterizing the drugs in the active site. 302-Pos Board B82 NMR Restrained Protein Structure Calculations in Implicit Water/ Membrane Environments Ye Tian1,2, Charles Schwieters3, Stanley Opella1, Francesca Marassi2. 1 Chemistry and Biochemistry, UCSD, La Jolla, CA 92093, CA, USA, 2 Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA, 3 Division of Computational Bioscience, National Institutes of Health, Bethesda, MD, USA. The aqueous and lipid membrane environments that make up the intra- and extra-cellular compartments of biological organisms are critical for supporting the structural and functional integrity of both soluble proteins and membrane proteins. NMR spectroscopy enables characterization of the structures of both types of proteins in their respective, near-native environments. However, most NMR structures are not calculated in physically realistic environments and de novo structure calculations in explicit solvent or explicit lipids are computationally expensive. Traditionally, NMR structures are calculated with a simplified repulsive term to prevent atom clashing. Such treatment accelerates the calculation but sacrifices non-bonded interactions. To facilitate NMR structure calculations in natural environments we are developing a computationally practical implicit solvent and membrane potential for Xplor-NIH. Here we show that this potential affords significant improvements in the conformational quality, accuracy and precision of the calculated structures. Further, it provides correct embedding of membrane proteins in lipid bilayers, as well as physically meaningful insights about residueresidue and protein-membrane interactions. We also describe extensions of the potential to bicelle and micelle sample environments as well as various membrane systems.
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mutations that still permit CEACAM recognition. To elucidate the nature of this binding process, we combine electron paramagnetic resonance experiments and molecular dynamics simulation as follows. Double electron electron resonance (DEER) distance measurements between Opa and CEACAM residues are used to inform molecular dynamics simulations of the Opa-CEACAM complex. We incorporate DEER measurements by adding spring potentials to the system Hamiltonian. In order to thoroughly sample the system’s free energy landscape, we use a MARTINI coarse-grained model, cluster the resultant trajectories, and begin additional atomistic simulations from cluster centers. The benefits of this approach are twofold. First, we can direct DEER experiments by identifying under-determined regions of phase space. We identify these regions via a maximum-information-minimum-redundancy approach: the mutual information is computed between each pair Opa-CEACAM residue pair and the system’s configuration, and then highly informative pairs are measured using DEER. Second, once the model is validated by DEER, we can use the same clustering methods to identify which conformational states and specific interactions contribute to Opa-CEACAM binding. 301-Pos Board B81 Computational Study of Anthracycline Interactions with MembraneEmbedded P-Glycoprotein Eric K. Wong, J. Alfredo Freites, Douglas J. Tobias. University of California, Irvine, Irvine, CA, USA. P-glycoprotein (P-gp), an ATP-dependent efflux pump, is responsible for multidrug resistance when overexpressed in cancer cells due to its broad substrate specificity. A clear understanding of the P-gp binding pathway and specificity is necessary for the design of effective anti-tumor agents. Daunorubicin and idarubicin are chemotherapeutic agents susceptible to P-gp-mediated efflux. Although both drugs are structurally similar, idarubicin is four times more lipophilic and is resistant to P-gp-mediated efflux. A combination of microsecond timescale atomistic molecular dynamics (MD) simulations and adaptive biasing force simulations were used to sample conformations of the protein and drugs in a lipid bilayer. Our simulations show that a lipid-solvated cavity in P-gp more accurately reproduces the inward-facing conformation found in the crystal structures. More importantly the substrate portal, the gate between the cavity and the membrane inner leaflet, remains open to drug entry when it has been previously observed to close in simulation. Unrestrained MD simulations of daunorubicin embedded in a lipid bilayer show drug diffusion from bulk lipid to this substrate portal. Potential of mean force (PMF) calculations of the drugs along the membrane normal show minima in the hydrophobic core of the membrane. Though the drug centers-of-mass are at similar membrane depths, the methoxy functional group in daunorubicin results in the drug orienting close to the head group region of the membrane. Idarubicin, which lacks the methoxy group, orients its hydrophobic ring system towards the membrane center. Continuing studies are aimed at determining the consequences of drug orientation in the membrane as they enter the protein cavity through the substrate portal as well as characterizing the drugs in the active site. 302-Pos Board B82 NMR Restrained Protein Structure Calculations in Implicit Water/ Membrane Environments Ye Tian1,2, Charles Schwieters3, Stanley Opella1, Francesca Marassi2. 1 Chemistry and Biochemistry, UCSD, La Jolla, CA 92093, CA, USA, 2 Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA, 3 Division of Computational Bioscience, National Institutes of Health, Bethesda, MD, USA. The aqueous and lipid membrane environments that make up the intra- and extra-cellular compartments of biological organisms are critical for supporting the structural and functional integrity of both soluble proteins and membrane proteins. NMR spectroscopy enables characterization of the structures of both types of proteins in their respective, near-native environments. However, most NMR structures are not calculated in physically realistic environments and de novo structure calculations in explicit solvent or explicit lipids are computationally expensive. Traditionally, NMR structures are calculated with a simplified repulsive term to prevent atom clashing. Such treatment accelerates the calculation but sacrifices non-bonded interactions. To facilitate NMR structure calculations in natural environments we are developing a computationally practical implicit solvent and membrane potential for Xplor-NIH. Here we show that this potential affords significant improvements in the conformational quality, accuracy and precision of the calculated structures. Further, it provides correct embedding of membrane proteins in lipid bilayers, as well as physically meaningful insights about residueresidue and protein-membrane interactions. We also describe extensions of the potential to bicelle and micelle sample environments as well as various membrane systems.