Biology subjects

Madej, M. G.

Publications and source records attributed to Madej, M. G..

3 recordsLinked to original sources

bioRxiv2024

Ion channel function of polycystin-2/polycystin-1 heteromer revealed by structure-guided mutagenesis

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations affecting polycystin-1 (PC1) or polycystin-2 (PC2). Recent structural data suggest that PC1 and PC2 can form heterotetrameric ion channels with a 3:1 stoichiometry, with the channel in a closed state. In this hetero-oligomeric formation three PC1 residues (R4100, R4107, and H4111) would work together with two PC2 residues (L677, N681) to block the ion permeation pathway. Here, we provide functional evidence supporting this model. When the pore-blocking residues are replaced by alanines, there is a significant gain-of-function (GOF) effect on PC2/PC1 currents. These PC2/PC1 GOF channels show different selectivity for monovalent cations and reduced Ca2+ permeability compared to PC2 homomers. By varying the expression ratio of PC1 and PC2, we found that PC2 preferentially forms heteromeric complexes with PC1 over homomers. Additionally, a re-interpretation of published PC2/PC1 cryo-electron microscopy data, combined with cysteine modification experiments, suggests that the pore-forming domain of PC1 adopts a canonical TRP-like conformation. The novel PC2/PC1 GOF construct offers a promising approach to investigate the functional impact of mutations linked to ADPKD.

biophysics↗

bioRxiv2022

Osmotic stress response in BetP: How lipids and K+ team up to overcome downregulation

The trimeric betaine symporter BetP senses an osmotic upshift via its osmosensory C-terminal domain and responds rapidly with a steep increase in transport rate. Full activation requires both an elevated internal K+ concentration and a yet unknown membrane stimulus. Moreover, the molecular mechanisms of stress sensing and upregulation remain unclear. Here, we show that K+ binding to BetP in vivo is highly cooperative. Using X-ray crystallography, we identify four putative K+ interaction sites at the C-terminal domains of BetP. Single particle CryoEM on BetP reconstituted in amphipols (AMP-BetP), in the absence of K+, revealed that the three C-terminal domains are oriented symmetrically near the membrane surface, representing a new downregulated state. The presence of K+ resulted in asymmetric partial unfolding of the C-terminal domains, which was assigned as an intermediate between the downregulated state and the conformation observed in crystal structures. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in 2D crystals of BetP reveal glutamate/aspartate and tyrosine responses to K+, in agreement with the identified K+ interaction sites, as well as specific unfolding events in the C-terminal domain upon activation. A rearrangement of the relative protomer orientations confers upregulation through key structural elements involved in the alternating access of BetP affecting sodium and betaine binding affinities. Although K+-specific regulation is unique to BetP we discuss unfolding/refolding of sensory domains as a unifying element in hyperosmotic stress response of osmoregulated transporters.

biophysics↗

bioRxiv2020

CopR, a global regulator of transcription to maintain copper homeostasis in Pyrococcus furiosus

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analysed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterise the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 M copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the complex regulatory network that enables Pyrococcus to respond to increased copper concentrations.

microbiology↗