Biology subjects

Gourdon, P.

Publications and source records attributed to Gourdon, P..

4 recordsLinked to original sources

bioRxiv2022

FK506-binding protein 2 participates in proinsulin folding

Apart from chaperoning, disulphide bond formation and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94, and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high molecular weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic {beta}-cells increased apoptosis without detectable upregulation of ER-stress response genes. Interestingly, FKBP2 mRNA was overexpressed in {beta}-cells from pancreatic islets of T2D patients. Based on molecular modelling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B chain (P28) is the substrate of FKBP2s isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

molecular biology↗

bioRxiv2022

Copper binding leads to increased dynamics in the regulatory N-terminal domain of full-length human copper transporter ATP7B

ATP7B is a human copper-transporting P1B-type ATPase that is involved in copper homeostasis and resistance to platinum drugs in cancer cells. ATP7B consists of a copper-transporting core and a regulatory N-terminal tail that contains six metal-binding domains (MBD1-6) connected by linker regions. The MBDs can bind copper, which changes the dynamics of the regulatory domain and activates the protein, but the underlying mechanism remains unknown. To identify possible copper-specific structural dynamics involved in transport regulation, we constructed a model of ATP7B spanning the N-terminal tail and core catalytic domains and performed molecular dynamics (MD) simulations with (holo) and without (apo) copper ions bound to the MBDs. In the holo protein, MBD2, MBD3 and MBD5 showed enhanced mobilities, which resulted in a more extended N-terminal regulatory region. The observed separation of MBD2 and MBD3 from the core protein supports a mechanism where copper binding activates the ATP7B protein by reducing interactions among MBD1-3 and between MBD1-3 and the core protein. Instead, an increased interaction between MBD5 and the core protein was observed that brought the copper-binding site of MBD5 closer to the high-affinity internal copper-binding site in the core protein. The simulation results assign specific, mechanistic roles to the metal-binding domains involved in ATP7B regulation that are testable in experimental settings.

biophysics↗

bioRxiv2021

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.2 [A] structure, revealing a classical {beta}-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion conducting pathway as also supported by metal binding quantification via ICP-MS and MD simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrated that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.

biochemistry↗

bioRxiv2021

Structure and ion-release mechanism of PIB-4-type ATPases

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here we present structures and complementary functional analyses of an archetypal PIB-4-ATPases, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy metal binding domains, and provides fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release, and as an internal counter ion, of an invariant, central histidine. We also establish that the turn-over of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in e.g. drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.

biochemistry↗