Biology subjects

Marrink, S.-J.

Publications and source records attributed to Marrink, S.-J..

26 records · Page 2Linked to original sources

bioRxiv2023

Lateral membrane organization as target of an antimicrobial peptidomimetic compound

Antimicrobial resistance is one of the leading concerns in medical care. Here we resolve the functional mechanism of the antimicrobial action of the cationic tripeptide AMC-109 by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on the negatively charged plasma membrane of Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates with specificity for negatively charged membranes. Second, by incorporation into the S. aureus membrane the lateral membrane organization is affected, dissolving membrane nanodomains. Domain dissolution affects membrane functions such as protein sorting and cell wall synthesis, and is suggested to cause a loss of resistance of methicillin-resistant S. aureus (MRSA) to methicillin. As the AMC-109 mode of action is similar to the activity of the disinfectant benzalkonium chloride (BAK), a broad applicability, but with low cytotoxicity to human cells, is expected.

biophysics↗

bioRxiv2021

A synthetic membrane shaper for controlled liposome deformation

Shape defines the structure and function of cellular membranes. In cell division, the cell membrane deforms into a dumbbell shape, while organelles such as the autophagosome exhibit stomatocyte shapes. Bottom-up in vitro reconstitution of protein machineries that stabilize or resolve the membrane necks in such deformed liposome structures is of considerable interest to characterize their function. Here we develop a DNA-nanotechnology-based approach that we call Synthetic Membrane Shaper (SMS), where cholesterol-linked DNA structures attach to the liposome membrane to reproducibly generate high yields of stomatocytes and dumbbells. In silico simulations confirm the shape-stabilizing role of the SMS. We show that the SMS is fully compatible with protein reconstitution by assembling bacterial divisome proteins (DynaminA, FtsZ:ZipA) at the catenoidal neck of these membrane structures. The SMS approach provides a general tool for studying protein binding to complex membrane geometries that will greatly benefit synthetic cell research.

synthetic biology↗

bioRxiv2020

Bottom-up fabrication of a multi-component nanopore sensor that unfolds, processes and recognizes single proteins

Transmembrane channels and pores have many biotechnological applications, notably in the single-molecule sequencing of DNA. Small synthetic nanopores have been designed using amphipathic peptides, or by assembling computationally designed transmembrane helices. The fabrication of more complex transmembrane devices has yet to be reported. In this work, we fabricated in two steps a multi-protein transmembrane device that addresses some of the main challenges in nanopore protein sequencing. In the first step, artificial nanopores are created from soluble proteins with toroid shapes. This design principle will allow fabricating a variety of nanopores for single-molecule analysis. In the second step one -subuinit of the 20S proteasome from Thermoplasma acidophilum is genetically integrated into the artificial nanopore, and a 28-component nanopore-proteasome is co-assembled in E. coli cells. This multi-component molecular machine opens the door to two new approaches in protein sequencing, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then identified by the nanopore sensor either as intact or fragmented polypeptides. The ability to integrate molecular devices directly onto a nanopore sensors allows creating next-generation protein sequencing devices, and will shed new lights on the fundamental processes of biological nanomachines.

bioengineering↗

bioRxiv2020

Structure of the Human Signal Peptidase Complex Reveals the Determinants for Signal Peptide Cleavage

The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. This unique architecture generates specificity for thousands of SPs based on the length of their hydrophobic segments.

biochemistry↗

bioRxiv2020

A second PI(4,5)P2 binding site determines PI(4,5)P2 sensitivity of the tubby domain

Phosphosinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. Such domains are also used widely as fluorescence-coupled biosensors for cellular PIs. For PI(4,5)P2, the dominant PI of the plasma membrane (PM), only two recognition domains have been characterized in detail and used as sensors. One of them, the tubby domain, which is conserved in the tubby-like protein (TULP) family, is essential for targeting proteins into cilia in a process involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic. Here we used coarse-grained molecular dynamics (MD) simulations to explore PI(4,5)P2 binding by the prototypic tubby domain (tubbyCT). While the MD simulations showed a comparatively low PI(4,5)P2 affinity of the previously described canonical binding site, they unexpectedly revealed an adjacent second binding site, consisting of a conserved cationic cluster at the protein-membrane interface. Population of this second site dramatically increased membrane association of tubbyCT. Although less specific than the canonical binding pocket, this second site preferred binding of PI(4,5)P2 over PI(4)P and phosphatidyl serine. Mutations in this site impaired PI(4,5)P2-dependent PM localization in living cells and PI(4,5)P2 interaction in silico. Thus, the second binding site essentially contributes to the effective affinity and hence PM association of the tubby domain. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of TULPs that underlies delivery of ciliary cargo.

biophysics↗

bioRxiv2020

Semi-processive hyperglycosylation of adhesin by bacterial protein N-glycosyltransferases

Processivity is an important feature of enzyme families such as DNA polymerases, polysaccharide synthases and protein kinases, to ensure high fidelity in biopolymer synthesis and modification. Here we reveal processive character in the family of cytoplasmic protein N-glycosyltransferases (NGTs). Through various activity assays, intact protein mass spectrometry and proteomics analysis, we established that NGTs from non-typeable Haemophilus influenzae and Actinobacillus pleuropneumoniae modify an adhesin protein fragment in a semi-processive manner. Molecular modeling studies suggest that the processivity arises from the shallow substrate binding groove in NGT, that promotes the sliding of the adhesin over the surface to allow further glycosylations without temporary dissociation. We hypothesize that the processive character of these bacterial protein glycosyltransferases is the mechanism to ensure multisite glycosylation of adhesins in vivo, thereby creating the densely glycosylated proteins necessary for bacterial self-aggregation and adherence to human cells, as a first step towards infection.

biochemistry↗

bioRxiv2019

Piezo1 Induces Local Curvature in a Mammalian Membrane and Forms Specific Protein-Lipid Interactions

Touch, hearing, and blood pressure control require mechanically-gated ion channels that convert mechanical stimuli into electrical currents. Piezo1 and Piezo2 were recently identified as essential eukaryotic mechanically-gated ion channels, yet how they respond to physical forces remains poorly understood. Here we use a multi-disciplinary approach to interrogate the interaction of Piezo1 with its lipid environment. We show that individual Piezo1 channels induce significant local curvature in the membrane that is magnified in a cooperative manner to generate larger curved Piezo1 pits. Curvature decreases under lateral membrane tension, consistent with a hypothesis that force detection can involve sensing changes to local curvature. The protein alters its local membrane composition, enriching specific lipids and forming essential binding sites for phosphoinositides and cholesterol that are functionally relevant and often related to Piezo1-mediated pathologies. Finally, we show that Piezo1 alters the expression of lipid-regulating proteins and modifies the cellular lipidome. In short, we find that lipids influence Piezo1 activity and Piezo1 influences the local morphology and composition of the bilayer as well as the cellular lipidome.

biophysics↗

bioRxiv2017

Glucose limitation in Lactococcus shapes a single-peaked fitness landscape exposing membrane occupancy as a constraint

A central theme in biology is to understand the molecular basis of fitness: which strategies succeed under which conditions; how are they mechanistically implemented; and which constraints shape trade-offs between alternative strategies. We approached these questions with parallel bacterial evolution experiments in chemostats. Chemostats provide a constant environment with a defined resource limitation (glucose), in which the growth rate can be controlled. Using Lactococcus lactis, we found a single mutation in a global regulator of carbon metabolism, CcpA, to confer predictable fitness improvements across multiple growth rates. In silico protein structural analysis complemented with biochemical and phenotypic assays, show that the mutation reprograms the CcpA regulon, specifically targeting transporters. This supports that membrane occupancy, rather than biosynthetic capacity, is the dominant constraint for the observed fitness enhancement. It also demonstrates that cells can modulate a pleiotropic regulator to work around limiting constraints.

genomics↗