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Bip Binding Affects Integration of Transmembrane Domains
500a
Wednesday, February 15, 2017
of Alzheimer’s disease (AD) plaques in the brain. Three consecutive GXXXG motifs and one GXXXA motif on the transmembrane domain (TM) of APP have been reported to be one or two important sites for mediating APP homodimerization in different experimental conditions, the difference are likely due to the membrane mimetic. We and several other groups recently demonstrated the feasibility to directly characterize recombinant proteins in native Escherichia coli (E. coli) membranes using magic-angle spinning (MAS) solid-state nuclear magnetic resonance (ssNMR)(2, 3). Here, we will present several strategies to further improve spectral sensitivity and resolution, and suppress background signals of E. coli proteins and lipids, including preparation of E. coli inner membranes, reverse 13C- and 15N-isotope labeling, and selective signal filtering with the frequency-selective REDOR pulse sequence. These improvements have allowed us to characterize the structure and dimerization of APP TM and its variants in a native membrane environment. 2460-Pos Board B67 Structural and Dynamical Basis of Protein Kinase C Alpha Regulation by the C-Terminal Tail Yuan Yang1, Julia A. Callender2, Alexandra C. Newton2, Tatyana I. Igumenova1. 1 Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA, 2Department of Pharmacology, University of California at San Diego, La Jolla, CA, USA. Protein Kinase C (PKC) isoenzymes are dynamic multi-modular proteins that regulate signal transduction processes at the membrane surface. When activated by second messengers, such as Ca2þ and diacylglycerol, PKC undergoes a drastic conformational transition from the inactive cytosolic state to the activated membrane-bound state. The structure of either state of PKC is not known. Using NMR, we demonstrate that the isolated Ca2þ-sensing lipid-binding C2 domain of PKCa interacts with the C-terminal tail of the kinase. We determined the structure of the complex between the C2 domain and the peptide corresponding to the phosphorylated hydrophobic motif of the C-terminal tail. The structure shows that the hydrophobic motif binds to the conserved lysine-rich cluster region of C2. The interface is stabilized by electrostatic interactions and stacking of the aromatic sidechains. We validated our structural model by mutating the interacting residues in full-length PKC, and characterizing these variants using in vitro FRET experiments and FRET-detected membrane translocation experiments in live cells. In addition, NMR-detected binding studies revealed that the hydrophobic motif and Ca2þ synergistically enhance each other’s affinities to C2. We propose a model where the C-terminal tail plays a dual role in PKC regulation: auto-inhibitory, through its interaction with the lysine-rich cluster of the C2 domain; and activating, through sensitization of PKC to intracellular Ca2þ oscillations. Support: Welch Foundation grant A-1784 (TII), NSF CAREER award CHE-1151435 (TII), NIH GM108998 (TII), and NIH GM43154 (ACN). 2461-Pos Board B68 Site-Directed Spin Labeling EPR Spectroscopy of the Cytoplasmic Tail of Influenza a M2 Alice L. Herneisen, Grace Kim, Kathleen P. Howard. Swarthmore College, Swarthmore, PA, USA. The M2 protein is a 97 residue homotetrameric, multifunctional ion channel that plays critical roles during the influenza infection cycle. While a variety of high-resolution biophysical techniques have been used to characterize the transmembrane domain (residues 22-46) and the juxtamembrane C-terminal region (46-62), less is known about the conformation and dynamics of the remaining residues of the C-terminal cytoplasmic tail. Here, we use site-directed spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to probe the secondary structure and membrane topology of cytoplasmic tail residues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is essential for the role the C-terminal domain of the M2 protein plays in viral budding. SDSL-EPR data is collected in both in the presence and absence of cholesterol. 2462-Pos Board B69 Polarity and Charge as Determinants for Translocase Requirement for Membrane Protein Insertion Balasubramani Hariharan1, Raunak Soman2, Ross E. Dalbey3. 1 Biophysics Graduate program, The Ohio State University, Columbus, OH, USA, 2OSBP, The Ohio State University, Columbus, OH, USA, 3The Ohio State University, Columbus, OH, USA. Membrane protein biogenesis in bacteria is a well-studied but poorly understood area in the field of membrane proteins. In bacteria, the Sec translocon and the YidC insertase promote the membrane insertion process. Both proteins are essential, and universally conserved, and together insert about 95% of the proteins in E.coli. Membrane insertion of SecYEG-
dependent proteins likely occurs at the lateral gate of SecY, the channelforming subunit of the bacterial Sec translocon. On the other hand, YidC catalyzes insertion by possessing a hydrophilic groove, which can accommodate the hydrophilic region of the substrate that inserts across the membrane. Recently, Soman et al (2014) have reported that increasing the charge of the translocated region of procoat can route substrates from the YidC pathway into the Sec pathway. Here, we have further tested this polarity/charge hypothesis by showing that the major coat protein of bacteriophage M13 can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of charged residues. We also show that adding hydrophobic amino acids to highly polar or charged loop can decrease the Secdependence of the otherwise strictly Sec-dependent substrates. Additionally we demonstrate that increasing the driving force of insertion by adding four leucyl residues to the transmembrane segment leads to translocation of a highly charged region that was not inserted by native TM segment. Finally, we show that the length of procoat loop is a determinant for Sec dependent insertion. The combined results support the polarity/charge hypothesis and is consistent with the notion that membrane insertion occurs at the interface of SecYEG and YidC. 2463-Pos Board B70 Bip Binding Affects Integration of Transmembrane Domains Mirjam Zimmermann1,2, Marco Janoschke2, Martin Spiess2. 1 Molecular and Membrane Biophysics, Institute of Biophysics, Linz, Austria, 2 Biozentrum, Basel, Switzerland. The Sec61 translocon mediates translocation of proteins into the endoplasmic reticulum (ER) and enables integration of hydrophobic (H) segments into the lipid bilayer via its lateral gate. The luminal chaperone BiP (Kar2 in yeast) has been shown to act as a molecular ratchet, recruited to the emerging polypeptide by the J-domain of Sec63, and to be involved in post- and cotranslational translocation. In this study, we address the question whether BiP binding to the nascent chain affects the process of membrane integration vs. translocation, i.e. whether integration is governed by thermodynamic equilibrium between pore and membrane. To determine the hydrophobicity threshold of membrane integration, a series of model proteins based on the sequence of dipeptidylaminopeptidase B (DPAPB) with oligo-alanine H-segments containing increasing numbers of leucines. The translocated loop preceding the H-segments was replaced by a sequence of similar length containing multiple copies of segments known to bind BiP with high affinity or not to interact with BiP. While H-segments preceded by the DPAPB sequence required ~3.7 Leu for 50% integration, this threshold was reduced to ~2.1 Leu with the BiP-binding sequence and increased to ~5.2 Leu with the non-binding sequence. These results show that membrane integration of an H-segment is influenced by the already translocated sequence preceding it and suggest that BiP binding promotes membrane integration. We observed changes of the apparent free energy in integration between 1 to 3 kcal/mol. We propose a model in which Brownian motion of the translocated loop with bound chaperones preferentially promotes release of the H-segment into the lipid bilayer. The work was supported by grant W0125 of the Austrian science fund (FWF). 2464-Pos Board B71 Elucidating the Uncoupling of ATP Hydrolysis and Ca2D Transport in SERCA by Sarcolipin Erin Birdsall1, Alysha Dicke1, Gianluigi Veglia1,2. 1 BMBB, University of Minnesota, Minneapolis, MN, USA, 2Chemistry, University of Minnesota, Minneapolis, MN, USA. The activity of the sarco(endo)plasmic reticulum Ca2þ -ATPase (SERCA), which plays a key role in muscle contraction and relaxation, is regulated by sarcolipin (SLN), a 31-residue transmembrane peptide. Sarcolipin has been shown to uncouple ATP hydrolysis and Ca2þ transport in SERCA and is largely found in skeletal muscle and the atria of the heart. The interaction between SLN and SERCA is thought to play an important role in energy metabolism. SLN is composed of a transmembrane helix flanked by two short, unstructured Nand C-termini. The N-terminus is hypothesized to play a role in the uncoupling of SERCA, while the role of the C-terminus has been shown to be largely responsible for SERCA inhibition. In this study, we made two constructs of SLN, with either the C- or N-terminus deleted. We then used coupledenzyme activity assays to measure the effect of the SLN constructs on SERCA ATPase activity and isothermal titration calorimetry (ITC) to monitor the change in heat release due to ATP hydrolysis. We found through these methods that the deletion of the termini led effected the uncoupling function in SLN. The results of this study, in combination with forthcoming solid-state NMR studies, will help to establish the mechanism by which SLN regulates SERCA activity.
Wednesday, February 15, 2017
of Alzheimer’s disease (AD) plaques in the brain. Three consecutive GXXXG motifs and one GXXXA motif on the transmembrane domain (TM) of APP have been reported to be one or two important sites for mediating APP homodimerization in different experimental conditions, the difference are likely due to the membrane mimetic. We and several other groups recently demonstrated the feasibility to directly characterize recombinant proteins in native Escherichia coli (E. coli) membranes using magic-angle spinning (MAS) solid-state nuclear magnetic resonance (ssNMR)(2, 3). Here, we will present several strategies to further improve spectral sensitivity and resolution, and suppress background signals of E. coli proteins and lipids, including preparation of E. coli inner membranes, reverse 13C- and 15N-isotope labeling, and selective signal filtering with the frequency-selective REDOR pulse sequence. These improvements have allowed us to characterize the structure and dimerization of APP TM and its variants in a native membrane environment. 2460-Pos Board B67 Structural and Dynamical Basis of Protein Kinase C Alpha Regulation by the C-Terminal Tail Yuan Yang1, Julia A. Callender2, Alexandra C. Newton2, Tatyana I. Igumenova1. 1 Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA, 2Department of Pharmacology, University of California at San Diego, La Jolla, CA, USA. Protein Kinase C (PKC) isoenzymes are dynamic multi-modular proteins that regulate signal transduction processes at the membrane surface. When activated by second messengers, such as Ca2þ and diacylglycerol, PKC undergoes a drastic conformational transition from the inactive cytosolic state to the activated membrane-bound state. The structure of either state of PKC is not known. Using NMR, we demonstrate that the isolated Ca2þ-sensing lipid-binding C2 domain of PKCa interacts with the C-terminal tail of the kinase. We determined the structure of the complex between the C2 domain and the peptide corresponding to the phosphorylated hydrophobic motif of the C-terminal tail. The structure shows that the hydrophobic motif binds to the conserved lysine-rich cluster region of C2. The interface is stabilized by electrostatic interactions and stacking of the aromatic sidechains. We validated our structural model by mutating the interacting residues in full-length PKC, and characterizing these variants using in vitro FRET experiments and FRET-detected membrane translocation experiments in live cells. In addition, NMR-detected binding studies revealed that the hydrophobic motif and Ca2þ synergistically enhance each other’s affinities to C2. We propose a model where the C-terminal tail plays a dual role in PKC regulation: auto-inhibitory, through its interaction with the lysine-rich cluster of the C2 domain; and activating, through sensitization of PKC to intracellular Ca2þ oscillations. Support: Welch Foundation grant A-1784 (TII), NSF CAREER award CHE-1151435 (TII), NIH GM108998 (TII), and NIH GM43154 (ACN). 2461-Pos Board B68 Site-Directed Spin Labeling EPR Spectroscopy of the Cytoplasmic Tail of Influenza a M2 Alice L. Herneisen, Grace Kim, Kathleen P. Howard. Swarthmore College, Swarthmore, PA, USA. The M2 protein is a 97 residue homotetrameric, multifunctional ion channel that plays critical roles during the influenza infection cycle. While a variety of high-resolution biophysical techniques have been used to characterize the transmembrane domain (residues 22-46) and the juxtamembrane C-terminal region (46-62), less is known about the conformation and dynamics of the remaining residues of the C-terminal cytoplasmic tail. Here, we use site-directed spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to probe the secondary structure and membrane topology of cytoplasmic tail residues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is essential for the role the C-terminal domain of the M2 protein plays in viral budding. SDSL-EPR data is collected in both in the presence and absence of cholesterol. 2462-Pos Board B69 Polarity and Charge as Determinants for Translocase Requirement for Membrane Protein Insertion Balasubramani Hariharan1, Raunak Soman2, Ross E. Dalbey3. 1 Biophysics Graduate program, The Ohio State University, Columbus, OH, USA, 2OSBP, The Ohio State University, Columbus, OH, USA, 3The Ohio State University, Columbus, OH, USA. Membrane protein biogenesis in bacteria is a well-studied but poorly understood area in the field of membrane proteins. In bacteria, the Sec translocon and the YidC insertase promote the membrane insertion process. Both proteins are essential, and universally conserved, and together insert about 95% of the proteins in E.coli. Membrane insertion of SecYEG-
dependent proteins likely occurs at the lateral gate of SecY, the channelforming subunit of the bacterial Sec translocon. On the other hand, YidC catalyzes insertion by possessing a hydrophilic groove, which can accommodate the hydrophilic region of the substrate that inserts across the membrane. Recently, Soman et al (2014) have reported that increasing the charge of the translocated region of procoat can route substrates from the YidC pathway into the Sec pathway. Here, we have further tested this polarity/charge hypothesis by showing that the major coat protein of bacteriophage M13 can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of charged residues. We also show that adding hydrophobic amino acids to highly polar or charged loop can decrease the Secdependence of the otherwise strictly Sec-dependent substrates. Additionally we demonstrate that increasing the driving force of insertion by adding four leucyl residues to the transmembrane segment leads to translocation of a highly charged region that was not inserted by native TM segment. Finally, we show that the length of procoat loop is a determinant for Sec dependent insertion. The combined results support the polarity/charge hypothesis and is consistent with the notion that membrane insertion occurs at the interface of SecYEG and YidC. 2463-Pos Board B70 Bip Binding Affects Integration of Transmembrane Domains Mirjam Zimmermann1,2, Marco Janoschke2, Martin Spiess2. 1 Molecular and Membrane Biophysics, Institute of Biophysics, Linz, Austria, 2 Biozentrum, Basel, Switzerland. The Sec61 translocon mediates translocation of proteins into the endoplasmic reticulum (ER) and enables integration of hydrophobic (H) segments into the lipid bilayer via its lateral gate. The luminal chaperone BiP (Kar2 in yeast) has been shown to act as a molecular ratchet, recruited to the emerging polypeptide by the J-domain of Sec63, and to be involved in post- and cotranslational translocation. In this study, we address the question whether BiP binding to the nascent chain affects the process of membrane integration vs. translocation, i.e. whether integration is governed by thermodynamic equilibrium between pore and membrane. To determine the hydrophobicity threshold of membrane integration, a series of model proteins based on the sequence of dipeptidylaminopeptidase B (DPAPB) with oligo-alanine H-segments containing increasing numbers of leucines. The translocated loop preceding the H-segments was replaced by a sequence of similar length containing multiple copies of segments known to bind BiP with high affinity or not to interact with BiP. While H-segments preceded by the DPAPB sequence required ~3.7 Leu for 50% integration, this threshold was reduced to ~2.1 Leu with the BiP-binding sequence and increased to ~5.2 Leu with the non-binding sequence. These results show that membrane integration of an H-segment is influenced by the already translocated sequence preceding it and suggest that BiP binding promotes membrane integration. We observed changes of the apparent free energy in integration between 1 to 3 kcal/mol. We propose a model in which Brownian motion of the translocated loop with bound chaperones preferentially promotes release of the H-segment into the lipid bilayer. The work was supported by grant W0125 of the Austrian science fund (FWF). 2464-Pos Board B71 Elucidating the Uncoupling of ATP Hydrolysis and Ca2D Transport in SERCA by Sarcolipin Erin Birdsall1, Alysha Dicke1, Gianluigi Veglia1,2. 1 BMBB, University of Minnesota, Minneapolis, MN, USA, 2Chemistry, University of Minnesota, Minneapolis, MN, USA. The activity of the sarco(endo)plasmic reticulum Ca2þ -ATPase (SERCA), which plays a key role in muscle contraction and relaxation, is regulated by sarcolipin (SLN), a 31-residue transmembrane peptide. Sarcolipin has been shown to uncouple ATP hydrolysis and Ca2þ transport in SERCA and is largely found in skeletal muscle and the atria of the heart. The interaction between SLN and SERCA is thought to play an important role in energy metabolism. SLN is composed of a transmembrane helix flanked by two short, unstructured Nand C-termini. The N-terminus is hypothesized to play a role in the uncoupling of SERCA, while the role of the C-terminus has been shown to be largely responsible for SERCA inhibition. In this study, we made two constructs of SLN, with either the C- or N-terminus deleted. We then used coupledenzyme activity assays to measure the effect of the SLN constructs on SERCA ATPase activity and isothermal titration calorimetry (ITC) to monitor the change in heat release due to ATP hydrolysis. We found through these methods that the deletion of the termini led effected the uncoupling function in SLN. The results of this study, in combination with forthcoming solid-state NMR studies, will help to establish the mechanism by which SLN regulates SERCA activity.