Biology subjects

Marassi, F. M.

Publications and source records attributed to Marassi, F. M..

8 recordsLinked to original sources

bioRxiv2026

Structure-activity of outer membrane proteins in native bacterial membrane vesicles by solid-state NMR

The outer membranes (OMs) of bacterial pathogens are potent virulence factors and serve as the first line of defense against host immunity. Their striking bilayer asymmetry is essential for function but poses an exceptional challenge for reconstitution in vitro, limiting structure-activity analysis to artificial non-native platforms that can interfere with structure and function. Here, we describe bacterial OM vesicles (OMVs), natively secreted from the cellular OM during bacterial cell growth and development, as an effective vehicle for structure-activity analysis in situ based on solid-state nuclear magnetic resonance (NMR). We show that E. coli OMVs may be engineered to express a range of isotopically labeled target OM proteins, and isolated for solid-state NMR magic angle spinning (MAS) experiments. High resolution NMR spectra are obtained for three bacterial virulence factors: the adhesion invasion locus (Ail) and plasminogen activator protease (Pla) from Yersinia pestis, and the major porin (OmpF) from E. coli. The spectra reflect the native protein structures, report on the specific OMV membrane environment, and may be used to map protein interactions with their human host ligands, specifically the multifunctional glycoprotein Vitronectin (Vn) which binds Ail as part of its serum protection activity. Notably, OMVs support protein functionality, enabling structure and activity to be correlated in situ. OMVs expressing plasmid-encoded Ail recruit human Vn and confer serum protection to wild-type E. coli cells, while OMVs expressing plasmid-encoded Pla support the proteolytic activity of Pla. Taken together, the data establish OMVs as a robust new platform for structure-activity analysis of OM proteins in situ, offer new insights about the complexity of the bacterial OM, and reveal additional functional aspects of OMVs as key ancillary units of bacterial infection.

biophysics↗

bioRxiv2025

Structural basis for lipid binding by the blood protein vitronectin, a component of HDL

Vitronectin (Vn) is a multifunctional blood glycoprotein involved in cell adhesion and migration, blood coagulation, and inflammation. It is a component of the high-density lipoprotein (HDL) proteome, and often found associated with the calcified, lipid-rich, protein deposits that are a hallmark of age-related macular degeneration, Alzheimers disease, atherosclerosis and other aging-related diseases. Here we explored the molecular basis for lipid binding by Vn using isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) and all-atom molecular dynamics (MD) simulations. The data reveal a hydrophobic groove on the surface of the hemopexin-like (HX) domain of Vn, that is capable of binding phosphatidylcholine (PC). Conformational landscape analyses of multiple, independent MD simulations identify key structural motifs and intermolecular contacts mediating the association of Vn with PC, and show that lipid binding is guided by interactions with positively charged and hydrophobic residues that organize the lipids in a tail-to-tail bilayer-like arrangement within the groove. Collectively, the data establish a comprehensive structural model for Vn association with HDL and provide mechanistic insight into its accumulation within lipid-rich deposits characteristic of age-related pathologies.

biophysics↗

bioRxiv2025

In situ NMR reveals a pH sensor motif in an outer membrane protein that drives bacterial vesicle production

The outer membrane vesicles (OMVs) produced by diderm bacteria have important roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella enterica Typhimurium (STm) activates OMV biogenesis inside the acidic vacuoles of host cells by upregulating the expression of the outer membrane (OM) protein PagC, one of the most robustly activated genes in a host environment. Here, we used solid-state nuclear magnetic resonance (NMR) and electron microscopy (EM), with native bacterial OMVs, to demonstrate that three histidines, essential for the OMV biogenic function of PagC, constitute a key pH-sensing motif. The NMR spectra of PagC in OMVs show that they become protonated around pH 6, and His protonation is associated with specific perturbations of select regions of PagC. The use of bacterial OMVs is an essential aspect of this work enabling NMR structural studies in the context of the physiological environment. PagC expression upregulates OMV production in E. coli, replicating its function in STm. Moreover, the presence of PagC drives a striking aggregation of OMVs and increases bacterial cell pellicle formation at acidic pH, pointing to a potential role as an adhesin active in biofilm formation. The data provide experimental evidence for a pH-dependent mechanism of OMV biogenesis and aggregation driven by an outer membrane protein. SignificanceThis work sheds light on the mechanism for extracellular vesicle biogenesis by Gram negative bacteria. It shows that the Salmonella surface protein PagC, a major driver of extracellular vesicle formation, harbors a set of pH-sensitive histidines that become protonated at acidic pH, increasing vesicle production, and promoting bacterial cell aggregation. NMR analysis of PagC in natively secreted bacterial vesicles is introduced as a new important tool for in situ structural analysis of bacterial membrane proteins. The results have important implications for understanding the molecular factors that drive the formation of bacterial extracellular vesicles, their functions in human infection, as well as their roles as vaccine, drug delivery and nanotechnology platforms.

biophysics↗

bioRxiv2024

Solid state NMR spectral editing of histidine, arginine and lysine using Hadamard encoding

The NMR signals from protein sidechains are rich in information about intra- and inter-molecular interactions, but their detection can be complicated due to spectral overlap as well as conformational and hydrogen exchange. In this work, we demonstrate a protocol for multi-dimensional solid-state NMR spectral editing of signals from basic sidechains based on Hadamard matrix encoding. The Hadamard method acquires multi-dimensional experiments in such a way that both the backbone and under-sampled sidechain signals can be decoded for unambiguous editing in the 15N spectral frequency dimension. All multi-dimensional 15N-edited solid-state NMR experiments can be acquired using this strategy, thereby accelerating the acquisition of spectra spanning broad frequency bandwidth. Application of these methods to the ferritin nanocage, reveals signals from N atoms from His, Arg, Lys and Trp sidechains, as well as their tightly bound, ordered water molecules. The Hadamard approach adds to the arsenal of spectroscopic approaches for protein NMR signal detection.

biophysics↗

bioRxiv2023

Dynamic nuclear polarization illuminates key protein-lipid interactions in the native bacterial cell envelope

Elucidating the structure and interactions of proteins in native environments has become a fundamental goal of structural biology. Nuclear magnetic resonance (NMR) spectroscopy is well suited for this task but often suffers from low sensitivity, especially in complex biological settings. Here, we use a sensitivity-enhancement technique called dynamic nuclear polarization (DNP) to overcome this challenge. We apply DNP to capture the membrane interactions of the outer membrane protein Ail, a key component of the host invasion pathway of Yersinia pestis. We show that the DNP-enhanced NMR spectra of Ail in native bacterial cell envelopes are well resolved and enriched in correlations that are invisible in conventional solid-state NMR experiments. Furthermore, we demonstrate the ability of DNP to capture elusive interactions between the protein and the surrounding lipopolysaccharide layer. Our results support a model where the extracellular loop arginine residues remodel the membrane environment, a process that is crucial for host invasion and pathogenesis.

biochemistry↗

bioRxiv2020

Structural basis for the association of PLEKHA7 with membrane-embedded phosphatidylinositol lipids

PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids, are critical for proper cellular localization and function, and their inhibition is an attractive target for anti-cancer therapy. While structural data can provide insights in this area, little is known about the way in which PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we report atomic-resolution structures of the PLEHA7 PH domain and describe the molecular mechanism for its recognition of membrane-bound PIPs. Using X-ray crystallography, nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC), we show - in atomic-level detail - that the interaction of PLEKHA7 with PIPs is multivalent and induces PIP clustering. The PIP binding mechanism is distinct from a discrete one-to-one interaction. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for the design of PLEKHA7-PIP inhibitors.

biochemistry↗

bioRxiv2020

Resolving atomic site interactions of the Y. pestis outer membrane protein Ail with human serum in the bacterial cell envelope

Understanding microbe-host interactions at the molecular level is a major goal of fundamental biology and therapeutic drug development. Structural biology strives to capture biomolecular structures in action, but the samples are often highly simplified versions of the complex native environment. Here we present an E. coli model system that allows us to probe the structure and function of Ail, the major surface protein of the deadly pathogen Yersinia pestis. We show that cell surface expression of Ail produces Y. pestis virulence phenotypes in E. coli, including resistance to human serum, co-sedimentation of human vitronectin and pellicle formation. Moreover, isolated bacterial cell envelopes, encompassing inner and outer membranes, yield high-resolution solid-state nuclear magnetic resonance (NMR) spectra that reflect the structure of Ail and reveal Ail sites that are sensitive to the bacterial membrane environment and involved in the interactions with human serum components. The data capture the structure and function of Ail in a bacterial outer membrane and set the stage for probing its interactions with the complex milieu of immune response proteins present in human serum. SIGNIFICANCEAil is a critical virulence factor of Y. pestis, and its interactions with human serum are central for promoting the immune resistance of bacteria to the human host defenses. Here we capture the action of Ail in a functional bacterial environment and set the stage for probing its interactions with the complex milieu of immune response proteins present in human serum. The development of an E. coli model system of Y. pestis for biophysical studies is new and biologically important. Finally, the work extends the range in-situ NMR spectroscopy to include models of microbial infection.

biochemistry↗

bioRxiv2020

Conformational states of the cytoprotective protein Bcl-xL

Bcl-xL is a major inhibitor of apoptosis, a fundamental homeostatic process of programmed cell death that is highly conserved across evolution. Because it plays prominent roles in cancer, Bcl-xL is a major target for anti-cancer therapy and for studies aimed at understanding its structure and activity. While Bcl-xL is active primarily at intracellular membranes, most studies have focused on soluble forms of the protein lacking both the membrane-anchoring C-terminal tail and the intrinsically disordered loop, and this has resulted in a fragmented view of the proteins biological activity. Here we describe how these segments affect the proteins conformation and ligand binding activity in both its soluble and membrane-anchored states. The combined data from nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC) provide information about the molecular basis for the proteins functionality and a view of its complex molecular mechanisms. SIGNIFICANCEThe human protein Bcl-xL is a key regulator of programmed cell death in health and disease. Structural studies, important for understating the molecular basis for its functions, have advanced primarily by deleting both the long disordered loop that regulates its activity and the C-terminal tail that anchors the protein to intracellular membranes Here we describe the preparation and conformations of full-length Bcl-xL in both its water-soluble and membrane-anchored states. The study provides new biophysical insights about Bcl-xL and its greater Bcl-2 protein family.

biophysics↗