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

Abstract

Guanylate-binding proteins (GBPs) are a group of GTPases that are induced by interferon-$$\gamma$$ γ and are crucial components of cell-autonomous immunity against intracellular pathogens. Here, we examine murine GBP2 (mGBP2), which we have previously shown to be an essential effector protein for the control of Toxoplasma gondii replication, with its recruitment through the membrane of the parasitophorous vacuole and its involvement in the destruction of this membrane likely playing a role. The overall aim of our work is to provide a molecular-level understanding of the mutual influences of mGBP2 and the parasitophorous vacuole membrane. To this end, we performed lipid-binding assays which revealed that mGBP2 has a particular affinity for cardiolipin. This observation was confirmed by fluorescence microscopy using giant unilamellar vesicles of different lipid compositions. To obtain an understanding of the protein dynamics and how this is affected by GTP binding, mGBP2 dimerization, and membrane binding, assuming that each of these steps are relevant for the function of the protein, we carried out standard as well as replica exchange molecular dynamics simulations with an accumulated simulation time of more than 30 μs. The main findings from these simulations are that mGBP2 features a large-scale hinge motion in its M/E domain, which is present in each of the studied protein states. When bound to a cardiolipin-containing membrane, this hinge motion is particularly pronounced, leading to an up and down motion of the M/E domain on the membrane, which did not occur on a membrane without cardiolipin. Our prognosis is that this up and down motion has the potential to destroy the membrane following the formation of supramolecular mGBP2 complexes on the membrane surface.

Integrative modeling of guanylate binding protein dimers

“…The protein was always placed in a rectangle simulation box of 10 × 10 × 18 nm 3 dimensions, ~56,000 water molecules were added, as well as 10 (apo) or 13 Na$$$ {}^{+} $$$ (GTP) for the neutralization of the systems, resulting in a total number of ~178,000 atoms. A similar HREMD protocol as applied in our study of mGBP2 (Loschwitz et al, 2023) was used and is described in detail in the Supporting information.…”

“…For mGBP7, we utilize a combination of protein–protein docking, small‐angle x‐ray scattering (SAXS), and crosslinking mass spectrometry (XL‐MS) to establish potential dimer structures. We evaluate the stability of these dimer models through molecular dynamics (MD) simulations, allowing us to compare GBP monomers (Barz et al, 2019 ; Loschwitz et al, 2023 ) and dimers while identifying critical residues at the dimerization interfaces. Our findings suggest that mGBP7 exhibits distinct dimerization preferences compared to hGBP1 and mGBP2, in line with variations in their sequences and dynamics.…”

Revealing innovative JAK1 and JAK3 inhibitors: a comprehensive study utilizing QSAR, 3D-Pharmacophore screening, molecular docking, molecular dynamics, and MM/GBSA analyses

“…The difference between RMSD and RMSF is that the latter is averaged over time, giving a value for each particle i, while for the RMSD the average is taken over the particles, giving time-specific values ( Loschwitz et al, 2023 , p. 2).…”

Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions

“…Mammalian GBP proteins vary in size from ~65 to 73 kDa and are mainly localized to the cytosol ( 5 , 8 ). They possess a large GTPase domain at the N-terminus representing motifs for guanine nucleotide binding, specifically GxxxxGK and x(V/L)RD ( 9 – 13 ), followed by a middle domain and the GTPase effector domain at the C-terminus ( 14 ). Human GBP1, 2 and 5 also harbor a CaaX motif at the C-terminus, which is important for isoprenylation and enables membrane anchoring ( 14 ).…”

“…Within Lagomorpha, there are two families, Leporidae (hares and rabbits) and Ochotonidae (pikas), which diverged approximately ~37 million years ago (MYA) ( 18 ). The Ochotonidae family is restricted to the genus Ochotona , which is further divided into four subgenera ( Pika, Logotona, Conothoa and Ochotona ) and the divergence time between these subgenera is ~7 to 14 MYA ( 13 , 19 – 21 ). The Leporidae family is divided into two groups, hares and rabbits, which diverged around 12 MYA ( 22 ).…”