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Molecular Determinants of Partial agonist Affinity in Adult Neuromuscular Acetylcholine Receptors
202a
Monday, February 29, 2016
and are implicated in many neurological disorders. Available x-ray structures of prokaryotic and eukaryotic Cys-loop receptors provide tremendous insights into the binding of agonists, the subsequent opening of the ion channel, and the mechanism of channel activation1-8. Yet the mechanism of inactivation ˚ x-ray structure by antagonists remains unknown. Here we present a 3.0 A of the human glycine receptor a3 homopentamer in complex with a high affinity, high specificity antagonist strychnine. Our structure allows us to explore in great detail the molecular recognition of antagonists. Comparisons with previous structures reveal a mechanism of inactivating Cys-loop receptors by antagonist binding. This work has been published in Nature Sep 28. http://dx.doi.org/10.1038/ nature14972. [Epub ahead of print] (2015) 1007-Plat Molecular Determinants of Partial agonist Affinity in Adult Neuromuscular Acetylcholine Receptors Iva Bruhova, Anthony Auerbach. Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY, USA. Adult neuromuscular acetylcholine receptor-channels have two agonist binding sites located at a-d and a-ε subunit interfaces. In a, W149, Y190, and Y198 (known as the aromatic ‘triad’) are the main determinants of acetylcholine affinity (Purohit et al., 2012). Ligands resembling the components of ACh, tetramethylammonium (TMA) and choline (Cho), are partial agonists. It is unknown what interactions at the binding site distinguish full vs. partial agonists. We used single-channel electrophysiology to estimate the binding energy (resting affinity) of mutants of the triad and non-a residues εY104/ dY106, εS117/dT119 and εL119/dL121 activated by partial agonists. All triad mutations weakened the affinity of the 3 agonists, in the order of aY190>aW149zaY198. aY190F produced the same, large reduction in affinity for ACh, TMA, Cho and 3 other choline derivatives. Dissimilarities between agonists were observed with aW149F and triad Ala mutants. ACh affinity depends on the type of aromatic residue at position 149, but not Cho. The removal of the aromatic group (to Ala) had a greater effect on ACh and TMA vs. Cho. The smaller change in binding energy of Cho with aW149 mutants is consistent with the hypothesis that Cho forms an H-bond with the aW149 backbone (Bruhova et al., 2013). On the non-a subunits, εY104/ dY106 mutations (A, S and L) enhanced ACh, TMA and Cho affinities, but εS117/dT119 or εL119/dL121 mutations weakly reduced affinity. The role of εY104 is explained by a molecular model in which εY104 p-stacks with aW149 to weaken the interaction of this indole with the agonist’s quaternary amine. In agreement, experiments showed strong energy coupling in the double mutant εY104A-aW149A. This also explains, in part, why the fetal receptor containing a Leu at gY104 has a higher affinity for ACh and Cho. Supported by CIHR and NS-64969.
Platform: Protein-Small Molecule Interactions 1008-Plat 2-Deoxy-ATP Enhances Multiple Kinetic Parameters to Improve Cardiac Function Ivan B. Tomasic, Marcus Henze, Ferdinand Evangelista, Anu R. Anto, Hector Rodriguez, Sadie R. Bartholomew. MyoKardia Inc., South San Francisco, CA, USA. Hypertrophic cardiomyopathy (HCM) is a form of genetic heart disease often caused by point mutations in sarcomeric proteins. As the underlying mechanisms of genetic HCM continue to be unraveled, developing novel ways to modulate the actomyosin contractile apparatus is of growing interest and importance. The nucleotide analog 2-deoxyadenosine triphosphate (dATP) has recently garnered interest as potentially having therapeutic benefit for treatment of systolic and/or diastolic heart failure. dATP has been previously reported to enhance cardiac contractility, increase þdP/dt, and improve diastolic relaxation parameters in transgenic mice with elevated levels of dATP in the heart (Korte, 2011). When compared to ATP, dATP has been reported to increase steady state ATPase activity, and sliding velocities in the in vitro motility assay up to 50% (Regnier et al 2000). To better understand potential therapeutic benefits of dATP on actomyosin, we characterize the mechanism of action for dATP using bovine cardiac myosin subfragments S1 and HMM in a variety of steady-state, transient, and single-molecule experiments. We report a 40% increase in unloaded in vitro motility sliding velocities, as well as increased ATPase activity, ADP release rates, and actin-binding affinities with dATP compared to ATP. The combination of transient kinetic rates and equilibrium constants of the actomyosin ATPase cycle, as well as basal myosin parameters, implicate ADP release as the primary contributor to the differences observed between the two nucleotides. We propose a model by
which enhancing both cardiac contraction and relaxation kinetics can improve cardiac function and potentially serve as a therapeutic for genetic heart disease. 1009-Plat Examination of ClpB Quaternary Structure and Linkage to Nucleotide Binding JiaBei Lin, Aaron L. Lucius. Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA. E. coli ClpB is a heat shock protein that belongs to the AAAþ protein family. Studies have shown that ClpB and its eukaryotic homologue, Hsp104, can disaggregate denatured proteins by themselves or cooperate with the DnaK chaperone system in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity and ClpB majorly exist as hexamer in the presence of nucleoside triphosphate. In contrast to this conclusion, our sedimentation velocity data show that ClpB resides in a monomer-dimer-tetramer-hexamer equilibrium in the presence of ATPYS (a slowly hydrolysable ATP analog). ClpB hexamers exhibit fast subunit exchange in the absence of nucleoside triphosphate, while the exchange rates decrease when the binding of nucleotide approaching to saturation. For the first time, we determined the binding constants and stoichiometries for ATPYS to each ClpB oligomer. The monomer is only able to bind one nucleotide whereas all twelve sites in the hexameric ring are bound by nucleotide. Interestingly, dimers and tetramers exhibit stoichiometries of ~3 and 7, respectively, which is one fewer than the maximum number of binding sites, which suggest an open conformation for the intermediates. We also determined the assembly constants for dimers, tetramers, and hexamers and their dependencies on nucleotide. These interaction constants make it possible to predict the concentration of hexamers present and able to bind to co-chaperones and polypeptide substrates. We anticipate our studies on ClpB assembly to be a starting point for understanding how ClpB hexamers disaggregate protein aggregates. 1010-Plat Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-Dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation Jiawen Li1, Daniel Deredge2, Patrick L. Wintrode2, Kenneth A. Johnson1. 1 Molecular Biosciences, University of Texas at Austin, Austin, TX, USA, 2 Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA. Hepatitis C virus (HCV) is a small, single stranded, positive-sense (þ) RNA virus that replicates its viral genome by an RNA-dependent RNA polymerase (RdRp), encoded as nonstructural protein 5B (NS5B), which is the primary target for anti-HCV treatment. Non-nucleoside inhibitors binding to the thumb site-2 (NNI2) of NS5B have shown success in clinical trials and although numerous crystal structures have been solved of NS5B with small molecule nonnucleoside inhibitors, little is known about the mechanism of inhibition. NS5B catalyzes de novo RNA initiation; thus, the RNA replication occurs in two stages, where abortive initiation is followed by highly processive elongation. Our kinetic analysis shows that NNI2 inhibitors do not significantly block initiation or elongation of RNA synthesis; rather they block the transition from the initiation to the elongation mode, which is thought to proceed with significant structural rearrangement with displacement of the beta-loop from the active site. We have also mapped the effect of three NNI2 inhibitors on the conformational dynamics of the enzyme using hydrogen/deuterium exchange. ˚ from the All three inhibitors rigidify an allosteric network extending up to 40 A binding site, providing a structural rationale for the disruption of the transition from initiation to elongation. Two of the inhibitors also suppress slow cooperative unfolding in the fingers extension-thumb interface and primer grip and appear to modestly inhibit translocation during RNA elongation. These results establish the allosteric mechanisms by which NNI2 inhibitors act, reveal important conformational changes underlying polymerase function, and point the way to the design of more effective allosteric inhibitors that exploit this new information. 1011-Plat Molecular Simulations Integrated with Experiments Unravel the Key Factors of Lipid Selection in Fatty Acid Amide Hydrolase and Suggest A General Mechanism of Lipid-Processing in the Parent Enzymes Giulia Palermo1, Inga Bauer2, Pablo Campomanes3, Andrea Cavalli2, Andrea Armirotti2, Stefania Girotto2, Marco De Vivo2, Ursula Rothlisberger1. 1 Institute of chemical science and engineering, EPF - Lausanne, Lausanne, Switzerland, 2Italian Institute of Technology, Genova, Italy, 3Structure et Re´activite´ des Systemes Mole´culaires Complexes, Universite´ de Lorraine, Vandoeuvre-le´s-Nancy, France.
Monday, February 29, 2016
and are implicated in many neurological disorders. Available x-ray structures of prokaryotic and eukaryotic Cys-loop receptors provide tremendous insights into the binding of agonists, the subsequent opening of the ion channel, and the mechanism of channel activation1-8. Yet the mechanism of inactivation ˚ x-ray structure by antagonists remains unknown. Here we present a 3.0 A of the human glycine receptor a3 homopentamer in complex with a high affinity, high specificity antagonist strychnine. Our structure allows us to explore in great detail the molecular recognition of antagonists. Comparisons with previous structures reveal a mechanism of inactivating Cys-loop receptors by antagonist binding. This work has been published in Nature Sep 28. http://dx.doi.org/10.1038/ nature14972. [Epub ahead of print] (2015) 1007-Plat Molecular Determinants of Partial agonist Affinity in Adult Neuromuscular Acetylcholine Receptors Iva Bruhova, Anthony Auerbach. Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY, USA. Adult neuromuscular acetylcholine receptor-channels have two agonist binding sites located at a-d and a-ε subunit interfaces. In a, W149, Y190, and Y198 (known as the aromatic ‘triad’) are the main determinants of acetylcholine affinity (Purohit et al., 2012). Ligands resembling the components of ACh, tetramethylammonium (TMA) and choline (Cho), are partial agonists. It is unknown what interactions at the binding site distinguish full vs. partial agonists. We used single-channel electrophysiology to estimate the binding energy (resting affinity) of mutants of the triad and non-a residues εY104/ dY106, εS117/dT119 and εL119/dL121 activated by partial agonists. All triad mutations weakened the affinity of the 3 agonists, in the order of aY190>aW149zaY198. aY190F produced the same, large reduction in affinity for ACh, TMA, Cho and 3 other choline derivatives. Dissimilarities between agonists were observed with aW149F and triad Ala mutants. ACh affinity depends on the type of aromatic residue at position 149, but not Cho. The removal of the aromatic group (to Ala) had a greater effect on ACh and TMA vs. Cho. The smaller change in binding energy of Cho with aW149 mutants is consistent with the hypothesis that Cho forms an H-bond with the aW149 backbone (Bruhova et al., 2013). On the non-a subunits, εY104/ dY106 mutations (A, S and L) enhanced ACh, TMA and Cho affinities, but εS117/dT119 or εL119/dL121 mutations weakly reduced affinity. The role of εY104 is explained by a molecular model in which εY104 p-stacks with aW149 to weaken the interaction of this indole with the agonist’s quaternary amine. In agreement, experiments showed strong energy coupling in the double mutant εY104A-aW149A. This also explains, in part, why the fetal receptor containing a Leu at gY104 has a higher affinity for ACh and Cho. Supported by CIHR and NS-64969.
Platform: Protein-Small Molecule Interactions 1008-Plat 2-Deoxy-ATP Enhances Multiple Kinetic Parameters to Improve Cardiac Function Ivan B. Tomasic, Marcus Henze, Ferdinand Evangelista, Anu R. Anto, Hector Rodriguez, Sadie R. Bartholomew. MyoKardia Inc., South San Francisco, CA, USA. Hypertrophic cardiomyopathy (HCM) is a form of genetic heart disease often caused by point mutations in sarcomeric proteins. As the underlying mechanisms of genetic HCM continue to be unraveled, developing novel ways to modulate the actomyosin contractile apparatus is of growing interest and importance. The nucleotide analog 2-deoxyadenosine triphosphate (dATP) has recently garnered interest as potentially having therapeutic benefit for treatment of systolic and/or diastolic heart failure. dATP has been previously reported to enhance cardiac contractility, increase þdP/dt, and improve diastolic relaxation parameters in transgenic mice with elevated levels of dATP in the heart (Korte, 2011). When compared to ATP, dATP has been reported to increase steady state ATPase activity, and sliding velocities in the in vitro motility assay up to 50% (Regnier et al 2000). To better understand potential therapeutic benefits of dATP on actomyosin, we characterize the mechanism of action for dATP using bovine cardiac myosin subfragments S1 and HMM in a variety of steady-state, transient, and single-molecule experiments. We report a 40% increase in unloaded in vitro motility sliding velocities, as well as increased ATPase activity, ADP release rates, and actin-binding affinities with dATP compared to ATP. The combination of transient kinetic rates and equilibrium constants of the actomyosin ATPase cycle, as well as basal myosin parameters, implicate ADP release as the primary contributor to the differences observed between the two nucleotides. We propose a model by
which enhancing both cardiac contraction and relaxation kinetics can improve cardiac function and potentially serve as a therapeutic for genetic heart disease. 1009-Plat Examination of ClpB Quaternary Structure and Linkage to Nucleotide Binding JiaBei Lin, Aaron L. Lucius. Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA. E. coli ClpB is a heat shock protein that belongs to the AAAþ protein family. Studies have shown that ClpB and its eukaryotic homologue, Hsp104, can disaggregate denatured proteins by themselves or cooperate with the DnaK chaperone system in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity and ClpB majorly exist as hexamer in the presence of nucleoside triphosphate. In contrast to this conclusion, our sedimentation velocity data show that ClpB resides in a monomer-dimer-tetramer-hexamer equilibrium in the presence of ATPYS (a slowly hydrolysable ATP analog). ClpB hexamers exhibit fast subunit exchange in the absence of nucleoside triphosphate, while the exchange rates decrease when the binding of nucleotide approaching to saturation. For the first time, we determined the binding constants and stoichiometries for ATPYS to each ClpB oligomer. The monomer is only able to bind one nucleotide whereas all twelve sites in the hexameric ring are bound by nucleotide. Interestingly, dimers and tetramers exhibit stoichiometries of ~3 and 7, respectively, which is one fewer than the maximum number of binding sites, which suggest an open conformation for the intermediates. We also determined the assembly constants for dimers, tetramers, and hexamers and their dependencies on nucleotide. These interaction constants make it possible to predict the concentration of hexamers present and able to bind to co-chaperones and polypeptide substrates. We anticipate our studies on ClpB assembly to be a starting point for understanding how ClpB hexamers disaggregate protein aggregates. 1010-Plat Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-Dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation Jiawen Li1, Daniel Deredge2, Patrick L. Wintrode2, Kenneth A. Johnson1. 1 Molecular Biosciences, University of Texas at Austin, Austin, TX, USA, 2 Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA. Hepatitis C virus (HCV) is a small, single stranded, positive-sense (þ) RNA virus that replicates its viral genome by an RNA-dependent RNA polymerase (RdRp), encoded as nonstructural protein 5B (NS5B), which is the primary target for anti-HCV treatment. Non-nucleoside inhibitors binding to the thumb site-2 (NNI2) of NS5B have shown success in clinical trials and although numerous crystal structures have been solved of NS5B with small molecule nonnucleoside inhibitors, little is known about the mechanism of inhibition. NS5B catalyzes de novo RNA initiation; thus, the RNA replication occurs in two stages, where abortive initiation is followed by highly processive elongation. Our kinetic analysis shows that NNI2 inhibitors do not significantly block initiation or elongation of RNA synthesis; rather they block the transition from the initiation to the elongation mode, which is thought to proceed with significant structural rearrangement with displacement of the beta-loop from the active site. We have also mapped the effect of three NNI2 inhibitors on the conformational dynamics of the enzyme using hydrogen/deuterium exchange. ˚ from the All three inhibitors rigidify an allosteric network extending up to 40 A binding site, providing a structural rationale for the disruption of the transition from initiation to elongation. Two of the inhibitors also suppress slow cooperative unfolding in the fingers extension-thumb interface and primer grip and appear to modestly inhibit translocation during RNA elongation. These results establish the allosteric mechanisms by which NNI2 inhibitors act, reveal important conformational changes underlying polymerase function, and point the way to the design of more effective allosteric inhibitors that exploit this new information. 1011-Plat Molecular Simulations Integrated with Experiments Unravel the Key Factors of Lipid Selection in Fatty Acid Amide Hydrolase and Suggest A General Mechanism of Lipid-Processing in the Parent Enzymes Giulia Palermo1, Inga Bauer2, Pablo Campomanes3, Andrea Cavalli2, Andrea Armirotti2, Stefania Girotto2, Marco De Vivo2, Ursula Rothlisberger1. 1 Institute of chemical science and engineering, EPF - Lausanne, Lausanne, Switzerland, 2Italian Institute of Technology, Genova, Italy, 3Structure et Re´activite´ des Systemes Mole´culaires Complexes, Universite´ de Lorraine, Vandoeuvre-le´s-Nancy, France.