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.

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Human guanylate-binding protein 1 (hGBP-1) shows a dimer-induced acceleration of the GTPase activity yielding GDP as well as GMP. While the head-to-head dimerization of the large GTPase (LG) domain is well understood, the role of the rest of the protein, particularly of the GTPase effector domain (GED), in dimerization and GTP hydrolysis is still obscure. In this study, with truncations and point mutations on hGBP-1 and by means of biochemical and biophysical methods, we demonstrate that the intramolecular communication between the LG domain and the GED (LG:GED) is crucial for protein dimerization and dimer-stimulated GTP hydrolysis. In the course of GTP binding and c-phosphate cleavage, conformational changes within hGBP-1 are controlled by a chain of amino acids ranging from the region near the nucleotide-binding pocket to the distant LG:GED interface and lead to the release of the GED from the LG domain. This opening of the structure allows the protein to form GED:GED contacts within the dimer, in addition to the established LG: LG interface. After releasing the cleaved c-phosphate, the dimer either dissociates yielding GDP as the final product or it stays dimeric to further cleave the b-phosphate yielding GMP. The second phosphate cleavage step, that is, the formation of GMP, is even more strongly coupled to structural changes and thus more sensitive to structural restraints imposed by the GED. Altogether, we depict a comprehensive mechanism of GTP hydrolysis catalyzed by hGBP-1, which provides a detailed molecular understanding of the enzymatic activity connected to large structural rearrangements of the protein.

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