Farnesylation of human guanylate‐binding protein 1 as safety mechanism preventing structural rearrangements and uninduced dimerization

Abstract

Human guanylate‐binding protein 1 (hGBP1) belongs to the family of dynamin‐like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post‐translationally modified by attachment of a farnesyl group yielding farn‐hGBP1. In this study, hydrodynamic differences in farn‐hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size‐exclusion chromatography (SEC). In addition, we performed small‐angle X‐ray scattering (SAXS) experiments coupled with a SEC setup (SEC‐SAXS) to investigate structural properties of nonmodified hGBP1 and farn‐hGBP1 in solution. SEC‐SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal‐like conformation in nucleotide‐free solution, whereas unmodified hGBP1 forms a monomer–dimer equilibrium both in the inactive ground state in nucleotide‐free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn‐hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified protein are prerequisites for further oligomer assemblies of farn‐hGBP1 that occur in the presence of nucleotides. Database All SEC‐SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8.

Domain motions, dimerization, and membrane interactions of the murine guanylate binding protein 2

“…Upon GTP binding, the key residues of the mGBP2's G domain interact with the nucleotide, as was previously demonstrated for hGBP1 28,36 . The decreased flexibility of the guanine cap following GTP binding seen here for the mGBP2 can explain why this shifts the monomer-dimer equilibrium to the dimeric state, like in hGBP1 24,33,37 , as this should reduce the penalty for dimer formation arising from the loss of conformational entropy of the guanine cap. In the dimeric interaction interface, we identified two residues in the guanine cap, R238/R244, which could have the same function as R240/R244 in the human orthologue 33 .…”

Guanylate-Binding Protein 1: An Emerging Target in Inflammation and Cancer

“…The regulation of GBP1 function is complicated by its isoprenylation site at the C-terminal CaaX motif, where farnesylation is necessary for lipid membrane localization of GBP1 (24,25) (Figure 2). GBP1 is a member of the dynamin superfamily, which drives vesicle cleavage through self-assembly at the membrane surface (26).…”

Integrative modeling of guanylate binding protein dimers

“…Common observations for the dimer models are that the intramolecular salt bridges locking α4′ and α12/13 never completely dissolved, due to a redundancy of up to four salt bridges of which at least one is always present with $$$ \gtrsim $$$50% occupancy. Only in one of the chains of the hGBP1 dimer, all salt bridges between α4′ and α12 completely dissolved, which on longer time scales could give rise to a folding out of the E domain, as was already observed for hGBP1 multimers (Lorenz et al, 2020; Sistemich et al, 2020, 2021). The G domain dimers (hGBP1, mGBP2, and model 1 for mGBP7) show an increase in the stability of the intramolecular salt bridge within the guanine cap, which coincides with a structural stabilization of the guanine cap, while this salt bridge is not stabilized in the stalk dimers of mGBP7 (models 2 and 3).…”