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In Silico Identification of Binding Sites Responsible for Species Specificity on Human CD81 and Hepatitis C Virus E2 Protein
528a
Wednesday, March 2, 2016
would be particularly useful for the treatment of T cell mediated autoimmune diseases like multiple sclerosis, psoriasis and rheumatoid arthritis. In fact, the Kv1.3 blocking peptide ShK-186 has recently been found to be effective in Phase-1b study for psoriatic arthritis. We hypothesized that Kv1.3 blockers might also be useful for reducing microglia activation in ischemic stroke and other neurological diseases accompanied by neuroinflammation. Starting with cultured microglia, we observed that Kv1.3 expression is up-regulated in pro-inflammatory M1-like microglia and that Kv1.3 blockers preferentially reduce the production of IL-1beta and TNF-alpha without affecting IL-10 production. In organotypic hippocampal slices exposed to hypoxia/aglycemia Kv1.3 blockers significantly reduced microglia activation and increased neuronal survival. We further observed strong Kv1.3 expression on iNOS expressing inflammatory microglia in human stroke biopsies validating Kv1.3 as potential target. In both mouse and rat models of ischemic stroke the small molecule Kv1.3 blocker PAP-1 significantly reduced infarct area and improved neurological deficit 7 days after reperfusion when administered 12 hours after reperfusion. In the mouse model, PAP-1 selectively reduced brain levels of the inflammatory cytokines IL1-beta and IFN-gamma without affecting IL-10 and BDNF. Based on these findings we propose Kv1.3 inhibitors as potential therapeutic agents for preferentially inhibiting proinflammatory M1 microglia functions in ischemic stroke and other neurological diseases. Supported by GM076063 and AG043788.
Platform: Protein Plasticity & Binding 2600-Plat A Structural Characterization of the Kv7.2-Kv7.3 M Channel Proximal C-Terminus/Cam Complex Roi Strulovich1, William Tobelaim2, Bernard Attali2, Joel A. Hirsch1. 1 Biochemistry, Tel Aviv University, Tel Aviv, Israel, 2Physiology and pharmacology, Tel Aviv University, Tel Aviv, Israel. The Kv7 (KCNQ) family plays major roles in fine-tuning neuronal and cardiomyocyte excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminus (CT) bound constitutively by calmodulin (CaM), responsible for tetramerization, trafficking, and gating properties. The CT contains four helices, dubbed A through D. CaM embraces the proximal CT, comprised of anti-parallel helices A and B. An X-ray structure of Kv7.1-CT/CaM showed that the CaM C-lobe in apo form binds helix A, while Ca2þ/N-lobe binds helix B. Concatameric channels demonstrated that CaM binds both helices within the same Kv7.1 subunit. Kv7.2 and Kv7.3 are the predominant members found in neural tissues. Together they form the heterotetrameric M channel, named after the IM current it conducts. IM yields larger current amplitudes compared to currents generated by Kv7.2 homomers. Kv7.3 channel conductance is difficult to distinguish from background level and may not exist as a homomeric channel in vivo. Mutations in Kv7.2 or Kv7.3 cause neonatal epileptic encephalopathies and myokymia. We characterized Kv7.2, Kv7.3 and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by light-scattering and SAXS at various Ca2þ concentrations. We tested the chimeric channel and found it to be functional. We subsequently determined the crystal structure of the Q3A˚ resolution. Our results indicate Q2B/CaM complex at high [Ca2þ] to 2.0 A unique interactions responsible for the inter-subunit pairing of Q3A and Q2B while suggesting that Q3A-Q2B/CaM binding may be similar to that observed for Kv7.1-CT/CaM and unlike the model suggested for Kv7.4-CT/CaM. The Q3A-Q2B/CaM structure can be used to rationalize various Kv7.2 channelopathic mutants. Other implications for M-channel structure-function will be discussed. 2601-Plat Lessons in Protein Design from Combined Evolution and Conformational Dynamics Margaret S. Cheung1, Swarnendu Tripathi1, M.N. Waxham2, Yin Liu2. 1 Physics, University of Houston, Houston, TX, USA, 2Neurobiology and Anatomy, University of Texas HSC, Houston, TX, USA. Protein-protein interactions play important roles in the control of every cellular process. How natural selection has optimized protein design to produce molecules capable of binding to many partner proteins is a fascinating problem but not well understood. Here, we performed a combinatorial analysis of protein sequence evolution and conformational dynamics to study how calmodulin (CaM), which plays essential roles in calcium signaling
pathways, has adapted to bind to a large number of partner proteins. We discovered that amino acid residues in CaM can be partitioned into unique classes according to their degree of evolutionary conservation and local stability. Holistically, categorization of CaM residues into these classes reveals enriched physico-chemical interactions required for binding to diverse targets, balanced against the need to maintain the folding and structural modularity of CaM to achieve its overall function. The sequence-structurefunction relationship of CaM provides a concrete example of the general principle of protein design. We have demonstrated the synergy between the fields of molecular evolution and protein biophysics and created a generalizable framework broadly applicable to the study of protein-protein interactions. 2602-Plat In Silico Identification of Binding Sites Responsible for Species Specificity on Human CD81 and Hepatitis C Virus E2 Protein Chun-Chun Chang1,2, Hao-Jen Hsu3, Je-Wen Liou1. 1 Institute of Medical Science, Tzu-Chi University, Hualien, Taiwan, 2 Departments of Laboratory Medicine, Buddhist Tzu Chi Medical Center, Hualien, Taiwan, 3Department of Life Sciences, Tzu-Chi University, Hualien, Taiwan. Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to that the treatments for HCV infection are still limited, understanding the interactions between this virus and its host cells in details is required. Previous studies showed that HCV binds to human cells with high specificity via the interactions between its E2 protein and host cell CD81. In this study, we compared the structural differences between human and rat CD81s, and performed molecular docking of the HCV E2 protein onto the CD81s to figure out the species specificity for HCV E2 binding. The docking results showed that short helices of HCV E2 bind to the head regions of dimeric human CD81 via hydrophobic interactions, while these interactions are altered when HCV E2 are docked to rat CD81. Binding free energy calculations showed that HCV E2 bind to human CD81 with better affinity as compared to that with rat CD81. Based on the simulations, two peptides with their sequences taken from HCV E2 protein responsible for CD81 binding were designed and synthesized for the measurements of surface plasmon resonance (SPR). The results showed that one of the two peptide was able to bind to both human and rat CD81s, while the second peptide only bound to human CD81, indicating that the second peptide is involved in the species specific interactions to human CD81 binding regions, which also agreed well with our simulations. The SPR results were further confirmed at cellular levels by flow cytometry of peptide treated human and rat cells. The study demonstrates potential method of combining molecular simulations and in vitro experiments to develop strategies to treat virus infections. 2603-Plat Single-Molecule Experiments to Resolve Structural and Mechanical Properties of Condensin Jorine Eeftens1, Allard Katan1, Marc Kschonsak2, Markus Hassler2, Essam Dief1, Liza de Wilde1, Christian Haering2, Cees Dekker1. 1 Cees Dekker lab, Bionanoscience, TU Delft, Delft, Netherlands, 2Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany. Single-molecule experiments have proven to be an excellent technique to study mechanistic aspects of DNA-protein interactions. Techniques such as atomic force spectroscopy (AFM) and magnetic tweezers can probe properties at the single-molecule level that normally remain hidden in bulk ensemble experiments. Here, we employ high-speed AFM as well as magnetic tweezers to study the structural and mechanical properties of the condensin complex. The condensin complex plays a central role in chromosomal organisation. The complex contains an Smc2-Smc4 heterodimer, which forms two 45nm long coiled coils. The coils are joined at one end at a ‘hinge’ domain and each have an ATPase ‘head’ domain at the other end. Presently, no consensus exists on the precise structure and conformation of SMC dimers, which is likely coupled to the protein’s function. In fact, it is largely unknown how the condensin complex configures DNA at the mechanistic level. With high-speed AFM, we probe the structural arrangement and dynamics of Saccharomyces cerevisiae Smc2/4-dimers. We show that the dimers adopt various conformations, and that these conformations change reversibly over time. Interestingly, we show that the coiled coils of both the Smc2 and the Smc4 are very flexible. Furthermore, with magnetic tweezers, we observe compaction of DNA by condensin.
Wednesday, March 2, 2016
would be particularly useful for the treatment of T cell mediated autoimmune diseases like multiple sclerosis, psoriasis and rheumatoid arthritis. In fact, the Kv1.3 blocking peptide ShK-186 has recently been found to be effective in Phase-1b study for psoriatic arthritis. We hypothesized that Kv1.3 blockers might also be useful for reducing microglia activation in ischemic stroke and other neurological diseases accompanied by neuroinflammation. Starting with cultured microglia, we observed that Kv1.3 expression is up-regulated in pro-inflammatory M1-like microglia and that Kv1.3 blockers preferentially reduce the production of IL-1beta and TNF-alpha without affecting IL-10 production. In organotypic hippocampal slices exposed to hypoxia/aglycemia Kv1.3 blockers significantly reduced microglia activation and increased neuronal survival. We further observed strong Kv1.3 expression on iNOS expressing inflammatory microglia in human stroke biopsies validating Kv1.3 as potential target. In both mouse and rat models of ischemic stroke the small molecule Kv1.3 blocker PAP-1 significantly reduced infarct area and improved neurological deficit 7 days after reperfusion when administered 12 hours after reperfusion. In the mouse model, PAP-1 selectively reduced brain levels of the inflammatory cytokines IL1-beta and IFN-gamma without affecting IL-10 and BDNF. Based on these findings we propose Kv1.3 inhibitors as potential therapeutic agents for preferentially inhibiting proinflammatory M1 microglia functions in ischemic stroke and other neurological diseases. Supported by GM076063 and AG043788.
Platform: Protein Plasticity & Binding 2600-Plat A Structural Characterization of the Kv7.2-Kv7.3 M Channel Proximal C-Terminus/Cam Complex Roi Strulovich1, William Tobelaim2, Bernard Attali2, Joel A. Hirsch1. 1 Biochemistry, Tel Aviv University, Tel Aviv, Israel, 2Physiology and pharmacology, Tel Aviv University, Tel Aviv, Israel. The Kv7 (KCNQ) family plays major roles in fine-tuning neuronal and cardiomyocyte excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminus (CT) bound constitutively by calmodulin (CaM), responsible for tetramerization, trafficking, and gating properties. The CT contains four helices, dubbed A through D. CaM embraces the proximal CT, comprised of anti-parallel helices A and B. An X-ray structure of Kv7.1-CT/CaM showed that the CaM C-lobe in apo form binds helix A, while Ca2þ/N-lobe binds helix B. Concatameric channels demonstrated that CaM binds both helices within the same Kv7.1 subunit. Kv7.2 and Kv7.3 are the predominant members found in neural tissues. Together they form the heterotetrameric M channel, named after the IM current it conducts. IM yields larger current amplitudes compared to currents generated by Kv7.2 homomers. Kv7.3 channel conductance is difficult to distinguish from background level and may not exist as a homomeric channel in vivo. Mutations in Kv7.2 or Kv7.3 cause neonatal epileptic encephalopathies and myokymia. We characterized Kv7.2, Kv7.3 and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by light-scattering and SAXS at various Ca2þ concentrations. We tested the chimeric channel and found it to be functional. We subsequently determined the crystal structure of the Q3A˚ resolution. Our results indicate Q2B/CaM complex at high [Ca2þ] to 2.0 A unique interactions responsible for the inter-subunit pairing of Q3A and Q2B while suggesting that Q3A-Q2B/CaM binding may be similar to that observed for Kv7.1-CT/CaM and unlike the model suggested for Kv7.4-CT/CaM. The Q3A-Q2B/CaM structure can be used to rationalize various Kv7.2 channelopathic mutants. Other implications for M-channel structure-function will be discussed. 2601-Plat Lessons in Protein Design from Combined Evolution and Conformational Dynamics Margaret S. Cheung1, Swarnendu Tripathi1, M.N. Waxham2, Yin Liu2. 1 Physics, University of Houston, Houston, TX, USA, 2Neurobiology and Anatomy, University of Texas HSC, Houston, TX, USA. Protein-protein interactions play important roles in the control of every cellular process. How natural selection has optimized protein design to produce molecules capable of binding to many partner proteins is a fascinating problem but not well understood. Here, we performed a combinatorial analysis of protein sequence evolution and conformational dynamics to study how calmodulin (CaM), which plays essential roles in calcium signaling
pathways, has adapted to bind to a large number of partner proteins. We discovered that amino acid residues in CaM can be partitioned into unique classes according to their degree of evolutionary conservation and local stability. Holistically, categorization of CaM residues into these classes reveals enriched physico-chemical interactions required for binding to diverse targets, balanced against the need to maintain the folding and structural modularity of CaM to achieve its overall function. The sequence-structurefunction relationship of CaM provides a concrete example of the general principle of protein design. We have demonstrated the synergy between the fields of molecular evolution and protein biophysics and created a generalizable framework broadly applicable to the study of protein-protein interactions. 2602-Plat In Silico Identification of Binding Sites Responsible for Species Specificity on Human CD81 and Hepatitis C Virus E2 Protein Chun-Chun Chang1,2, Hao-Jen Hsu3, Je-Wen Liou1. 1 Institute of Medical Science, Tzu-Chi University, Hualien, Taiwan, 2 Departments of Laboratory Medicine, Buddhist Tzu Chi Medical Center, Hualien, Taiwan, 3Department of Life Sciences, Tzu-Chi University, Hualien, Taiwan. Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to that the treatments for HCV infection are still limited, understanding the interactions between this virus and its host cells in details is required. Previous studies showed that HCV binds to human cells with high specificity via the interactions between its E2 protein and host cell CD81. In this study, we compared the structural differences between human and rat CD81s, and performed molecular docking of the HCV E2 protein onto the CD81s to figure out the species specificity for HCV E2 binding. The docking results showed that short helices of HCV E2 bind to the head regions of dimeric human CD81 via hydrophobic interactions, while these interactions are altered when HCV E2 are docked to rat CD81. Binding free energy calculations showed that HCV E2 bind to human CD81 with better affinity as compared to that with rat CD81. Based on the simulations, two peptides with their sequences taken from HCV E2 protein responsible for CD81 binding were designed and synthesized for the measurements of surface plasmon resonance (SPR). The results showed that one of the two peptide was able to bind to both human and rat CD81s, while the second peptide only bound to human CD81, indicating that the second peptide is involved in the species specific interactions to human CD81 binding regions, which also agreed well with our simulations. The SPR results were further confirmed at cellular levels by flow cytometry of peptide treated human and rat cells. The study demonstrates potential method of combining molecular simulations and in vitro experiments to develop strategies to treat virus infections. 2603-Plat Single-Molecule Experiments to Resolve Structural and Mechanical Properties of Condensin Jorine Eeftens1, Allard Katan1, Marc Kschonsak2, Markus Hassler2, Essam Dief1, Liza de Wilde1, Christian Haering2, Cees Dekker1. 1 Cees Dekker lab, Bionanoscience, TU Delft, Delft, Netherlands, 2Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany. Single-molecule experiments have proven to be an excellent technique to study mechanistic aspects of DNA-protein interactions. Techniques such as atomic force spectroscopy (AFM) and magnetic tweezers can probe properties at the single-molecule level that normally remain hidden in bulk ensemble experiments. Here, we employ high-speed AFM as well as magnetic tweezers to study the structural and mechanical properties of the condensin complex. The condensin complex plays a central role in chromosomal organisation. The complex contains an Smc2-Smc4 heterodimer, which forms two 45nm long coiled coils. The coils are joined at one end at a ‘hinge’ domain and each have an ATPase ‘head’ domain at the other end. Presently, no consensus exists on the precise structure and conformation of SMC dimers, which is likely coupled to the protein’s function. In fact, it is largely unknown how the condensin complex configures DNA at the mechanistic level. With high-speed AFM, we probe the structural arrangement and dynamics of Saccharomyces cerevisiae Smc2/4-dimers. We show that the dimers adopt various conformations, and that these conformations change reversibly over time. Interestingly, we show that the coiled coils of both the Smc2 and the Smc4 are very flexible. Furthermore, with magnetic tweezers, we observe compaction of DNA by condensin.