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Petri Kursula - One of the best experts on this subject based on the ideXlab platform.
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cryo em x ray diffraction and atomistic simulations reveal determinants for the formation of a supramolecular myelin like proteolipid lattice
Journal of Biological Chemistry, 2020Co-Authors: Salla Ruskamo, Tuomo Nieminen, Arne Raasakka, Ilpo Vattulainen, M. Lehtimaki, Oda C Krokengen, Julia Kowal, Venkata P Dandey, Henning Stahlberg, Petri KursulaAbstract:Myelin Protein P2 is a peripheral membrane Protein of the fatty acid–binding Protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot-Marie-Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered three-dimensional (3D) lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ∼3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral Protein organization, and surface mutagenesis of P2 coupled with structure-function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg-88 is critical for P2-membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 β-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane Protein from human myelin. This is an important step toward deciphering the 3D assembly of a mature myelin sheath at the molecular level.
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determinants for forming a supramolecular myelin like proteolipid lattice
bioRxiv, 2020Co-Authors: Salla Ruskamo, Tuomo Nieminen, Arne Raasakka, Ilpo Vattulainen, M. Lehtimaki, Oda C Krokengen, Julia Kowal, Venkata P Dandey, Henning Stahlberg, Petri KursulaAbstract:Myelin Protein P2 is a peripheral membrane Protein of the fatty acid binding Protein family. It functions in the formation and maintenance of the peripheral nerve myelin sheath, and several P2 mutations causing human Charot-Marie-Tooth neuropathy have been reported. Here, electron cryomicroscopy of myelin-like proteolipid multilayers revealed a three-dimensionally ordered lattice of P2 molecules between stacked lipid bilayers, visualizing its possible assembly at the myelin major dense line. A single layer of P2 is inserted between two bilayers in a tight intermembrane space of ~3 nm, implying direct interactions between P2 and two membrane surfaces. Further details on lateral Protein organization were revealed through X-ray diffraction from bicelles stacked by P2. Surface mutagenesis of P2 coupled to structural and functional experiments revealed a role for both the portal region and the opposite face of P2 in membrane interactions. Atomistic molecular dynamics simulations of P2 on myelin-like and model membrane surfaces suggested that Arg88 is an important residue for P2-membrane interactions, in addition to the helical lid domain on the opposite face of the molecule. Negatively charged myelin lipid headgroups anchor P2 stably on the bilayer surface. Membrane binding may be accompanied by opening of the P2 β barrel structure and ligand exchange with the apposing lipid bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane Protein from human myelin. This is an important step towards deciphering the 3-dimensional assembly of a mature myelin sheath at the molecular level.
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Production, crystallization and neutron diffraction of fully deuterated human myelin peripheral membrane Protein P2.
Acta crystallographica. Section F Structural biology communications, 2015Co-Authors: Saara Laulumaa, Arne Raasakka, Matthew P Blakeley, Martine Moulin, Michael Härtlein, Petri KursulaAbstract:The molecular details of the formation of the myelin sheath, a multilayered membrane in the nervous system, are to a large extent unknown. P2 is a peripheral membrane Protein from peripheral nervous system myelin, which is believed to play a role in this process. X-ray crystallographic studies and complementary experiments have provided information on the structure-function relationships in P2. In this study, a fully deuterated sample of human P2 was produced. Crystals that were large enough for neutron diffraction were grown by a ten-month procedure of feeding, and neutron diffraction data were collected to a resolution of 2.4 Å from a crystal of 0.09 mm(3) in volume. The neutron crystal structure will allow the positions of H atoms in P2 and its fatty-acid ligand to be visualized, as well as shedding light on the fine details of the hydrogen-bonding networks within the P2 ligand-binding cavity.
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Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering--A Comparison between Wild-Type Protein and a Hinge Mutant.
PLOS ONE, 2015Co-Authors: Saara Laulumaa, Petri Kursula, Michael Marek Koza, Tuomo Nieminen, Ilpo Vattulainen, M. Lehtimaki, Shweta Aggarwal, Mikael Simons, Francesca NataliAbstract:Myelin Protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 Protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated Protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the Protein. For a deeper insight into P2 structure-function relationships, information on Protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from Protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 Protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 Protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding Protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.
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Neutron scattering studies on Protein dynamics using the human myelin peripheral membrane Protein P2
EPJ Web of Conferences, 2015Co-Authors: Saara Laulumaa, Petri Kursula, Francesca NataliAbstract:Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin Protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin Protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant Protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect Protein dynamics, and in the case of P2, they may explain functional differences between the two Proteins.
Pang-chui Shaw - One of the best experts on this subject based on the ideXlab platform.
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Structural and Functional Investigation and Pharmacological Mechanism of Trichosanthin, a Type 1 Ribosome-Inactivating Protein
MDPI AG, 2018Co-Authors: Wei-wei Shi, Kam-bo Wong, Pang-chui ShawAbstract:Trichosanthin (TCS) is an RNA N-glycosidase that depurinates adenine-4324 in the conserved α-sarcin/ricin loop (α-SRL) of rat 28 S ribosomal RNA (rRNA). TCS has only one chain, and is classified as type 1 ribosome-inactivating Protein (RIP). Our structural studies revealed that TCS consists of two domains, with five conserved catalytic residues Tyr70, Tyr111, Glu160, Arg163 and Phe192 at the active cleft formed between them. We also found that the structural requirements of TCS to interact with the ribosomal stalk Protein P2 C-terminal tail. The structural analyses suggest TCS attacks ribosomes by first binding to the C-terminal domain of ribosomal P Protein. TCS exhibits a broad spectrum of biological and pharmacological activities including anti-tumor, anti-virus, and immune regulatory activities. This review summarizes an updated knowledge in the structural and functional studies and the mechanism of its multiple pharmacological effects
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Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2
Toxins, 2016Co-Authors: Wei-wei Shi, Kam-bo Wong, Yun-sang Tang, See-yuen Sze, Zhen-ning Zhu, Pang-chui ShawAbstract:Ricin is a type 2 ribosome-inactivating Protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits Protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 A crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk Protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P Protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.
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maize ribosome inactivating Protein uses lys158 lys161 to interact with ribosomal Protein P2 and the strength of interaction is correlated to the biological activities
PLOS ONE, 2012Co-Authors: Yuen-ting Wong, Amanda Nga-sze Mak, Kong-hung Sze, Kam-bo Wong, Pang-chui ShawAbstract:Ribosome-inactivating Proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk Protein P2 via Lys158–Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the ‘FGLFD’ motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (KD) of their interaction, which are 1037.50±65.75 µM, 611.70±28.13 µM and 194.84±9.47 µM respectively. Despite MOD and TCS target at the same ribosomal Protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal Proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal Proteins for targeting the sarcin-ricin loop and the importance of Protein-Protein interaction for ribosome-inactivating activity.
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Solution structure of the dimerization domain of ribosomal Protein P2 provides insights for the structural organization of eukaryotic stalk.
Nucleic Acids Research, 2010Co-Authors: Ka-ming Lee, Kong-hung Sze, Pang-chui Shaw, Denise So-bik Chan, Teddy Yu-hin Chiu, Guang Zhu, Kam-bo WongAbstract:The lateral stalk of ribosome is responsible for kingdom-specific binding of translation factors and activation of GTP hydrolysis that drives Protein synthesis. In eukaryotes, the stalk is composed of acidic ribosomal Proteins P0, P1 and P2 that constitute a pentameric P-complex in 1: 2: 2 ratio. We have determined the solution structure of the N-terminal dimerization domain of human P2 (NTD-P2), which provides insights into the structural organization of the eukaryotic stalk. Our structure revealed that eukaryotic stalk Protein P2 forms a symmetric homodimer in solution, and is structurally distinct from the bacterial counterpart L12 homodimer. The two subunits of NTD-P2 form extensive hydrophobic interactions in the dimeric interface that buries 2400 A(2) of solvent accessible surface area. We have showed that P1 can dissociate P2 homodimer spontaneously to form a more stable P1/P2 1 : 1 heterodimer. By homology modelling, we identified three exposed polar residues on helix-3 of P2 are substituted by conserved hydrophobic residues in P1. Confirmed by mutagenesis, we showed that these residues on helix-3 of P1 are not involved in the dimerization of P1/P2, but instead play a vital role in anchoring P1/P2 heterodimer to P0. Based on our results, models of the eukaryotic stalk complex were proposed.
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Solution structure of an active mutant of maize ribosome-inactivating Protein (MOD) and its interaction with the ribosomal stalk Protein P2.
Journal of Molecular Biology, 2010Co-Authors: Yinhua Yang, Amanda Nga-sze Mak, Pang-chui Shaw, Kong-hung SzeAbstract:Ribosome-inactivating Proteins (RIPs) are N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of ribosomal RNA. This modification renders the ribosome unable to bind the elongation factors, thereby inhibiting the Protein synthesis. Maize RIP, a type III RIP, is unique compared to the other type I and type II RIPs because it is synthesized as a precursor with a 25-residue internal inactivation region, which is removed in order to activate the Protein. In this study, we describe the first solution structure of this type of RIP, a 28-kDa active mutant of maize RIP (MOD). The overall Protein structure of MOD is comparable to those of the other type I RIPs and the A-chain of type II RIPs but shows significant differences in specific regions, including (1) shorter β6 and αB segments, probably for accommodating easier substrate binding, and (2) an α-helix instead of an antiparallel β-sheet in the C-terminal domain, which has been reported to be involved in binding ribosomal Protein P2 in some RIPs. Furthermore, NMR chemical shift perturbation experiments revealed that the P2 binding site on MOD is located at the N-terminal domain near the internal inactivation region. This relocation of the P2 binding site can be rationalized by concerted changes in the electrostatic surface potential and 3D structures on the MOD Protein and provides vital clues about the underlying molecular mechanism of this unique type of RIP.
Salla Ruskamo - One of the best experts on this subject based on the ideXlab platform.
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cryo em x ray diffraction and atomistic simulations reveal determinants for the formation of a supramolecular myelin like proteolipid lattice
Journal of Biological Chemistry, 2020Co-Authors: Salla Ruskamo, Tuomo Nieminen, Arne Raasakka, Ilpo Vattulainen, M. Lehtimaki, Oda C Krokengen, Julia Kowal, Venkata P Dandey, Henning Stahlberg, Petri KursulaAbstract:Myelin Protein P2 is a peripheral membrane Protein of the fatty acid–binding Protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot-Marie-Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered three-dimensional (3D) lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ∼3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral Protein organization, and surface mutagenesis of P2 coupled with structure-function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg-88 is critical for P2-membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 β-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane Protein from human myelin. This is an important step toward deciphering the 3D assembly of a mature myelin sheath at the molecular level.
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determinants for forming a supramolecular myelin like proteolipid lattice
bioRxiv, 2020Co-Authors: Salla Ruskamo, Tuomo Nieminen, Arne Raasakka, Ilpo Vattulainen, M. Lehtimaki, Oda C Krokengen, Julia Kowal, Venkata P Dandey, Henning Stahlberg, Petri KursulaAbstract:Myelin Protein P2 is a peripheral membrane Protein of the fatty acid binding Protein family. It functions in the formation and maintenance of the peripheral nerve myelin sheath, and several P2 mutations causing human Charot-Marie-Tooth neuropathy have been reported. Here, electron cryomicroscopy of myelin-like proteolipid multilayers revealed a three-dimensionally ordered lattice of P2 molecules between stacked lipid bilayers, visualizing its possible assembly at the myelin major dense line. A single layer of P2 is inserted between two bilayers in a tight intermembrane space of ~3 nm, implying direct interactions between P2 and two membrane surfaces. Further details on lateral Protein organization were revealed through X-ray diffraction from bicelles stacked by P2. Surface mutagenesis of P2 coupled to structural and functional experiments revealed a role for both the portal region and the opposite face of P2 in membrane interactions. Atomistic molecular dynamics simulations of P2 on myelin-like and model membrane surfaces suggested that Arg88 is an important residue for P2-membrane interactions, in addition to the helical lid domain on the opposite face of the molecule. Negatively charged myelin lipid headgroups anchor P2 stably on the bilayer surface. Membrane binding may be accompanied by opening of the P2 β barrel structure and ligand exchange with the apposing lipid bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane Protein from human myelin. This is an important step towards deciphering the 3-dimensional assembly of a mature myelin sheath at the molecular level.
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structure and dynamics of a human myelin Protein P2 portal region mutant indicate opening of the β barrel in fatty acid binding Proteins
BMC Structural Biology, 2018Co-Authors: Saara Laulumaa, Tuomo Nieminen, Erik I. Hallin, Arne Raasakka, Oda C Krokengen, Anushik Safaryan, Guillaume Brysbaert, Marc F Lensink, Salla RuskamoAbstract:Myelin is a multilayered proteolipid sheath wrapped around selected axons in the nervous system. Its constituent Proteins play major roles in forming of the highly regular membrane structure. P2 is a myelin-specific Protein of the fatty acid binding Protein (FABP) superfamily, which is able to stack lipid bilayers together, and it is a target for mutations in the human inherited neuropathy Charcot-Marie-Tooth disease. A conserved residue that has been proposed to participate in membrane and fatty acid binding and conformational changes in FABPs is Phe57. This residue is thought to be a gatekeeper for the opening of the portal region upon ligand entry and egress. We performed a structural characterization of the F57A mutant of human P2. The mutant Protein was crystallized in three crystal forms, all of which showed changes in the portal region and helix α2. In addition, the behaviour of the mutant Protein upon lipid bilayer binding suggested more unfolding than previously observed for wild-type P2. On the other hand, membrane binding rendered F57A heat-stable, similarly to wild-type P2. Atomistic molecular dynamics simulations showed opening of the side of the discontinuous β barrel, giving important indications on the mechanism of portal region opening and ligand entry into FABPs. The results suggest a central role for Phe57 in regulating the opening of the portal region in human P2 and other FABPs, and the F57A mutation disturbs dynamic cross-correlation networks in the portal region of P2. Overall, the F57A variant presents similar properties to the P2 patient mutations recently linked to Charcot-Marie-Tooth disease. Our results identify Phe57 as a residue regulating conformational changes that may accompany membrane surface binding and ligand exchange in P2 and other FABPs.
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Structure and dynamics of a human myelin Protein P2 portal region mutant indicate opening of the β barrel in fatty acid binding Proteins
BMC Structural Biology, 2018Co-Authors: Saara Laulumaa, Salla Ruskamo, Tuomo Nieminen, Erik I. Hallin, Arne Raasakka, Oda C Krokengen, Anushik Safaryan, Guillaume Brysbaert, Marc F Lensink, Ilpo VattulainenAbstract:Background Myelin is a multilayered proteolipid sheath wrapped around selected axons in the nervous system. Its constituent Proteins play major roles in forming of the highly regular membrane structure. P2 is a myelin-specific Protein of the fatty acid binding Protein (FABP) superfamily, which is able to stack lipid bilayers together, and it is a target for mutations in the human inherited neuropathy Charcot-Marie-Tooth disease. A conserved residue that has been proposed to participate in membrane and fatty acid binding and conformational changes in FABPs is Phe57. This residue is thought to be a gatekeeper for the opening of the portal region upon ligand entry and egress. Results We performed a structural characterization of the F57A mutant of human P2. The mutant Protein was crystallized in three crystal forms, all of which showed changes in the portal region and helix α2. In addition, the behaviour of the mutant Protein upon lipid bilayer binding suggested more unfolding than previously observed for wild-type P2. On the other hand, membrane binding rendered F57A heat-stable, similarly to wild-type P2. Atomistic molecular dynamics simulations showed opening of the side of the discontinuous β barrel, giving important indications on the mechanism of portal region opening and ligand entry into FABPs. The results suggest a central role for Phe57 in regulating the opening of the portal region in human P2 and other FABPs, and the F57A mutation disturbs dynamic cross-correlation networks in the portal region of P2. Conclusions Overall, the F57A variant presents similar properties to the P2 patient mutations recently linked to Charcot-Marie-Tooth disease. Our results identify Phe57 as a residue regulating conformational changes that may accompany membrane surface binding and ligand exchange in P2 and other FABPs.
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Molecular mechanisms of Charcot-Marie-Tooth neuropathy linked to mutations in human myelin Protein P2
Scientific Reports, 2017Co-Authors: Salla Ruskamo, Tuomo Nieminen, Cecilie Katrin Kristiansen, Guro Helén Vatne, Anne Baumann, Erik I. Hallin, Arne Raasakka, Päivi Joensuu, Ulrich Bergmann, Ilpo VattulainenAbstract:Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies. Recently, three CMT1-associated point mutations (I43N, T51P, and I52T) were discovered in the abundant peripheral myelin Protein P2. These mutations trigger abnormal myelin structure, leading to reduced nerve conduction velocity, muscle weakness, and distal limb atrophy. P2 is a myelin-specific Protein expressed by Schwann cells that binds to fatty acids and membranes, contributing to peripheral myelin lipid homeostasis. We studied the molecular basis of the P2 patient mutations. None of the CMT1-associated mutations alter the overall folding of P2 in the crystal state. P2 disease variants show increased aggregation tendency and remarkably reduced stability, T51P being most severe. In addition, P2 disease mutations affect Protein dynamics. Both fatty acid binding by P2 and the kinetics of its membrane interactions are affected by the mutations. Experiments and simulations suggest opening of the β barrel in T51P, possibly representing a general mechanism in fatty acid-binding Proteins. Our findings demonstrate that altered biophysical properties and functional dynamics of P2 may cause myelin defects in CMT1 patients. At the molecular level, a few malformed hydrogen bonds lead to structural instability and misregulation of conformational changes related to ligand exchange and membrane binding.
Kam-bo Wong - One of the best experts on this subject based on the ideXlab platform.
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Structural and Functional Investigation and Pharmacological Mechanism of Trichosanthin, a Type 1 Ribosome-Inactivating Protein
MDPI AG, 2018Co-Authors: Wei-wei Shi, Kam-bo Wong, Pang-chui ShawAbstract:Trichosanthin (TCS) is an RNA N-glycosidase that depurinates adenine-4324 in the conserved α-sarcin/ricin loop (α-SRL) of rat 28 S ribosomal RNA (rRNA). TCS has only one chain, and is classified as type 1 ribosome-inactivating Protein (RIP). Our structural studies revealed that TCS consists of two domains, with five conserved catalytic residues Tyr70, Tyr111, Glu160, Arg163 and Phe192 at the active cleft formed between them. We also found that the structural requirements of TCS to interact with the ribosomal stalk Protein P2 C-terminal tail. The structural analyses suggest TCS attacks ribosomes by first binding to the C-terminal domain of ribosomal P Protein. TCS exhibits a broad spectrum of biological and pharmacological activities including anti-tumor, anti-virus, and immune regulatory activities. This review summarizes an updated knowledge in the structural and functional studies and the mechanism of its multiple pharmacological effects
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Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2
Toxins, 2016Co-Authors: Wei-wei Shi, Kam-bo Wong, Yun-sang Tang, See-yuen Sze, Zhen-ning Zhu, Pang-chui ShawAbstract:Ricin is a type 2 ribosome-inactivating Protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits Protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 A crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk Protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P Protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.
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maize ribosome inactivating Protein uses lys158 lys161 to interact with ribosomal Protein P2 and the strength of interaction is correlated to the biological activities
PLOS ONE, 2012Co-Authors: Yuen-ting Wong, Amanda Nga-sze Mak, Kong-hung Sze, Kam-bo Wong, Pang-chui ShawAbstract:Ribosome-inactivating Proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk Protein P2 via Lys158–Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the ‘FGLFD’ motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (KD) of their interaction, which are 1037.50±65.75 µM, 611.70±28.13 µM and 194.84±9.47 µM respectively. Despite MOD and TCS target at the same ribosomal Protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal Proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal Proteins for targeting the sarcin-ricin loop and the importance of Protein-Protein interaction for ribosome-inactivating activity.
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Solution structure of the dimerization domain of ribosomal Protein P2 provides insights for the structural organization of eukaryotic stalk.
Nucleic Acids Research, 2010Co-Authors: Ka-ming Lee, Kong-hung Sze, Pang-chui Shaw, Denise So-bik Chan, Teddy Yu-hin Chiu, Guang Zhu, Kam-bo WongAbstract:The lateral stalk of ribosome is responsible for kingdom-specific binding of translation factors and activation of GTP hydrolysis that drives Protein synthesis. In eukaryotes, the stalk is composed of acidic ribosomal Proteins P0, P1 and P2 that constitute a pentameric P-complex in 1: 2: 2 ratio. We have determined the solution structure of the N-terminal dimerization domain of human P2 (NTD-P2), which provides insights into the structural organization of the eukaryotic stalk. Our structure revealed that eukaryotic stalk Protein P2 forms a symmetric homodimer in solution, and is structurally distinct from the bacterial counterpart L12 homodimer. The two subunits of NTD-P2 form extensive hydrophobic interactions in the dimeric interface that buries 2400 A(2) of solvent accessible surface area. We have showed that P1 can dissociate P2 homodimer spontaneously to form a more stable P1/P2 1 : 1 heterodimer. By homology modelling, we identified three exposed polar residues on helix-3 of P2 are substituted by conserved hydrophobic residues in P1. Confirmed by mutagenesis, we showed that these residues on helix-3 of P1 are not involved in the dimerization of P1/P2, but instead play a vital role in anchoring P1/P2 heterodimer to P0. Based on our results, models of the eukaryotic stalk complex were proposed.
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interaction between trichosanthin a ribosome inactivating Protein and the ribosomal stalk Protein P2 by chemical shift perturbation and mutagenesis analyses
Nucleic Acids Research, 2007Co-Authors: Denise S B Chan, Kong-hung Sze, Pang-chui Shaw, Ka-ming Lee, Guang Zhu, Laion Chu, Priscilla Hiumei Too, Kam-bo WongAbstract:Trichosanthin (TCS) is a type I ribosome-inactivating Protein that inactivates ribosome by enzymatically depurinating the A4324 at the α-sarcin/ricin loop of 28S rRNA. We have shown in this and previous studies that TCS interacts with human acidic ribosomal Proteins P0, P1 and P2, which constitute the lateral stalk of eukaryotic ribosome. Deletion mutagenesis showed that TCS interacts with the C-terminal tail of P2, the sequences of which are conserved in P0, P1 and P2. The P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation experiments. Scanning charge-to-alanine mutagenesis has shown that K173, R174 and K177 in the C-terminal domain of TCS are involved in interacting with the P2, presumably through forming charge–charge interactions to the conserved DDD motif at the C-terminal tail of P2. A triple-alanine variant K173A/R174A/K177A of TCS, which fails to bind P2 and ribosomal stalk in vitro, was found to be 18-fold less active in inhibiting translation in rabbit reticulocyte lysate, suggesting that interaction with P-Proteins is required for full activity of TCS. In an analogy to the role of stalk Proteins in binding elongation factors, we propose that interaction with acidic ribosomal stalk Proteins help TCS to locate its RNA substrate.
T F Murphy - One of the best experts on this subject based on the ideXlab platform.
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Antibodies directed at a conserved motif in loop 6 of outer membrane Protein P2 of nontypeable Haemophilus influenzae recognize multiple strains in immunoassays
Fems Immunology and Medical Microbiology, 2006Co-Authors: John M. Neary, T F MurphyAbstract:The P2 porin is the most abundant Protein in the outer membrane of nontypeable Haemophilus influenzae. Analysis of P2 sequences from a limited number of strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. We have previously shown that antibodies raised against the loop 6 sequence of P2 from strain 5657 are bactericidal against multiple isolates. In this study, we determined the nucleotide sequence of the loop 6 region of the P2 molecule from 108 strains of nontypeable H. influenzae in order to assess more rigorously the degree of conservation of loop 6. Based on this analysis, we identified a conserved sequence, different from that of strain 5657, that occurs in approximately one-third of the strains sequenced. To assess the potential of this peptide as a vaccine antigen, antibodies raised to a multiple antigenic peptide corresponding to this sequence were characterized with respect to specificity for the P2 molecule and reactivity with heterologous strains in immunoblot assay, flow cytometry and bactericidal assays. Antibodies were reactive to the P2 molecule of 16 of 20 strains tested by immunoblot assay. Antibodies recognized nine of the 20 strains in a flow cytometry assay, and 13 of 20 demonstrated complement-mediated killing in bactericidal assays. These results support the concept of using conserved regions of the P2 Protein as a vaccine antigen.
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horizontal transfer of the gene encoding outer membrane Protein P2 of nontypeable haemophilus influenzae in a patient with chronic obstructive pulmonary disease
The Journal of Infectious Diseases, 2003Co-Authors: Thomas J. Hiltke, Sanjay Sethi, Andrew T Schiffmacher, Antoinette J Dagonese, T F MurphyAbstract:An adult with chronic obstructive pulmonary disease was monitored prospectively for 2 years. Nontypeable Haemophilus influenzae was isolated from sputum cultures at 22 of 23 monthly clinic visits. Analysis of the isolates, by pulsed-field gel electrophoresis (PFGE), revealed that the patient was colonized by 3 different strains during the 2-year period. The gene encoding outer-membrane Protein (OMP) P2, ompP2, was amplified from sputum samples and selected strains obtained from this patient. Analysis of the ompP2 sequences, in combination with the PFGE patterns, indicated that ompP2 horizontal transfer between 2 strains occurred in the respiratory tract, between clinic visits 13 and 14. Observation of ompP2 horizontal transfer in the human respiratory tract has important implications for both the understanding of ompP2 diversity among strains and the future design of OMP P2-based vaccines.
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Sequence Stability of the Gene Encoding Outer Membrane Protein P2 of Nontypeable Haemophilus influenzae in the Human Respiratory Tract
The Journal of Infectious Diseases, 2002Co-Authors: Thomas J. Hiltke, Sanjay Sethi, T F MurphyAbstract:Nontypeable Haemophilus influenzae (NTHI) is an important cause of lower respiratory tract infections in patients with chronic obstructive pulmonary disease. Recent findings suggest that the major outer membrane Protein P2 should be reconsidered as a vaccine candidate for NTHI. A P2-based vaccine would require a relative degree of sequence stability of the gene encoding P2 (ompP2) during colonization. To characterize the sequence stability of ompP2 during colonization of the human respiratory tract, ompP2 genes from 13 sets of isolates that persisted in patients with chronic obstructive pulmonary disease (mean colonization, 7 months) were sequenced. In 9 sets of isolates, ompP2 did not change. Sequence changes were noted in 4 sets of isolates. Most of these changes occurred within areas of repetitive DNA, suggesting that this type of DNA has a role in antigenic variation of P2. The sequence of ompP2 is relatively stable during persistence of NTHI in the human host.
