ABSTRACT
Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor.
Subject(s)
Axons/physiology , Chondroitin Sulfate Proteoglycans/metabolism , Heparan Sulfate Proteoglycans/metabolism , Receptor-Like Protein Tyrosine Phosphatases, Class 2/chemistry , Receptor-Like Protein Tyrosine Phosphatases, Class 2/metabolism , Sensory Receptor Cells/physiology , Amino Acid Sequence , Animals , Binding Sites , Cell Membrane/metabolism , Cells, Cultured , Chondroitin Sulfate Proteoglycans/chemistry , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/metabolism , Crystallography, X-Ray , Extracellular Matrix , Ganglia, Spinal , Glypicans/metabolism , Growth Cones/metabolism , Heparan Sulfate Proteoglycans/chemistry , Heparitin Sulfate/analogs & derivatives , Heparitin Sulfate/chemistry , Heparitin Sulfate/metabolism , Humans , Mice , Models, Biological , Models, Molecular , Molecular Sequence Data , Neurites/physiology , Neurocan/metabolism , Protein Conformation , Protein Multimerization , Protein Structure, TertiaryABSTRACT
Ionotropic glutamate receptors are functionally diverse but have a common architecture, including the 400-residue amino-terminal domain (ATD). We report a 1.8-A resolution crystal structure of human GluR2-ATD. This dimeric structure provides a mechanism for how the ATDs can drive receptor assembly and subtype-restricted composition. Lattice contacts in a 4.1-A resolution crystal form reveal a tetrameric (dimer-dimer) arrangement consistent with previous cellular and cryo-electron microscopic data for full-length AMPA receptors.
Subject(s)
Receptors, AMPA/chemistry , Crystallography, X-Ray , Humans , Models, Molecular , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, TertiaryABSTRACT
The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.
Subject(s)
Bacteriophage PRD1/chemistry , Bacteriophage PRD1/ultrastructure , Viral Structural Proteins/chemistry , Virion/chemistry , Virus Assembly , Amino Acid Sequence , Capsid/chemistry , Capsid/ultrastructure , Cryoelectron Microscopy , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Protein Subunits/chemistry , Viral Structural Proteins/ultrastructure , Virion/ultrastructureABSTRACT
Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 A resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 A resolution, allows the first detailed analysis of a membrane-containing virus. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.
Subject(s)
Bacteriophage PRD1/chemistry , Bacteriophage PRD1/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , DNA, Viral/metabolism , Viral Proteins/metabolism , Bacteriophage PRD1/genetics , Capsid/chemistry , Capsid/metabolism , Crystallography, X-Ray , DNA, Viral/chemistry , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Viral Proteins/chemistry , Virus AssemblyABSTRACT
The packaging of genomic RNA in members of the Cystoviridae is performed by P4, a hexameric protein with NTPase activity. Across family members such as Phi6, Phi8 and Phi13, the P4 proteins show low levels of sequence identity, but presumably have similar atomic structures. Initial structure-determination efforts for P4 from Phi6 and Phi8 were hampered by difficulties in obtaining crystals that gave ordered diffraction. Diffraction from crystals of full-length P4 showed a variety of disorder and anisotropy. Subsequently, crystals of Phi13 P4 were obtained which yielded well ordered diffraction to 1.7 A. Comparison of the packing arrangements of P4 hexamers in different crystal forms and analysis of the disorder provides insights into the flexibility of this family of proteins, which might be an integral part of their biological function.
