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.

Belonging to the dynamin superfamily of large GTPases, human guanylate-binding proteins (hGBPs) comprise a family of seven isoforms (hGBP-1 to hGBP-7) that are strongly upregulated in response to interferon-γ and other cytokines. Accordingly, several hGBPs are found to exhibit various cellular functions encompassing inhibitory effects on cell proliferation, tumor suppression as well as antiviral and antibacterial activity; however, their mechanism of action is only poorly understood. Often, cellular functions of dynamin-related proteins are closely linked to their ability to form nucleotide-dependent oligomers, a feature that also applies to hGBP-1 and hGBP-5. hGBPs are described as monomers, dimers, tetramers, and higher oligomeric species, the function of which is not clearly established. Therefore, this work focused on the oligomerization capability of hGBP-1 and hGBP-5, which are reported to assemble to homodimers and homotetramers. Employing independent methods such as size-exclusion chromatography, which relies on the hydrodynamic radius, and multiangle light scattering, which relies on the mass of the protein, revealed that previous interpretations regarding the size of the proteins and their complexes have to be revised. Additional studies using inter- and intramolecular Förster resonance energy transfer demonstrated that nucleotide-triggered intramolecular structural changes lead to a more extended shape of hGBP-1 being responsible for the appearance of larger oligomeric species. Thus, previously reported tetrameric and dimeric species of hGBP-1 and hGBP-5 were unmasked as dimers and monomers, respectively, with their shapes depending on both the bound nucleotide and the ionic strength of the solution.

We show for the first time that bisphenol A (10) has the capacity to interact directly with K-Ras and that Rheb weakly binds to bisphenol A (10) and 4,4'-biphenol derivatives. We have characterized these interactions at atomic resolution suggesting that these compounds sterically interfere with the Sos-mediated nucleotide exchange in H- and K-Ras. We show that 4,4'-biphenol (5) selectively inhibits Rheb signaling and induces cell death suggesting that this compound might be a novel candidate for treatment of tuberous sclerosis-mediated tumor growth. Our results propose a new mode of action for bisphenol A (10) that advocates a reduced exposure to this compound in our environment. Our data may lay the foundation for the future design of GTPase-selective antagonists with higher affinity to benefit of the treatment of cancer because K-Ras inhibition is regarded to be a promising strategy with a potential therapeutic window for targeting Sos in Ras-driven tumors.

g Gamma interferon (IFN-␥) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-␥ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-␥-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-␥-dependent defense strategies.

The human guanylate-binding protein 1 (hGBP1) is a large GTP-binding protein belonging to the dynamin family, a common feature of which is nucleotide-dependent assembly to homotypic oligomers. Assembly leads to stimulation of GTPase activity, which, in the case of dynamin, is responsible for scission of vesicles from membranes. By yeast two-hybrid and biochemical experiments we addressed intermolecular interactions between all subdomains of hGBP1 and identified the C-terminal subdomain, a12 ⁄ 13, as a new interaction site for self-assembly. a12 ⁄ 13 represents a stable subdomain of hGBP1, as shown by CD spectroscopy. In addition to contacts between GTPase domains leading to dimer formation, the interaction between two a12 ⁄ 13 subdomains, in the course of GTP hydrolysis, results in tetramer formation of the protein. With the help of CD spectroscopy we showed coiled-coil formation of two a12 ⁄ 13 subdomains and concentration-dependent measurements allow estimating a value for the dissociation constant of 7.3 lM. We suggest GTP hydrolysis-driven release of the a12 ⁄ 13 subdomain, making it available for coiled-coil formation. Furthermore, we can demonstrate the biological relevance of hGBP1 tetramer formation in living cells by chemical cross-link experiments.Structured digital abstract l hGBP1 and hGBP1 bind by cross-linking study (View interaction) l hGBP1 and hGBP1 bind by molecular sieving (View Interaction: 1, 2) l hGBP1 physically interacts with hGBP1 by two hybrid (View Interaction: 1,2,3)