Nucleotide-dependent farnesyl switch orchestrates polymerization and membrane binding of human guanylate-binding protein 1.

Dynamin-like proteins (DLPs) mediate various membrane fusion and fission processes within the cell, which often require the polymerization of DLPs. An IFN-inducible family of DLPs, the guanylate-binding proteins (GBPs), is involved in antimicrobial and antiviral responses within the cell. Human guanylate-binding protein 1 (hGBP1), the founding member of GBPs, is also engaged in the regulation of cell adhesion and migration. Here, we show how the GTPase cycle of farnesylated hGBP1 (hGBP1F) regulates its self-assembly and membrane interaction. Using vesicles of various sizes as a lipid bilayer model, we show GTP-dependent membrane binding of hGBP1F In addition, we demonstrate nucleotide-dependent tethering ability of hGBP1F Furthermore, we report nucleotide-dependent polymerization of hGBP1F, which competes with membrane binding of the protein. Our results show that hGBP1F acts as a nucleotide-controlled molecular switch by modulating the accessibility of its farnesyl moiety, which does not require any supportive proteins.

Drosophila Ebony: a novel type of nonribosomal peptide synthetase related enzyme with unusually fast peptide bond formation kinetics.

Drosophila Ebony is a ß-alanyl biogenic amine synthetase with proven function in cuticle and in glia of the nervous system. It is closely related to nonribosomal peptide synthetases (NRPSs), which typically consist of at least an adenylation, a peptidyl carrier protein and a peptide bond forming condensation domain. Besides its role in cuticle formation, Ebony is in most glia of the brain thought to convert biogenic amines to ß-alanyl conjugates. If the metabolization of the neurotransmitter histamine to ß-alanyl histamine requires a fast reaction in visual signal transduction, Ebony must be able to fulfill this requirement. Since NRPSs are in general slowly acting multi-modular protein machineries, the enigma of how Ebony quickly facilitates this inactivation remains a key question for understanding its role in vision. To quantitatively analyze the reaction kinetics, we used phosphopantetheinylated holo-Ebony prepared from Baculovirus infected Sf9 cells. Kinetic parameters for the loading reaction, e.g. the formation of ß-alanyl-Ebony thioester, complied with those of slow NRPSs. In contrast, single-turnover analysis of the last reaction step, peptide bond formation between pre-activated ß-alanyl Ebony thioester and histamine, revealed a very rapid conjugation reaction. This biphasic nature of activity identifies Ebony as a novel type of NRPS related molecule that combines a slow amino acid activation phase with a very fast product formation step.

Mechanism of GTPase-activity-induced self-assembly of human guanylate binding protein 1.

Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of large GTPases (LGs). In the course of GTP hydrolysis, the protein undergoes structural changes leading to self-assembly of the protein, which is a characteristic property of all family members. For self-assembly, the protein employs two distinct interaction sites, one of which is located within the LG domain of the protein located at the N-terminus, and the second is located in the C-terminal alpha-helical domain. Here, we identify intramolecular contacts between the LG domain and the helical part of hGBP1, which relay nucleotide-dependent structural changes from the N-terminus to the C-terminus and thereby mediate tetramer formation of the protein through a second contact site at the C-terminus. Furthermore, we demonstrate the impact of this intramolecular communication on the enzymatic activity of hGBP1 and on its cellular localization.