- Email: [email protected]
Structure and Dynamics of Complexes of Interleukin-8 and its Receptor CXCR1 in Phospholipid Bilayers by Solid-State NMR
192a
Monday, February 29, 2016
956-Plat Structural Determinants and Binding Properties of the Neurite Outgrowth Inhibitor (NOGO) Melanie J. Cocco, Ali Alhoshani, Verna Vu, D’Artagnan Greene. Dept Mol Biol and Biochem, University of California, Irvine, CA, USA. Compelling evidence indicates that repair of damage to the central nervous system (CNS) is inhibited by the presence of protein factors within myelin that prevent axonal regrowth. Myelin growth inhibitors and their common receptor have been identified as targets in the treatment of spinal cord injury, MS, ALS and stroke. We have determined the NMR structure of one of the myelin growth inhibitors, the neurite outgrowth inhibitor (Nogo). We studied the structure of this protein alone and in the presence of dodecylphosphocholine micelles to mimic the natural cell membrane environment. To understand interactions between Nogo and lipid, we hypothesize that aromatic groups and a negative charge hyperconserved among this family of proteins drive the remarkably strong association of Nogo-66 with a phosphocholine surface. We modeled the docking of dodecylphosphocholine (DPC) with Nogo-66 and found that a lipid choline group could form a stable salt bridge with Glu26 and serve as a membrane anchor point. To test the role of the Glu26 anion in binding choline, we mutated this residue to alanine and assessed the structural consequences, the association with lipid and the affinity for the Nogo receptor. Paramagnetic probes allowed us to define portions of the growth inhibitor that are accessible to solvent (and consequently the Nogo receptor). Using computational docking methods, NMR data and mutagenesis results, we calculated the optimal protein-protein interface between our structure of Nogo and the Nogo receptor. This model has inspired the development of compounds that can bind and block Nogo. 957-Plat Structure and Dynamics of Complexes of Interleukin-8 and its Receptor CXCR1 in Phospholipid Bilayers by Solid-State NMR Sang Ho Park, Anna De Angelis, Jasmina Radoicic, Sabrina Berkamp, Zheng Long, Stanley J. Opella. Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA. CXCR1, a G protein-coupled receptor for chemokine interleukin-8 (IL-8) is a key mediator of immune and inflammatory responses and is involved in various diseases, including cancer. Here we describe proton-detected fast MAS solidstate NMR studies of IL-8 and CXCR1 complexes in phospholipid bilayers. More than half of the IL-8 backbone residues were immobilized by the receptor and their chemical shifts significantly perturbed, suggesting that both dynamics and conformation of IL-8 are affected by interactions with CXCR1. Comparison of IL-8 spectra interacting with N-terminal domain and first transmembrane domain (1TM1-72), N-terminal truncated (NT39-350), and wild-type (WT1350) CXCR1 constructs enabled mapping of IL-8 residues involved in interactions with N-terminal and extracellular regions of CXCR1, providing valuable insight into understanding the first step of the CXCR1 mediated signaling cascade. Long-range distance restraints obtained from intermolecular paramagnetic relaxation enhancement of unnatural amino acid HQA-incorporated CXCR1 constructs serve as crucial input for structure determination of IL-8CXCR1 complex. Progress toward the determination of the CXCR1 structure bound to Gai protein and the comparison of the structure of CXCR1 and CXCR2 will be presented. 958-Plat Functional, Dynamic and Structural Understanding of M2 Proton Channel from Influenza A and its Inhibition Timothy A. Cross1,2, Riqiang Fu1, E. Vindana Ekanayake1,2, Yimin Miao1,2, Joana Paulino1,3, Wright Anna1,3, Jian Dai3,4, Huan-Xiang Zhou3,4. 1 NHMFL/FSU, Tallahassee, FL, USA, 2Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA, 3Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA, 4Department of Physics, Florida State University, Tallahassee, FL, USA. While it has long been agreed that the M2 proton channel functions by shuttling protons on and off of the histidine tetrad near the mid plane of the lipid bilayer, the details have been hotly debated. Now details of the shuttling mechanism have been elucidated from solid state NMR spectroscopy. Short hydrogen bonds between the imidazole-imidazolium pairs are confirmed as well as their disruption by aqueous attack followed by N-H exchange and imidazoliumimidazole hydrogen bond reformation by either a deprotonation to the aqueous pore connected to the viral external environment (a futile proton cycle) or by deprotonation to a waters in the internal cavity between the His37 and Trp41 tetrads. For this latter option when the Trp41 gate opens the proton can be conducted into the viral interior (conductance cycle). The dynamics of the Trp41
residues and those of the secondary conductance gate (Val27) have been evaluated by wide-line solid state NMR. Solid state NMR REDOR and NCA experiments of the full length wild type M2 channel in complex with rimantadine enantiomers. The binding of these enantiomers to M2 shows a difference in binding affinity and a different set of binding interactions implying that stereospecific drugs may have biomedical benefits. M2 has multiple functions, among them is the facilitation of viral budding that takes advantage of the pyramidal shape of the M2 structure to induce membrane curvature. This shape is caused by the insertion of the juxtamembrane amphipathic helix in the lipid bilayer interface. We have now shown that the stability of this helix in the membrane is the result of cholesterol binding to this helix.
Platform: Cytoskeletal Motor Proteins 959-Plat On the Force-Generating Capacity of Disassembling Microtubules Jonathan W. Driver1, Elisabeth Geyer2, Luke M. Rice2, Charles L. Asbury1. 1 Physiology & Biophysics, University of Washington, Seattle, WA, USA, 2 Biophysics, UT Southwestern, Dallas, TX, USA. Microtubules can generate force independently of motor enzymes, especially at kinetochores where disassembling microtubules drive movement of mitotic chromosomes. A popular explanation for this microtubule-powered motility is the conformational wave model, where individual rows of tubulin subunits, the protofilaments, push or pull on a kinetochore as they curl outward from a disassembling microtubule tip. One pioneering study has demonstrated that curling protofilaments are capable of generating force, but the measured forces were far weaker (~0.24 pN) than forces generated at kinetochores in vivo (4 - 8 pN) or in vitro (~9 pN). Thus it has remained unclear whether the wave mechanism can contribute significantly to kinetochore force production. Here, by developing a wave assay using recombinant tubulin and a feedback-controlled laser trap, we show that the conformational wave can generate forces 50-fold higher than previously recorded. Measuring its full capacity for work output reveals that the wave carries at least three times the energy harnessed by kinetochores in vitro, and enables an estimate of the mechanical strain energy trapped per tubulin dimer in the microtubule lattice. Surprisingly, a b-tubulin mutation that allosterically enhances microtubule stability has little effect on wave energy. Our work indicates that the conformational wave mechanism can make a major contribution to kinetochore motility and it provides a new, direct way to examine tubulin mechanochemistry. 960-Plat A Structural Model of the Mitotic Kinesin-6 Mechanochemical Cycle Joseph Atherton1, I-Mei Yu2, Steven S. Rosenfeld3, Anne Houdusse2, Carolyn A. Moores1. 1 Biological Sciences, Birkbeck College, London, London, United Kingdom, 2 Structural Motility, Institut Curie, Paris, France, 3Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. MKLP2 is a member of the kinesin-6 family, with critical roles during the metaphase-anaphase transition and in cytokinesis. The MKLP2 motor domain contains the conserved nucleotide and microtubule binding sites characteristic of plus-end mitotic kinesins. However, it is ~60% larger than most other kinesins due to large inserts, the precise role(s) of which are unknown. Our biochemical studies show that the MKLP2 motor domain has nucleotideinsensitive sub-micromolar affinity for microtubules, except in the presence of the ATP analogue ADP.AlFx, which induces apparent nanomolar affinity. Consistently, we find that ATP binding kinetics appear to be uncoupled from microtubule binding. Using cryo-electron microscopy and structure determina˚ resolution), we have visualized the microtubule-bound MKLP2 tion (to ~5A motor domain at successive steps in its ATPase cycle. Its orientation with respect to the microtubule surface is conserved compared to other kinesins. ADP release induces small perturbations of the P-loop and loop11, described for transport kinesins and consistent with high levels of conservation in these regions. However, sequence divergence within the motor manifests itself at larger scales. Strikingly, ADP release does not induce major conformational changes around the nucleotide-binding site. However, in the presence of the ADP.AlFx, loop11 and loop9 are ordered, becoming compact around the bound nucleotide, forming a hydrolysis-competent conformation. The divergent loop2 also forms a contact with the microtubule surface, as do regions of the highly extended loop6. Along with these changes, the elongated kinesin-6 neck-linker is directed towards the microtubule plus-end but does not dock along the motor domain. The N-terminus of the motor is also disordered in all states and a cover neck bundle is not formed.
Monday, February 29, 2016
956-Plat Structural Determinants and Binding Properties of the Neurite Outgrowth Inhibitor (NOGO) Melanie J. Cocco, Ali Alhoshani, Verna Vu, D’Artagnan Greene. Dept Mol Biol and Biochem, University of California, Irvine, CA, USA. Compelling evidence indicates that repair of damage to the central nervous system (CNS) is inhibited by the presence of protein factors within myelin that prevent axonal regrowth. Myelin growth inhibitors and their common receptor have been identified as targets in the treatment of spinal cord injury, MS, ALS and stroke. We have determined the NMR structure of one of the myelin growth inhibitors, the neurite outgrowth inhibitor (Nogo). We studied the structure of this protein alone and in the presence of dodecylphosphocholine micelles to mimic the natural cell membrane environment. To understand interactions between Nogo and lipid, we hypothesize that aromatic groups and a negative charge hyperconserved among this family of proteins drive the remarkably strong association of Nogo-66 with a phosphocholine surface. We modeled the docking of dodecylphosphocholine (DPC) with Nogo-66 and found that a lipid choline group could form a stable salt bridge with Glu26 and serve as a membrane anchor point. To test the role of the Glu26 anion in binding choline, we mutated this residue to alanine and assessed the structural consequences, the association with lipid and the affinity for the Nogo receptor. Paramagnetic probes allowed us to define portions of the growth inhibitor that are accessible to solvent (and consequently the Nogo receptor). Using computational docking methods, NMR data and mutagenesis results, we calculated the optimal protein-protein interface between our structure of Nogo and the Nogo receptor. This model has inspired the development of compounds that can bind and block Nogo. 957-Plat Structure and Dynamics of Complexes of Interleukin-8 and its Receptor CXCR1 in Phospholipid Bilayers by Solid-State NMR Sang Ho Park, Anna De Angelis, Jasmina Radoicic, Sabrina Berkamp, Zheng Long, Stanley J. Opella. Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA. CXCR1, a G protein-coupled receptor for chemokine interleukin-8 (IL-8) is a key mediator of immune and inflammatory responses and is involved in various diseases, including cancer. Here we describe proton-detected fast MAS solidstate NMR studies of IL-8 and CXCR1 complexes in phospholipid bilayers. More than half of the IL-8 backbone residues were immobilized by the receptor and their chemical shifts significantly perturbed, suggesting that both dynamics and conformation of IL-8 are affected by interactions with CXCR1. Comparison of IL-8 spectra interacting with N-terminal domain and first transmembrane domain (1TM1-72), N-terminal truncated (NT39-350), and wild-type (WT1350) CXCR1 constructs enabled mapping of IL-8 residues involved in interactions with N-terminal and extracellular regions of CXCR1, providing valuable insight into understanding the first step of the CXCR1 mediated signaling cascade. Long-range distance restraints obtained from intermolecular paramagnetic relaxation enhancement of unnatural amino acid HQA-incorporated CXCR1 constructs serve as crucial input for structure determination of IL-8CXCR1 complex. Progress toward the determination of the CXCR1 structure bound to Gai protein and the comparison of the structure of CXCR1 and CXCR2 will be presented. 958-Plat Functional, Dynamic and Structural Understanding of M2 Proton Channel from Influenza A and its Inhibition Timothy A. Cross1,2, Riqiang Fu1, E. Vindana Ekanayake1,2, Yimin Miao1,2, Joana Paulino1,3, Wright Anna1,3, Jian Dai3,4, Huan-Xiang Zhou3,4. 1 NHMFL/FSU, Tallahassee, FL, USA, 2Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA, 3Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA, 4Department of Physics, Florida State University, Tallahassee, FL, USA. While it has long been agreed that the M2 proton channel functions by shuttling protons on and off of the histidine tetrad near the mid plane of the lipid bilayer, the details have been hotly debated. Now details of the shuttling mechanism have been elucidated from solid state NMR spectroscopy. Short hydrogen bonds between the imidazole-imidazolium pairs are confirmed as well as their disruption by aqueous attack followed by N-H exchange and imidazoliumimidazole hydrogen bond reformation by either a deprotonation to the aqueous pore connected to the viral external environment (a futile proton cycle) or by deprotonation to a waters in the internal cavity between the His37 and Trp41 tetrads. For this latter option when the Trp41 gate opens the proton can be conducted into the viral interior (conductance cycle). The dynamics of the Trp41
residues and those of the secondary conductance gate (Val27) have been evaluated by wide-line solid state NMR. Solid state NMR REDOR and NCA experiments of the full length wild type M2 channel in complex with rimantadine enantiomers. The binding of these enantiomers to M2 shows a difference in binding affinity and a different set of binding interactions implying that stereospecific drugs may have biomedical benefits. M2 has multiple functions, among them is the facilitation of viral budding that takes advantage of the pyramidal shape of the M2 structure to induce membrane curvature. This shape is caused by the insertion of the juxtamembrane amphipathic helix in the lipid bilayer interface. We have now shown that the stability of this helix in the membrane is the result of cholesterol binding to this helix.
Platform: Cytoskeletal Motor Proteins 959-Plat On the Force-Generating Capacity of Disassembling Microtubules Jonathan W. Driver1, Elisabeth Geyer2, Luke M. Rice2, Charles L. Asbury1. 1 Physiology & Biophysics, University of Washington, Seattle, WA, USA, 2 Biophysics, UT Southwestern, Dallas, TX, USA. Microtubules can generate force independently of motor enzymes, especially at kinetochores where disassembling microtubules drive movement of mitotic chromosomes. A popular explanation for this microtubule-powered motility is the conformational wave model, where individual rows of tubulin subunits, the protofilaments, push or pull on a kinetochore as they curl outward from a disassembling microtubule tip. One pioneering study has demonstrated that curling protofilaments are capable of generating force, but the measured forces were far weaker (~0.24 pN) than forces generated at kinetochores in vivo (4 - 8 pN) or in vitro (~9 pN). Thus it has remained unclear whether the wave mechanism can contribute significantly to kinetochore force production. Here, by developing a wave assay using recombinant tubulin and a feedback-controlled laser trap, we show that the conformational wave can generate forces 50-fold higher than previously recorded. Measuring its full capacity for work output reveals that the wave carries at least three times the energy harnessed by kinetochores in vitro, and enables an estimate of the mechanical strain energy trapped per tubulin dimer in the microtubule lattice. Surprisingly, a b-tubulin mutation that allosterically enhances microtubule stability has little effect on wave energy. Our work indicates that the conformational wave mechanism can make a major contribution to kinetochore motility and it provides a new, direct way to examine tubulin mechanochemistry. 960-Plat A Structural Model of the Mitotic Kinesin-6 Mechanochemical Cycle Joseph Atherton1, I-Mei Yu2, Steven S. Rosenfeld3, Anne Houdusse2, Carolyn A. Moores1. 1 Biological Sciences, Birkbeck College, London, London, United Kingdom, 2 Structural Motility, Institut Curie, Paris, France, 3Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. MKLP2 is a member of the kinesin-6 family, with critical roles during the metaphase-anaphase transition and in cytokinesis. The MKLP2 motor domain contains the conserved nucleotide and microtubule binding sites characteristic of plus-end mitotic kinesins. However, it is ~60% larger than most other kinesins due to large inserts, the precise role(s) of which are unknown. Our biochemical studies show that the MKLP2 motor domain has nucleotideinsensitive sub-micromolar affinity for microtubules, except in the presence of the ATP analogue ADP.AlFx, which induces apparent nanomolar affinity. Consistently, we find that ATP binding kinetics appear to be uncoupled from microtubule binding. Using cryo-electron microscopy and structure determina˚ resolution), we have visualized the microtubule-bound MKLP2 tion (to ~5A motor domain at successive steps in its ATPase cycle. Its orientation with respect to the microtubule surface is conserved compared to other kinesins. ADP release induces small perturbations of the P-loop and loop11, described for transport kinesins and consistent with high levels of conservation in these regions. However, sequence divergence within the motor manifests itself at larger scales. Strikingly, ADP release does not induce major conformational changes around the nucleotide-binding site. However, in the presence of the ADP.AlFx, loop11 and loop9 are ordered, becoming compact around the bound nucleotide, forming a hydrolysis-competent conformation. The divergent loop2 also forms a contact with the microtubule surface, as do regions of the highly extended loop6. Along with these changes, the elongated kinesin-6 neck-linker is directed towards the microtubule plus-end but does not dock along the motor domain. The N-terminus of the motor is also disordered in all states and a cover neck bundle is not formed.