guanosine-diphosphate and Genetic-Diseases--Inborn

The large GTPase atlastin belongs to the dynamin superfamily that has been widely implicated in facilitating membrane tubulation, fission, and in select cases, fusion. Mutations spread across atlastin isoform 1 (atlastin-1) have been identified in patients suffering from hereditary spastic paraplegia (HSP), a neurodegenerative disorder affecting motor neuron function in the lower extremities. On a molecular level, atlastin-1 associates with high membrane curvature and fusion events at the endoplasmic reticulum and cis-Golgi. Here we report crystal structures of atlastin-1 comprising the G and middle domains in two different conformations. Although the orientation of the middle domain relative to the G domain is different in the two structures, both reveal dimeric assemblies with a common, GDP-bound G domain dimer. In contrast, dimer formation in solution is observed only in the presence of GTP and transition state analogs, similar to other G proteins that are activated by nucleotide-dependent dimerization. Analyses of solution scattering data suggest that upon nucleotide binding, the protein adopts a somewhat extended, dimeric conformation that is reminiscent of one of the two crystal structures. These structural studies suggest a model for nucleotide-dependent regulation of atlastin with implications for membrane fusion. This mechanism is affected in several mutants associated with HSP, providing insights into disease pathogenesis.

Topics: Crystallography, X-Ray; Genetic Diseases, Inborn; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Membrane Fusion; Membrane Proteins; Models, Molecular; Mutation; Paraparesis, Spastic; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Protein Structure, Tertiary; Structure-Activity Relationship

Topics: Crystallography, X-Ray; Genetic Diseases, Inborn; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Membrane Fusion; Membrane Proteins; Models, Molecular; Mutation; Paraparesis, Spastic; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Protein Structure, Tertiary; Structure-Activity Relationship

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Topics: Amino Acid Motifs; Amino Acid Substitution; Cell Line; Genetic Diseases, Inborn; GTP-Binding Protein alpha Subunits; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hydrophobic and Hydrophilic Interactions; Hypogonadism; Point Mutation; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1