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Wonhwa Cho - One of the best experts on this subject based on the ideXlab platform.
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Genome-wide structural analysis reveals novel Membrane Binding properties of AP180 N-terminal homology (ANTH) domains.
The Journal of biological chemistry, 2011Co-Authors: Antonina Silkov, Robert V. Stahelin, Wonhwa Cho, Youngdae Yoon, Hunjoong Lee, Nikhil A. Gokhale, Emmanuel Adu-gyamfi, Diana MurrayAbstract:Abstract An increasing number of cytosolic proteins are shown to interact with Membrane lipids during diverse cellular processes, but computational prediction of these proteins and their Membrane Binding behaviors remains challenging. Here, we introduce a new combinatorial computation protocol for systematic and robust functional prediction of Membrane-Binding proteins through high throughput homology modeling and in-depth calculation of biophysical properties. The approach was applied to the genomic scale identification of the AP180 N-terminal homology (ANTH) domain, one of the modular lipid Binding domains, and prediction of their Membrane Binding properties. Our analysis yielded comprehensive coverage of the ANTH domain family and allowed classification and functional annotation of proteins based on the differences in local structural and biophysical features. Our analysis also identified a group of plant ANTH domains with unique structural features that may confer novel functionalities. Experimental characterization of a representative member of this subfamily confirmed its unique Membrane Binding mechanism and unprecedented Membrane deforming activity. Collectively, these studies suggest that our new computational approach can be applied to genome-wide functional prediction of other lipid Binding domains.
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Structural and Membrane Binding analysis of the Phox homology domain of Bem1p: basis of phosphatidylinositol 4-phosphate specificity.
The Journal of biological chemistry, 2007Co-Authors: Robert V. Stahelin, Diana Murray, Dimitrios Karathanassis, Roger Williams, Wonhwa ChoAbstract:Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and Membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of the diverse PI specificity of PX domains, we determined the crystal structure of the PX domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the Membrane Binding properties of the PX domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX domain contains a signature PI-Binding site optimized for PtdIns(4)P Binding and also harbors basic and hydrophobic residues on the Membrane-Binding surface. The Membrane Binding of the Bem1p PX domain is initiated by nonspecific electrostatic interactions between the cationic Membrane-Binding surface of the domain and anionic Membrane surfaces, followed by the Membrane penetration of hydrophobic residues. Unlike other PX domains, the Bem1p PX domain has high intrinsic Membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial Membrane penetration may occur before specific PtdIns(4)P Binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-Binding Bem1p PX domain provides new insight into the diverse PI specificities and Membrane-Binding mechanisms of PX domains.
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Structural Bioinformatics Prediction of Membrane-Binding Proteins
Journal of molecular biology, 2006Co-Authors: Nitin Bhardwaj, Robert V. Stahelin, Robert Langlois, Wonhwa ChoAbstract:Membrane-Binding peripheral proteins play important roles in many biological processes, including cell signaling and Membrane trafficking. Unlike integral Membrane proteins, these proteins bind the Membrane mostly in a reversible manner. Since peripheral proteins do not have canonical transMembrane segments, it is difficult to identify them from their amino acid sequences. As a first step toward genome-scale identification of Membrane-Binding peripheral proteins, we built a kernel-based machine learning protocol. Key features of known Membrane-Binding proteins, including electrostatic properties and amino acid composition, were calculated from their amino acid sequences and tertiary structures, which were then incorporated into the support vector machine to perform the classification. A data set of 40 Membrane-Binding proteins and 230 non-Membrane-Binding proteins was used to construct and validate the protocol. Cross-validation and holdout evaluation of the protocol showed that the accuracy of the prediction reached up to 93.7% and 91.6%, respectively. The protocol was applied to the prediction of Membrane-Binding properties of four C2 domains from novel protein kinases C. Although these C2 domains have 50% sequence identity, only one of them was predicted to bind the Membrane, which was verified experimentally with surface plasmon resonance analysis. These results suggest that our protocol can be used for predicting Membrane-Binding properties of a wide variety of modular domains and may be further extended to genome-scale identification of Membrane-Binding peripheral proteins.
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Membrane-Binding and activation mechanism of PTEN.
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Sudipto Das, Jack E. Dixon, Wonhwa ChoAbstract:PTEN is a tumor suppressor that reverses the action of phosphoinositide 3-kinase by catalyzing the removal of the 3' phosphate of phosphoinositides. Despite the critical role of PTEN in cell signaling and regulation, the mechanisms of its Membrane recruitment and activation is still poorly understood. PTEN is composed of an N-terminal phosphatase domain, a C2 domain, and a C-terminal tail region that contains the PSD-95/Dlg/ZO-1 homology (PDZ) domain-Binding sequence and multiple phosphorylation sites. Our in vitro surface plasmon resonance measurements using immobilized vesicles showed that both the phosphatase domain and the C2 domain, but not the C-terminal tail, are involved in electrostatic Membrane Binding of PTEN. Furthermore, the phosphorylation-mimicking mutation on the C-terminal tail of PTEN caused an approximately 80-fold reduction in its Membrane affinity, mainly by slowing the Membrane-association step. Subcellular localization studies of PTEN transfected into HEK293T and HeLa cells indicated that targeting of PTEN to the plasma Membrane is coupled with rapid degradation and that the phosphatase domain and the C2 domain are both necessary and sufficient for its Membrane recruitment. Results also indicated that the phosphorylation regulates the targeting of PTEN to the plasma Membrane not by blocking the PDZ domain-Binding site but by interfering with electrostatic Membrane Binding of PTEN. On the basis of these results, we propose a Membrane-Binding and activation mechanism for PTEN, in which the phosphorylation/dephosphorylation of the C-terminal region serves as an electrostatic switch that controls the Membrane translocation of the protein.
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Roles of calcium ions in the Membrane Binding of C2 domains
Biochemical Journal, 2001Co-Authors: Robert V. Stahelin, Wonhwa ChoAbstract:The C2 domain is a Membrane-targeting domain found in many cellular proteins involved in signal transduction or Membrane trafficking. The majority of C2 domains co-ordinate multiple Ca2+ ions and bind the Membrane in a Ca2+-dependent manner. To understand the mechanisms by which Ca2+ mediates the Membrane Binding of C2 domains, we measured the Membrane Binding of the C2 domains of group IV cytosolic phospholipase A2 (cPLA2) and protein kinase C-α (PKC-α) by surface plasmon resonance and lipid monolayer analyses. Ca2+ ions mainly slow the Membrane dissociation of cPLA2-C2, while modulating both Membrane association and dissociation rates for PKC-α-C2. Further studies with selected mutants showed that for cPLA2 a Ca2+ ion bound to the C2 domain of cPLA2 induces the intra-domain conformational change that leads to the Membrane penetration of the C2 domain whereas the other Ca2+ is not directly involved in Membrane Binding. For PKC-α, a Ca2+ ion induces the inter-domain conformational changes of the protein and the Membrane penetration of non-C2 residues. The other Ca2+ ion of PKC-α-C2 is involved in more complex interactions with the Membrane, including both non-specific and specific electrostatic interactions. Together, these studies of isolated C2 domains and their parent proteins allow for the determination of the distinct and specific roles of each Ca2+ ion bound to different C2 domains.
Eric O. Freed - One of the best experts on this subject based on the ideXlab platform.
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depletion of cellular cholesterol inhibits Membrane Binding and higher order multimerization of human immunodeficiency virus type 1 gag
Virology, 2007Co-Authors: Akira Ono, Abdul A Waheed, Eric O. FreedAbstract:Recent studies have suggested that the plasma Membrane contains cholesterol-enriched microdomains known as lipid rafts. HIV-1 Gag binds raft-rich regions of the plasma Membrane, and cholesterol depletion impairs HIV-1 particle production. In this study, we sought to define the block imposed by cholesterol depletion. We observed that Membrane Binding and higher-order multimerization of Gag were markedly reduced upon cholesterol depletion. Fusing to Gag a highly efficient, heterologous Membrane-Binding sequence reversed the defects in Gag-Membrane Binding and multimerization caused by cholesterol depletion, indicating that the impact of reducing the Membrane cholesterol content on Gag-Membrane Binding and multimerization can be circumvented by increasing the affinity of Gag for Membrane. Virus release efficiency of this Gag derivative was minimally affected by cholesterol depletion. Altogether, these results are consistent with the hypothesis that cholesterol-enriched Membrane microdomains promote HIV-1 particle production by facilitating both Gag-Membrane Binding and Gag multimerization.
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Relationship between human immunodeficiency virus type 1 Gag multimerization and Membrane Binding.
Journal of virology, 2000Co-Authors: Akira Ono, Dimiter G. Demirov, Eric O. FreedAbstract:The human immunodeficiency virus type 1 (HIV-1) Gag precursor, Pr55Gag, is necessary and sufficient for the assembly and release of viruslike particles. Binding of Gag to Membrane and Gag multimerization are both essential steps in virus assembly, yet the domains responsible for these events have not been fully defined. In addition, the relationship between Membrane Binding and Gag-Gag interaction remains to be elucidated. To investigate these issues, we analyzed, in vivo, the Membrane-Binding and assembly properties of a series of C-terminally truncated Gag mutants. Pr55Gag was truncated at the C terminus of matrix (MAstop), between the N- and C-terminal domains of capsid (CA146stop), at the C terminus of capsid (p41stop), at the C terminus of p2 (p43stop), and after the N-terminal 35 amino acids of nucleocapsid (NC35stop). The ability of these truncated Gag molecules to assemble and release viruslike particles and their capacity to copackage into particles when coexpressed with full-length Gag were determined. We demonstrate that the amount of truncated Gag incorporated into particles is incrementally increased by extension from CA146 to NC35, suggesting that multiple sites in this region are involved in Gag multimerization. Using Membrane flotation centrifugation, we observe that MA shows significantly reduced Membrane Binding relative to full-length Gag but that CA146 displays steady-state Membrane-Binding properties comparable to those of Pr55Gag. The finding that the CA146 mutant, which contains only matrix and the N-terminal domain of capsid, exhibits levels of steady-state Membrane Binding equivalent to those of full-length Gag indicates that strong Gag-Gag interaction domains are not required for the efficient Binding of HIV-1 Gag to Membrane.
Gregor Anderluh - One of the best experts on this subject based on the ideXlab platform.
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Membrane Binding of zebrafish actinoporin-like protein: AF domains, a novel superfamily of cell Membrane Binding domains
The Biochemical journal, 2006Co-Authors: Ion Gutiérrez-aguirre, Peter Trontelj, Peter Macek, Jeremy H. Lakey, Gregor AnderluhAbstract:Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing Membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen-Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the Membrane Binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays Membrane-Binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of Membrane Binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).
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Membrane Binding of zebrafish actinoporin-like protein: AF domains, a novel superfamily of cell Membrane Binding domains
Biochemical Journal, 2006Co-Authors: Ion Gutiérrez-aguirre, Peter Trontelj, Peter Macek, Jeremy H. Lakey, Gregor AnderluhAbstract:Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing Membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomson-Friedenreich antigen. In this study we found a number of sequences in public databases similar to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the Membrane Binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in E. coli. It displays Membrane-Binding behaviour, but does not have permeabilising activity nor sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit body lectin family comprise a novel superfamily of Membrane Binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).
Robert V. Stahelin - One of the best experts on this subject based on the ideXlab platform.
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Genome-wide structural analysis reveals novel Membrane Binding properties of AP180 N-terminal homology (ANTH) domains.
The Journal of biological chemistry, 2011Co-Authors: Antonina Silkov, Robert V. Stahelin, Wonhwa Cho, Youngdae Yoon, Hunjoong Lee, Nikhil A. Gokhale, Emmanuel Adu-gyamfi, Diana MurrayAbstract:Abstract An increasing number of cytosolic proteins are shown to interact with Membrane lipids during diverse cellular processes, but computational prediction of these proteins and their Membrane Binding behaviors remains challenging. Here, we introduce a new combinatorial computation protocol for systematic and robust functional prediction of Membrane-Binding proteins through high throughput homology modeling and in-depth calculation of biophysical properties. The approach was applied to the genomic scale identification of the AP180 N-terminal homology (ANTH) domain, one of the modular lipid Binding domains, and prediction of their Membrane Binding properties. Our analysis yielded comprehensive coverage of the ANTH domain family and allowed classification and functional annotation of proteins based on the differences in local structural and biophysical features. Our analysis also identified a group of plant ANTH domains with unique structural features that may confer novel functionalities. Experimental characterization of a representative member of this subfamily confirmed its unique Membrane Binding mechanism and unprecedented Membrane deforming activity. Collectively, these studies suggest that our new computational approach can be applied to genome-wide functional prediction of other lipid Binding domains.
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Structural and Membrane Binding analysis of the Phox homology domain of Bem1p: basis of phosphatidylinositol 4-phosphate specificity.
The Journal of biological chemistry, 2007Co-Authors: Robert V. Stahelin, Diana Murray, Dimitrios Karathanassis, Roger Williams, Wonhwa ChoAbstract:Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and Membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of the diverse PI specificity of PX domains, we determined the crystal structure of the PX domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the Membrane Binding properties of the PX domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX domain contains a signature PI-Binding site optimized for PtdIns(4)P Binding and also harbors basic and hydrophobic residues on the Membrane-Binding surface. The Membrane Binding of the Bem1p PX domain is initiated by nonspecific electrostatic interactions between the cationic Membrane-Binding surface of the domain and anionic Membrane surfaces, followed by the Membrane penetration of hydrophobic residues. Unlike other PX domains, the Bem1p PX domain has high intrinsic Membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial Membrane penetration may occur before specific PtdIns(4)P Binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-Binding Bem1p PX domain provides new insight into the diverse PI specificities and Membrane-Binding mechanisms of PX domains.
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Membrane Binding and subcellular targeting of c2 domains
Biochimica et Biophysica Acta, 2006Co-Authors: Robert V. StahelinAbstract:C2 domains are a ubiquitous structural module and many of them function in Ca 2+ -dependent Membrane Binding and thereby serve as Ca 2+ effectors for divergent Ca 2+ -mediated cellular processes. Extensive structural, biochemical, biophysical, and cellular studies of C2 domains and host proteins in the past decade have shown that due to their structural diversity C2 domains have disparate Ca 2+ sensitivity, lipid selectivity and Membrane Binding mechanisms. This review summarizes the basic structural and functional properties of C2 domains as well as recent findings on Ca 2+ and Membrane Binding, lipid selectivity, and subcellular localization of C2 domains and their host proteins.
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Structural Bioinformatics Prediction of Membrane-Binding Proteins
Journal of molecular biology, 2006Co-Authors: Nitin Bhardwaj, Robert V. Stahelin, Robert Langlois, Wonhwa ChoAbstract:Membrane-Binding peripheral proteins play important roles in many biological processes, including cell signaling and Membrane trafficking. Unlike integral Membrane proteins, these proteins bind the Membrane mostly in a reversible manner. Since peripheral proteins do not have canonical transMembrane segments, it is difficult to identify them from their amino acid sequences. As a first step toward genome-scale identification of Membrane-Binding peripheral proteins, we built a kernel-based machine learning protocol. Key features of known Membrane-Binding proteins, including electrostatic properties and amino acid composition, were calculated from their amino acid sequences and tertiary structures, which were then incorporated into the support vector machine to perform the classification. A data set of 40 Membrane-Binding proteins and 230 non-Membrane-Binding proteins was used to construct and validate the protocol. Cross-validation and holdout evaluation of the protocol showed that the accuracy of the prediction reached up to 93.7% and 91.6%, respectively. The protocol was applied to the prediction of Membrane-Binding properties of four C2 domains from novel protein kinases C. Although these C2 domains have 50% sequence identity, only one of them was predicted to bind the Membrane, which was verified experimentally with surface plasmon resonance analysis. These results suggest that our protocol can be used for predicting Membrane-Binding properties of a wide variety of modular domains and may be further extended to genome-scale identification of Membrane-Binding peripheral proteins.
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Roles of calcium ions in the Membrane Binding of C2 domains
Biochemical Journal, 2001Co-Authors: Robert V. Stahelin, Wonhwa ChoAbstract:The C2 domain is a Membrane-targeting domain found in many cellular proteins involved in signal transduction or Membrane trafficking. The majority of C2 domains co-ordinate multiple Ca2+ ions and bind the Membrane in a Ca2+-dependent manner. To understand the mechanisms by which Ca2+ mediates the Membrane Binding of C2 domains, we measured the Membrane Binding of the C2 domains of group IV cytosolic phospholipase A2 (cPLA2) and protein kinase C-α (PKC-α) by surface plasmon resonance and lipid monolayer analyses. Ca2+ ions mainly slow the Membrane dissociation of cPLA2-C2, while modulating both Membrane association and dissociation rates for PKC-α-C2. Further studies with selected mutants showed that for cPLA2 a Ca2+ ion bound to the C2 domain of cPLA2 induces the intra-domain conformational change that leads to the Membrane penetration of the C2 domain whereas the other Ca2+ is not directly involved in Membrane Binding. For PKC-α, a Ca2+ ion induces the inter-domain conformational changes of the protein and the Membrane penetration of non-C2 residues. The other Ca2+ ion of PKC-α-C2 is involved in more complex interactions with the Membrane, including both non-specific and specific electrostatic interactions. Together, these studies of isolated C2 domains and their parent proteins allow for the determination of the distinct and specific roles of each Ca2+ ion bound to different C2 domains.
M D Resh - One of the best experts on this subject based on the ideXlab platform.
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Differential Membrane Binding of the human immunodeficiency virus type 1 matrix protein.
Journal of virology, 1996Co-Authors: Wenjun Zhou, M D ReshAbstract:The human immunodeficiency virus type 1 matrix protein (p17MA) plays a central role at both the early and late stages of the virus life cycle. During viral assembly, the p17MA domain of Pr55gag promotes Membrane association, which is essential for the formation of viral particles. When viral infection occurs, the mature p17MA dissociates from the plasma Membrane and participates in the nuclear targeting process. Thus, p17MA contains a reversible Membrane Binding signal to govern its differential subcellular localization and biological functions. We previously identified a Membrane Binding signal within the amino-terminal 31 amino acids of the matrix domain of human immunodeficiency virus type 1 Gag, consisting of myristate and a highly basic region (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). Here we show that exposure of this Membrane Binding signal is regulated in different Gag protein contexts. Within full-length Pr55gag, the Membrane targeting signal is exposed and can direct Pr55gag as well as heterologous proteins to the plasma Membrane. However, in the context of p17MA alone, this signal is hidden and unable to confer plasma Membrane Binding. To investigate the molecular mechanism for regulation of Membrane Binding, a series of deletions within p17MA was generated by sequentially removing alpha-helical regions defined by the nuclear magnetic resonance structure. Removal of the last alpha helix (amino acids 97 to 109) of p17MA was associated with enhancement of Binding to biological Membranes in vitro and in vivo. Liposome Binding experiments indicated that the C-terminal region of p17MA exerts a negative effect on the N-terminal MA Membrane targeting domain by sequestering the myristate signal. We propose that mature p17MA adopts a conformation different from that of the p17MA domain within Pr55gag and present evidence to support this hypothesis. It is likely that such a conformational change results in an N-terminal myristyl switch which governs differential Membrane Binding.
