Biology subjects

Loschwitz, J.

Publications and source records attributed to Loschwitz, J..

6 recordsLinked to original sources

bioRxiv2026

Lipopolysaccharide chain length and coat assembly keep guanylate-binding proteins upright on bacterial membranes

Guanylate-binding proteins (GBPs) defend host cells by assembling on intracellular pathogen membranes. For human GBP1, this requires a switch from a compact, closed conformation to an extended, open one, whereas for murine orthologues the open state is not known. How the open state behaves at a membrane has remained inaccessible to experiment. Using all-atom and coarse-grained molecular dynamics simulations covering over 0.5 ms, we followed human GBP1 and murine GBP2 as monomers, dimers, and higher-order assemblies on phosphatidylcholine membranes, Shigella flexneri lipopolysaccharide (LPS), and phosphoinositide-containing bilayers. Closed GBPs dock through their prenyl anchor with minimal lipid selectivity. Open GBPs, by contrast, are intrinsically unstable: monomers fold back into membrane-bound resting states, and dimers tip over unless supported, either by long LPS O-antigen chains or by neighboring molecules within a coat. The open state thus appears to be maintained not by the nucleotide alone but by the protein's molecular environment.

biophysics↗

bioRxiv2026

Conformational dynamics of the membrane-anchored foldase LipH from Pseudomonas aeruginosa governs recognition and release of its client lipase

The lipase LipA from Pseudomonas aeruginosa is an extracellular enzyme that plays an important role in bacterial infections. Prior its export via the type II secretion system, LipA requires the cognate membrane-anchored foldase LipH for maturation in the periplasm. Though structural studies elucidated the architecture of the LipH:lipase complex, how the full-length, membrane-tethered foldase recognizes, folds, and releases its client has remained barely understood. Here, we combine in silico and in vitro analysis to resolve the conformational dynamics and function of the full-length LipH in a membrane context. Simulations reveal that the membrane-anchored LipH is highly dynamic, sampling a broad ensemble of conformations. The chaperoning cavity is transiently closed or occluded by both the proximal membrane and the linker polypeptide, which is further confirmed by structural analysis. Despite the steric hindrance, the full-length LipH reconstituted into lipid-based nanodiscs and amphipols efficiently activates LipA, though it displays substantially reduced affinity for the client. We propose that the negatively charged membrane promotes release of the folded client, enabling multiple chaperoning cycles. Hydrogen/deuterium exchange analysis reveals that the MD2 domain of LipH in engaged in stable interactions with the lipase, whereas MD1 contacts are transient. Consistently, LipH readily captures N-terminal fragments of LipA, indicating that initial recognition relies on local interactions via the MD2 domain. Together, our results show how membrane coupling and intrinsic conformational plasticity modulate the function of the steric chaperone, and suggest that the membrane-anchored LipH balances capture, folding, and release of the client LipA to enable its efficient secretion.

biochemistry↗

bioRxiv2024

Assembly and dynamics of the outer membrane exopolysaccharide transporter PelBC of Pseudomonas aeruginosa

The infamous opportunistic pathogen Pseudomonas aeruginosa enhances its virulence and antibiotic resistance upon formation of durable biofilms. The biofilm stability is mediated by its matrix built of secreted exopolysaccharides, eDNA, and structural proteins. Exopolysaccharides of P. aeruginosa - Pel, Psl and alginate - have the highest biomedical relevance, but the mechanisms behind their synthesis and secretion are poorly understood. Here, we employ cryogenic electron microscopy to resolve the 2.5 [A] structure of the outer membrane complex PelBC for Pel exopolysaccharide, which is uniquely composed of the membrane-embedded {beta}-barrel PelB and the asymmetrical ring of 12 lipoproteins PelC at the periplasmic interface. The assembly captured in the lipid-based nanodisc is stabilized by electrostatic contacts of PelC with the periplasmic loops of PelB and multiple interactions with PelB N-terminal helical domains. Within the membrane, the resolved acyl chains of the PelC lipoproteins are alternated by the tryptophan residues immersed into the lipid leaflet, thus offering a stable anchoring architecture. The highly anionic interior of the PelB {beta}-barrel is sealed by three loops at the extracellular side, where the short Plug-S loop is aligned with the periplasmic helical scaffold, being the potential gating element for the Pel exopolysaccharide tunneling. Molecular dynamic simulations of PelB in native-like membrane environments suggest that Plug-S is sufficiently flexible to open a tunnel, and so serve as a gate. The gating model is further supported by single-channel conductivity measurements, which identify two conductance states of PelB. Via mutational analysis we confirm that Plug-S mediates opening of a narrow tunnel, as required for the controlled exopolysaccharide transport. Our structural and functional analysis of the pathogenicity-relevant complex offer a detailed and comprehensive view on this unique machinery and suggest the route taken by the exopolysaccharide at the final secretion step.

biophysics↗

bioRxiv2022

Integrative modeling of guanylate binding protein dimers

Guanylate binding proteins (GBPs) are interferon-{gamma}-activated large GTPases, effective against intracellular pathogens like Toxoplasma gondii. Their host-protective functions require oligomerization, however, the oligomer structures have not been completely resolved yet. Here, we provide dimer models for hGBP1 and the murine GBPs 2 and 7 (mGBP2 and mGBP7) based on integrative modeling that involves the crystal structure of the G domain dimer of hGBP1, cross-linking mass spectrometry (XL-MS), small angle X-ray scattering (SAXS), protein-protein docking, and molecular dynamics (MD) simulations of hGBP1, mGBP2 and mGBP7. We first compare the sequences and protein dynamics of the monomeric hGBP1, mGBP2, and mGBP7, finding that the M/E domain of all three proteins is highly mobile featuring a hinge movement, yet this motion is less pronounced in mGBP7 while its GTPase (G) domain is more flexible. These differences can be explained by the variations in the sequences between mGBP7 and hGBP1/mGBP2 and extend to their dimers. While hGBP1 and its close orthologue mGBP2 dimerize via their G domains, mGBP7 shows a variety of possible dimer structures, among them parallel and crossed-stalk conformations. The G domain is only partly involved in mGBP7 dimerization, which provides a rational why mGBP7, unlike hGBP1 and mGBP2, can dimerize in the absence of GTP. The different GBP dimer structures, which still exhibit hinge movements to certain degrees, are expected to encode diverging functions, such as a destabilization of pathogenic membranes or fusion of the parasitophorous vacuole membrane with the autophagic machinery.

biochemistry↗

bioRxiv2022

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

Guanylate-binding proteins (GBPs) are a group of GTPases that are induced by interferon-{gamma} 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 s. 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.

biophysics↗

bioRxiv2019

Large-scale, dynamin-like motions of the human guanylate binding protein 1 revealed by multi-resolution simulations

Guanylate binding proteins (GBPs) belong to the dynamin-related superfamily and exhibit various functions in the fight against infections. The functions of the human guanylate binding protein 1 (hGBP1) are tightly coupled to GTP hydrolysis and dimerization. Despite known crystal structures of the hGBP1 monomer and GTPase domain dimer, little is known about the dynamics of hGBP1. To gain a mechanistic understanding of hGBP1, we performed sub-millisecond multi-resolution molecular dynamics simulations of both the hGBP1 monomer and dimer. We found that hGBP1 is a highly flexible protein that undergoes a hinge motion similar to the movements observed for other dynamin-like proteins. Another large-scale motion was observed for the C-terminal helix 13, providing a molecular view for the 13-13 distances previously reported for the hGBP1 dimer. Most of the loops of the GTPase domain were found to be flexible, disclosing why GTP binding is needed for hGBP1 dimerization to occur.\n\nAuthor summaryGunaylate binding proteins are key fighters against microbial and viral pathogens. In the human body there are seven types of such proteins, among which is the guanylate binding protein 1 (hGBP1). This protein is able to perform its function only once it is activated by binding and converting guanosinetriphosphat (GTP) to guanosinediphosphat and guanosinemonophosphat via hydrolysis. In concert with the conversion of GTP the dimerization of hGBP1 occurs, which can further interact with the lipid membrane of the pathogen and disrupt it. While the crystal structure of the protein is known, the activation and dimerization steps are not well understood at molecular level as studying them experimentally is difficult. An alternative approach is given by molecular simulations, allowing us to elucidate the protein dynamics closely connected to these steps. From our simulations applied to both the hGBP1 monomer and dimer we identified large-scale motions taking place in hGBP1 that had not been reported before. We discuss the relevance of these motions in terms of their biological function, such as possible membrane damage caused by one of the motions or locking the protein in the dimer state.

biophysics↗