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Influence of Familial Parkinson's Disease Mutations on Mitochondrial Localization and Secondary Structure of PINK1
230a
Monday, February 29, 2016
Solid-state 2H NMR spectra were acquired for different tilt angles of the aligned samples. The methyl group orientations relative to the membrane normal were calculated by fitting the experimental 2H NMR spectra using a static uniaxial distribution [3] for the protein embedded within lipid bilayers. We found that the orientation of the C9-methyl group obtained from 2 H NMR spectroscopy was similar to that from X-ray data. By contrast, the orientations of the C5- and C13- methyl groups were different versus the X-ray crystal structures. The retinal structure was analyzed using the three-plane model, as in previous studies of rhodopsin in the dark and the Meta-I states [4]. Moreover, a new approach was tested that combines 2H NMR and X-ray restraints for retinal together with the rhodopsin binding-pocket using simulated annealing. Our results yield new insights into formation of the activated state of the receptor in lipid membranes. [1] A.V. Struts et al. (2011) Nat. Struct. Mol. Biol. 18, 392-394. [2] A.V. Struts et al. (2015) Meth. Mol. Biol. 1271, 133-158. [3] A.A. Nevzorov et al. (1999) JACS 121, 7636-7643. [4] G.F.J. Salgado et al. (2006) JACS 128, 11067-11071. 1142-Pos Board B119 Understanding Structural and Functional Stability of two Rhomboid Proteases: HiGlpG and PsAarA Rashmi Panigrahi1, Elena Arutyunova1, Pankaj Panwar2, Katharina Gimpl3, Sandro Keller3, Joanne Lemieux1. 1 University of Alberta, Edmonton, AB, Canada, 2University of British Columbia, Vancouver, BC, Canada, 3University of Kaiserslautern, Kaiserslautern, Germany. Functions of proteins critically rely on their conformation, but understanding conformational transitions in membrane proteins poses challenges because of their hydrophobic and aggregation-prone nature. In the current study, we focus on the two topologically unique intramembrane rhomboid proteases: HiGlpG and PsAarA, with an aim to understand the relationship between the extent of unfolding upon exposure to a chemical denaturant or temperature, and regain of native activity in the reassembled form. Whereas thermal denaturation has irreversible destabilizing effects on both proteins, unfolding induced by guanidinium chloride is completely reversible for HiGlpG but only partly reversible for PsAarA. For both rhomboid proteases, circular dichroism spectroscopy as a function of denaturant concentration reveals a broad, gradual transition from the native, folded state to the denatured, unfolded state, thus arguing against a classical two-state model as found for many globular soluble proteins. Upon removal of denaturant, protein refolding and dimer re-association result in regain of native-like activity, as confirmed by fluorescence-based enzyme kinetics. This concerted biophysical and functional analysis demonstrates that the simple 6TM organization of HiGlpG is more robust than the 6þ1TM organization of PsAarA. 1143-Pos Board B120 Influence of Familial Parkinson’s Disease Mutations on Mitochondrial Localization and Secondary Structure of PINK1 Stephania Irwin, Rashmi Panigrahi, Elena Arutyunova, Nicolas Touret, M. Joanne Lemieux. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada. Mutations in the PARK6 gene, encoding for the protein PINK1 (PhosphataseTensin homologue (PTEN)-induced protein kinase), are associated with early-onset familial Parkinson’s disease (PD). PINK1 is found in multiple cellular locations, including the cytosol and both mitochondrial membranes (outer mitochondrial membrane, OMM; and inner mitochondrial membrane, IMM). PINK1 must enter into the IMM in order to be cleaved by PARL (Presenilin-associated rhomboid-like), an IMM protease. Cleavage of PINK1 by PARL activates the degradation of PINK1, indicating a healthy, polarized mitochondrion. Improper processing of PINK1 PD mutants leads to unprocessed PINK1 accumulating on the OMM which induces mitophagy, or the selective autophagy of mitochondria. We hypothesize that accumulation of unprocessed PINK1 on the OMM is either due to improper localization of PINK1 or due to the inability of PARL to cleave the mutated protein. Many traditional protein localization techniques including confocal microscopy cannot distinguish between the inner and outer mitochondrial membrane. Super-resolution microscopy will be used to determine the precise localization patterns to the appropriate mitochondrial membrane of both wildtype PINK1 and PINK1 with point mutations associated to PD. Preliminary confocal microscopy determined that PINK1 constructs are capable of being translated in vivo in HeLa cells. These preliminary results have proved to be promising and further experiments will be conducted using superresolution microscopy and structured illumination microscopy to determine precise mitochondrial localization patterns in order to determine if PD muta-
tions lead to improper localization of PINK1. Molecular dynamics simulations on PINK1 transmembrane segments harbouring familial Parkinson’s disease mutations will be conducted to determine if localization defects are due to structural perturbations. This work sets the stage for an understanding for the role of PINK1 mutations in Parkinson’s disease at a molecular level. 1144-Pos Board B121 Structural Basis for KCNQ1 Long-QT Syndrome Disease causing Mutations Keenan C. Taylor, Hui Huang, Brett Kroncke, Charles Sanders. Biochemistry, Vanderbilt, Nashville, TN, USA. The human potassium channel KCNQ1 is a polytopic a-helical membrane protein. KCNQ1 is expressed in both epithelial and heart tissue. In the heart, KCNQ1, in association with KCNE1, mediates the Iks current responsible for the repolarization of the cardiac action potential. Mutations that cause a loss-of-function of KCNQ1 result in a congenital condition known as longQT syndrome (LQTS). Congenital LQTS predisposes an individual to cardiac arrhythmia and can result in sudden death. While current homology models are sufficient to generate testable structure-function hypotheses, low sequence conservation between KCNQ1 and other potassium channel structures, especially in the voltage-sensing domain, highlight the need for an experimentally determined KCNQ1 structure. Our focus is on the KCNQ1 voltage-sensing domain (Q1-VSD) that mediates the voltage response of the ion channel. Isolated Q1VSDs exist in equilibrium between an open and closed state, likely favoring the open conformation. To facilitate nuclear magnetic resonance (NMR) structural investigation of the Q1-VSD, we employ state-locking mutations that provide a homogeneous reference spectrum. Our goal is to develop a biochemical model for the disease mechanism of specific LQTS mutations. The structural effects of specific LQTS disease-causing mutations are being evaluated in two ways (1) by comparison to the Q1-VSD ‘locked’ open- and closed-state NMR reference spectra and (2) in the context of current KCNQ1 homology models. Preliminary comparison of wild type Q1-VSD to a ‘locked’ closed-state construct by NMR in model membranes indicates a global conformational rearrangement. Ultimately we seek to leverage the closed-state stabilizing mutations to facilitate NMR structure determination of the Q1-VSD in its inactive ‘‘down’’ state. 1145-Pos Board B122 Energy Coupling Mechanisms and Lipid-Mediated Subunit Interactions of the Mitochondrial Protein Transport Machinery Nathan N. Alder1, Ketan Malhotra1, Murugappan Sathappa1, Shivangi Nangia1, Tyler Daman1, Dejana Mokranjac2, Eric May1. 1 Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA, 2 Physiological Chemistry, Ludwig-Maximilians-University of Munich, Munich, Germany. The TIM23 complex of the mitochondrial inner membrane mediates the translocation and integration of nuclear-encoded polypeptides that are targeted to the mitochondrion. Using a host of in organello and model membrane systems, we have analyzed the conformational alterations of key TIM23 subunits and site-specific interactions that occur between subunits during different steps of the transport process. First, using a fluorescence mapping strategy, we have analyzed the structural dynamics of the central voltage-gated channel-forming subunit, Tim23. We show dramatic alterations in the conformation of Tim23 transmembrane segments of the channel region that occur in a manner coupled to the magnitude of the proton-motive force across the inner membrane. Second, using a series of model membrane systems and reductionist approaches, we have analyzed site-specific interactions that occur between the Tim23 channel and the central receptor Tim50. Using Tim23 reconstituted into nanodiscs of different lipid composition, we identify the Tim50-interactive regions within the soluble and channel domains of Tim23. We find a critical role for cardiolipin, the signature anionic phospholipid of the mitochondrion, in promoting Tim23-Tim50 interactions. Using a combination of biochemical, mass spectrometry, and molecular dynamics approaches, we identify sites of the Tim50 receptor that bind to cardiolipin-containing lipid bilayers. The headgroup and acyl chain regions of cardiolipin that are critical in mediating Tim50 binding have been addressed using mitochondria from yeast strains defective in different steps of cardiolipin remodeling as well as model membranes of different lipid composition. Finally, we use small angle x-ray scattering (SAXS) to generate ab initio models of the Tim50 receptor domain and its lipid-interactive surface. These results illuminate the early receptormediated stages of protein translocation by the TIM23 complex and on the role of cardiolipin in promoting subunit interactions within the translocon.
Monday, February 29, 2016
Solid-state 2H NMR spectra were acquired for different tilt angles of the aligned samples. The methyl group orientations relative to the membrane normal were calculated by fitting the experimental 2H NMR spectra using a static uniaxial distribution [3] for the protein embedded within lipid bilayers. We found that the orientation of the C9-methyl group obtained from 2 H NMR spectroscopy was similar to that from X-ray data. By contrast, the orientations of the C5- and C13- methyl groups were different versus the X-ray crystal structures. The retinal structure was analyzed using the three-plane model, as in previous studies of rhodopsin in the dark and the Meta-I states [4]. Moreover, a new approach was tested that combines 2H NMR and X-ray restraints for retinal together with the rhodopsin binding-pocket using simulated annealing. Our results yield new insights into formation of the activated state of the receptor in lipid membranes. [1] A.V. Struts et al. (2011) Nat. Struct. Mol. Biol. 18, 392-394. [2] A.V. Struts et al. (2015) Meth. Mol. Biol. 1271, 133-158. [3] A.A. Nevzorov et al. (1999) JACS 121, 7636-7643. [4] G.F.J. Salgado et al. (2006) JACS 128, 11067-11071. 1142-Pos Board B119 Understanding Structural and Functional Stability of two Rhomboid Proteases: HiGlpG and PsAarA Rashmi Panigrahi1, Elena Arutyunova1, Pankaj Panwar2, Katharina Gimpl3, Sandro Keller3, Joanne Lemieux1. 1 University of Alberta, Edmonton, AB, Canada, 2University of British Columbia, Vancouver, BC, Canada, 3University of Kaiserslautern, Kaiserslautern, Germany. Functions of proteins critically rely on their conformation, but understanding conformational transitions in membrane proteins poses challenges because of their hydrophobic and aggregation-prone nature. In the current study, we focus on the two topologically unique intramembrane rhomboid proteases: HiGlpG and PsAarA, with an aim to understand the relationship between the extent of unfolding upon exposure to a chemical denaturant or temperature, and regain of native activity in the reassembled form. Whereas thermal denaturation has irreversible destabilizing effects on both proteins, unfolding induced by guanidinium chloride is completely reversible for HiGlpG but only partly reversible for PsAarA. For both rhomboid proteases, circular dichroism spectroscopy as a function of denaturant concentration reveals a broad, gradual transition from the native, folded state to the denatured, unfolded state, thus arguing against a classical two-state model as found for many globular soluble proteins. Upon removal of denaturant, protein refolding and dimer re-association result in regain of native-like activity, as confirmed by fluorescence-based enzyme kinetics. This concerted biophysical and functional analysis demonstrates that the simple 6TM organization of HiGlpG is more robust than the 6þ1TM organization of PsAarA. 1143-Pos Board B120 Influence of Familial Parkinson’s Disease Mutations on Mitochondrial Localization and Secondary Structure of PINK1 Stephania Irwin, Rashmi Panigrahi, Elena Arutyunova, Nicolas Touret, M. Joanne Lemieux. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada. Mutations in the PARK6 gene, encoding for the protein PINK1 (PhosphataseTensin homologue (PTEN)-induced protein kinase), are associated with early-onset familial Parkinson’s disease (PD). PINK1 is found in multiple cellular locations, including the cytosol and both mitochondrial membranes (outer mitochondrial membrane, OMM; and inner mitochondrial membrane, IMM). PINK1 must enter into the IMM in order to be cleaved by PARL (Presenilin-associated rhomboid-like), an IMM protease. Cleavage of PINK1 by PARL activates the degradation of PINK1, indicating a healthy, polarized mitochondrion. Improper processing of PINK1 PD mutants leads to unprocessed PINK1 accumulating on the OMM which induces mitophagy, or the selective autophagy of mitochondria. We hypothesize that accumulation of unprocessed PINK1 on the OMM is either due to improper localization of PINK1 or due to the inability of PARL to cleave the mutated protein. Many traditional protein localization techniques including confocal microscopy cannot distinguish between the inner and outer mitochondrial membrane. Super-resolution microscopy will be used to determine the precise localization patterns to the appropriate mitochondrial membrane of both wildtype PINK1 and PINK1 with point mutations associated to PD. Preliminary confocal microscopy determined that PINK1 constructs are capable of being translated in vivo in HeLa cells. These preliminary results have proved to be promising and further experiments will be conducted using superresolution microscopy and structured illumination microscopy to determine precise mitochondrial localization patterns in order to determine if PD muta-
tions lead to improper localization of PINK1. Molecular dynamics simulations on PINK1 transmembrane segments harbouring familial Parkinson’s disease mutations will be conducted to determine if localization defects are due to structural perturbations. This work sets the stage for an understanding for the role of PINK1 mutations in Parkinson’s disease at a molecular level. 1144-Pos Board B121 Structural Basis for KCNQ1 Long-QT Syndrome Disease causing Mutations Keenan C. Taylor, Hui Huang, Brett Kroncke, Charles Sanders. Biochemistry, Vanderbilt, Nashville, TN, USA. The human potassium channel KCNQ1 is a polytopic a-helical membrane protein. KCNQ1 is expressed in both epithelial and heart tissue. In the heart, KCNQ1, in association with KCNE1, mediates the Iks current responsible for the repolarization of the cardiac action potential. Mutations that cause a loss-of-function of KCNQ1 result in a congenital condition known as longQT syndrome (LQTS). Congenital LQTS predisposes an individual to cardiac arrhythmia and can result in sudden death. While current homology models are sufficient to generate testable structure-function hypotheses, low sequence conservation between KCNQ1 and other potassium channel structures, especially in the voltage-sensing domain, highlight the need for an experimentally determined KCNQ1 structure. Our focus is on the KCNQ1 voltage-sensing domain (Q1-VSD) that mediates the voltage response of the ion channel. Isolated Q1VSDs exist in equilibrium between an open and closed state, likely favoring the open conformation. To facilitate nuclear magnetic resonance (NMR) structural investigation of the Q1-VSD, we employ state-locking mutations that provide a homogeneous reference spectrum. Our goal is to develop a biochemical model for the disease mechanism of specific LQTS mutations. The structural effects of specific LQTS disease-causing mutations are being evaluated in two ways (1) by comparison to the Q1-VSD ‘locked’ open- and closed-state NMR reference spectra and (2) in the context of current KCNQ1 homology models. Preliminary comparison of wild type Q1-VSD to a ‘locked’ closed-state construct by NMR in model membranes indicates a global conformational rearrangement. Ultimately we seek to leverage the closed-state stabilizing mutations to facilitate NMR structure determination of the Q1-VSD in its inactive ‘‘down’’ state. 1145-Pos Board B122 Energy Coupling Mechanisms and Lipid-Mediated Subunit Interactions of the Mitochondrial Protein Transport Machinery Nathan N. Alder1, Ketan Malhotra1, Murugappan Sathappa1, Shivangi Nangia1, Tyler Daman1, Dejana Mokranjac2, Eric May1. 1 Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA, 2 Physiological Chemistry, Ludwig-Maximilians-University of Munich, Munich, Germany. The TIM23 complex of the mitochondrial inner membrane mediates the translocation and integration of nuclear-encoded polypeptides that are targeted to the mitochondrion. Using a host of in organello and model membrane systems, we have analyzed the conformational alterations of key TIM23 subunits and site-specific interactions that occur between subunits during different steps of the transport process. First, using a fluorescence mapping strategy, we have analyzed the structural dynamics of the central voltage-gated channel-forming subunit, Tim23. We show dramatic alterations in the conformation of Tim23 transmembrane segments of the channel region that occur in a manner coupled to the magnitude of the proton-motive force across the inner membrane. Second, using a series of model membrane systems and reductionist approaches, we have analyzed site-specific interactions that occur between the Tim23 channel and the central receptor Tim50. Using Tim23 reconstituted into nanodiscs of different lipid composition, we identify the Tim50-interactive regions within the soluble and channel domains of Tim23. We find a critical role for cardiolipin, the signature anionic phospholipid of the mitochondrion, in promoting Tim23-Tim50 interactions. Using a combination of biochemical, mass spectrometry, and molecular dynamics approaches, we identify sites of the Tim50 receptor that bind to cardiolipin-containing lipid bilayers. The headgroup and acyl chain regions of cardiolipin that are critical in mediating Tim50 binding have been addressed using mitochondria from yeast strains defective in different steps of cardiolipin remodeling as well as model membranes of different lipid composition. Finally, we use small angle x-ray scattering (SAXS) to generate ab initio models of the Tim50 receptor domain and its lipid-interactive surface. These results illuminate the early receptormediated stages of protein translocation by the TIM23 complex and on the role of cardiolipin in promoting subunit interactions within the translocon.