Biology subjects

Graewert, M. A.

Publications and source records attributed to Graewert, M. A..

4 recordsLinked to original sources

bioRxiv2025

SAXS reveals the molecular basis underlying pH-driven G3BP1 conformational dynamics: implications for stress granule formation

G3BP1 is the central node and molecular switch in stress granule (SG) assembly. However, structural insights into full-length G3BP1 remain elusive owing to its extensive intrinsically disordered regions (IDRs). Using size-exclusion chromatography-coupled small-angle X-ray scattering (SEC-SAXS), we have characterized the solution architecture and conformational dynamics of full-length G3BP1. Under physiological conditions, G3BP1 adopts an elongated, head-to-head antiparallel homodimeric conformation, whereas acidification induces a pronounced conformational compaction. Subsequent biophysical studies reveal that this compact state enables robust RNA-mediated and, notably, homotypic phase separation in vitro. Deletion of the RGG region abolishes this acidity-induced compaction and markedly impairs phase separation, establishing a causal link between the RGG-dependent conformational switch and phase separation propensity. By moving beyond hypothetical models to experimental solution-state data, our work fills a longstanding void in the field and provides critical insights into the structural plasticity that underlies G3BP1 function, offering a missing structural link essential for deciphering the molecular mechanism of SG formation. We propose that stress-associated physicochemical changes, specifically localized acidification coupled with mRNA accumulation, trigger this reversible structural reconfiguration of G3BP1, thereby facilitating phase separation.

molecular biology↗

bioRxiv2023

Vibrio cholerae's ToxRS Bile Sensing System

Cholera represents a diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence causing recurring sudden outbreaks is enabled by V. choleraes rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies e.g. bile resistance and virulence regulation. Currently, transcription factor ToxR is described as main environmental sensor for bile acid, whose activity is enhanced by binding to ToxS. Here, the presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unraveling the intricate mechanisms governing ToxRSs environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrios survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.

biochemistry↗

bioRxiv2020

The allosteric modulation of Complement C5 by knob domain peptides

To overcome limited germline combinatorial diversity, bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce affinity-maturated peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 [A] from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.

immunology↗

bioRxiv2020

Structural insights into F-actin regulation and sarcomere assembly via myotilin

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain containing protein myotilin provides structural integrity to Z-discs - the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and -actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. We used a combination of small angle X-ray scattering, cross-linking mass spectrometry, biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilins structural role in Z-discs. Significance StatementSarcomeres are the primary structural and functional unit of striated muscles, conferring movement in all animals. The Z-disk is the boundary between adjacent sarcomeres, where actin filaments (F-actin) are anchored. Z-disc protein myotilin, is a scaffold protein, which provides structural integrity to the Z-disc by multiple interactions to its central components, including F-actin and -actinin-2. Here we provide the structure of myotilin, revealing its structural plasticity in solution and the first integrative structural model of its complex with F-actin. We further show that myotilin displaces tropomyosin from F-actin, implying a novel role of myotilin in sarcomere biogenesis beyond being an interaction hub for Z-disk partners. Highlights Myotilin is structurally described as a dynamic ensemble Flanking regions enhance F-acting binding to tandem Ig domains Integrative structural model of myotilin bound to F-actin Myotilin displaces tropomyosin from F-actin, suggesting an organisational role in Z-disc

biochemistry↗