Biology subjects

Ghirlando, R.

Publications and source records attributed to Ghirlando, R..

8 recordsLinked to original sources

bioRxiv2026

Structural and functional insights into the Rcs phosphorelay

The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction with IgaA, but the precise activation mechanisms remain unclear. In this study, we determined the structures of IgaA and the IgaA/RcsF complex using Cryo-electron microscopy (Cryo-EM). We also determined the structures of RcsC and RcsD, which both form homodimers stabilized by hydrophobic interactions, creating ladder-like structures. Combining the Cryo-EM structures, AlphaFold3 structure predictions of IgaA/RcsD and RcsF/IgaA/RcsD, and genetic studies, we describe a model for how RcsF modifies the IgaA/RcsD interaction, lifting negative regulation and activating the Rcs phosphorelay. Our findings provide a high-resolution depiction of the Rcs stress response system and suggest potential targets for small molecule inhibitors.

biophysics↗

bioRxiv2025

APOE genotypes differentially remodel the astrocytic lipiddroplet-associated proteome to shape lipid droplet dynamics

The lipids and proteins that comprise lipid droplets regulate several cellular functions including lipid storage, stress responses, and inflammation. Glial lipid droplets have been implicated in the pathogenesis and progression of Alzheimers disease (AD), yet the mechanisms linking genetic risk to lipid droplet biology remain unclear. Here we examined how APOE, the strongest genetic modulator of late-onset AD, impacts lipid droplet composition and dynamics. We defined the lipid droplet-associated proteome and lipidome in human induced pluripotent stem cell-derived astrocytes harboring the three common APOE genotypes: APOE2 (protective), APOE3 (neutral), and APOE4 (risk). Each APOE variant displays distinct lipid droplet-associated proteins and lipids. These molecular changes yield differences in lipophagy; lipid droplets in APOE2 astrocytes undergo autophagic turnover, whereas those in APOE4 astrocytes are resistant to degradation. These findings suggest that impaired lipid droplet clearance, rather than accumulation, distinguishes APOE4-associated AD risk, and may present a new metabolic node for modulating risk.

cell biology↗

bioRxiv2025

Specificity and mechanism of the double-stranded RNA-specific J2 monoclonal antibody

Double-stranded (ds) RNAs are major structural components of the transcriptome, hallmarks of viral infection, and primary triggers of innate immune responses. The J2 monoclonal antibody is the gold-standard method to discover and map endogenous dsRNAs across subcellular locations and cell surfaces, detect exogenous RNAs in viral infection, and surveil mRNA prophylactics and therapeutics for inflammatory dsRNAs. To define its epitope, specificity, and mechanism, we determine a 2.85 [A] co-crystal structure of J2 antigen-binding fragment (Fab) bound to dsRNA. J2 uses its heavy and light chains in tandem to track the dsRNA minor groove, recognizing a staggered 8-bp duplex. J2 is exquisitely selective for dsRNAs, requires 14 bp for robust binding, and exhibits greatly diminished binding for GC-rich dsRNAs. J2 and R-loop-specific S9.6 antibody share a common recognition strategy distinct from intracellular dsRNA-binding proteins. This study provides mechanistic insights into dsRNA recognition and establishes a framework for reliable application and data interpretation of the J2 antibody in RNA discovery.

molecular biology↗

bioRxiv2025

Structural Insights into Cir-mediated Killing by the Antimicrobial Protein Microcin V

Drug-resistant bacterial infections have become a high-priority concern in the global healthcare landscape. Novel treatments for such infections are needed, but can be especially difficult to develop for Gram-negative species owing to the need to traverse the outer membrane to reach targets beneath. A promising solution is found in natural antibiotics such as albomycin, which can bind outer membrane siderophore receptors and co-opt them for import into the periplasm. Exploring this and similar mechanisms may open avenues for antibiotic development. An underappreciated class of natural antibiotics are the microcins, which are small antimicrobial proteins secreted by certain bacterial species as a means of inter-species competition. Once secreted, the microcins bind specific outer-membrane receptors of prey species and cross into the periplasm. Microcins have potent activity, bind specific targets, and have been shown to control pathobiont expansion and pathogen colonization in animal models. One such microcin, MccV, has previously been shown to utilize the E. coli colicin Ia receptor, Cir, for periplasmic import. Here, we report the first high-resolution structure of the Cir/MccV complex by Cryo-EM, revealing an interaction centered on an electropositive cavity within a usually occluded binding pocket in Cir. We also report the calculated affinity of MccV for Cir. We mutagenized putative interacting residues at this interface and identified key contacts that are critical to MccV binding and Cir-mediated import and bacteriolysis. Future efforts in this project will help us better understand the mechanisms of microcin killing, and will assess relationships between other microcins and their respective importers with the aim of understanding the potential for microcins to be used as treatment for drug-resistant pathogens.

molecular biology↗

bioRxiv2024

Cryo-EM structures of the E. coli Ton and Tol motor complexes

The Ton and Tol motor proteins use the proton gradient at the inner membrane of Gram-negative bacteria as an energy source. The generated force is transmitted through the periplasmic space to protein components associated with the outer membrane, either to maintain the outer membrane integrity for the Tol system, or to allow essential nutrients to enter the cell for Ton. We have solved the high-resolution structures of the E. coli TonB-ExbB- ExbD and TolA-TolQ-TolR complexes, revealing the inner membrane embedded engine parts of the Ton and Tol systems, and showing how TonB and TolA interact with the ExbBD and TolQR subcomplexes. Structural similarities between the two motor complexes suggest a common mechanism for the opening of the proton channel and the propagation of the proton motive force into movement of the TonB and TolA subunits. Because TonB and TolA bind at preferential ExbB or TolQ subunits, we propose a new mechanism of assembly of TonB and TolA with their respective ExbBD and TolQR subcomplexes and discuss its impact on the mechanism of action for the Ton and Tol systems.

molecular biology↗

bioRxiv2024

Structural details of helix-mediated TDP-43 C-terminal domain multimerization

The primarily disordered C-terminal domain (CTD) of TAR DNA binding protein-43 (TDP-43), a key nuclear protein in RNA metabolism, forms neuronal inclusions in several neurodegenerative diseases. A conserved region (CR, spanning residues 319-341) in CTD forms transient helix-helix contacts important for its higher-order oligomerization and function that are disrupted by ALS-associated mutations. However, the structural details of CR assembly and the explanation for several ALS-associated variants impact on phase separation and function remain unclear due to challenges in analyzing the dynamic association of TDP-43 CTD using traditional structural biology approaches. By employing an integrative approach, combining biophysical experiments, biochemical assays, AlphaFold2-Multimer (AF2-Multimer), and atomistic simulations, we generated structural models of helical oligomerization of TDP-43 CR. Using NMR, we first established that the native state of TDP-43 CR under physiological conditions is -helical. Next, alanine scanning mutagenesis revealed that while hydrophobic residues in the CR are important for CR assembly, phase separation and TDP-43 nuclear retention function, polar residues down regulate these processes. Finally, pairing AF2-Multimer modeling with AAMD simulations indicated that dynamic, oligomeric assemblies of TDP-43 that are stabilized by a methionine-rich core with specific contributions from a tryptophan/leucine pair. In conclusion, our results advance the structural understanding of the mechanisms driving TDP-43 function and provide a window into the initial stages of its conversion into pathogenic aggregates.

biochemistry↗

bioRxiv2022

Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells

Mechanistic regulation of DNA transposon systems in mammalian cells remains poorly understood. Using modeling, biochemical, and cell-based assays, we sought to extend the recent cryoEM structural insight into the piggyBac transpososome to evaluate the previously unexplained role of the transposase N-terminus, the need for asymmetric transposon ends, and the complexity of transposase tetramer formation for transposition in mammalian cells. We found that N-terminal phosphorylation by casein kinase II inhibits transposase-DNA interaction and designed deletion of this phosphorylated domain releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. N-terminal deletion enables transposition of symmetric transposon ends that was previously not achievable with piggyBac. The complex transposase tetramer needed for transposition of asymmetric transposon ends can be overcome via appending a second transposase C-terminal domain in combination with symmetric transposon ends overcoming the negative regulation by asymmetric ends. Our results demonstrate that N-terminal transposase phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposons in mammalian cells. These novel insights into mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.

molecular biology↗

bioRxiv2019

Biochemical and structural analysis reveals the Neurofibromin (NF1) protein forms a high-affinity dimer

Neurofibromin is the protein product of the NF1 gene which is mutated in the Rasopathy disease Neurofibromatosis Type I. Defects in NF1 lead to aberrant signaling through the RAS-MAPK pathway due to disruption of the Neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of the majority of Neurofibromin--to date, biochemical and structural data exist only for the GAP domain and the region containing a Sec-PH motif. To better understand the role of this large protein, we carried out a series of biochemical and biophysical studies which demonstrate that full length Neurofibromin forms a high-affinity dimer. Neurofibromin dimerization also occurs in cells, and likely has biological and clinical implications. Analysis of purified full-length and truncated variants of Neurofibromin by negative stain electron microscopy reveals the overall architecture of the dimer and predicts the potential interactions which contribute to the dimer interface. Structures resembling high-affinity full-length dimers could be reconstituted by mixing N- and C-terminal protein domains in vitro. Taken together these data suggest how Neurofibromin dimers might form and be stabilized within the cell.

biochemistry↗