Biology subjects

Barducci, A.

Publications and source records attributed to Barducci, A..

4 recordsLinked to original sources

bioRxiv2018

Interplay of protein disorder in retinoic acid receptor heterodimer and its corepressor regulates gene expression

The retinoic acid receptors (RARs) form heterodimers with retinoid X receptors (RXRs) and control gene transcription in response to ligand binding and via allosteric activation of the C-termini helix (helix H12) of its ligand-binding domain. Herein we show that in the absence of ligand, helices H12 of RXR and RAR are disordered. The selective RAR agonist, Am580, induces folding of H12, whereas in the presence of the inverse agonist BMS493, H12 stays mostly disordered. These results substantiate a link between the structural dynamics of H12 and RXR/RAR heterodimer biological functions, and highlight disordered-to-order transition as an essential mechanism for retinoic acid mediated regulation. Unliganded RAR exerts a strong repressive activity allowed by the recruitment of transcriptional corepressors and establishment of a corepressor complex in the promoter region of target genes. The human regulatory complex of the RAR bound to the full-length interaction domain of the corepressor N-CoR was studied by integrating several experimental (SAXS, X-ray crystallography, NMR, CD, AUC) and computational data. Unexpectedly, we found that, while mainly intrinsically disordered, the N-CoR presents partially evolutionary conserved structured regions that are involved in transient intramolecular contacts. In the presence of RXR/RAR, we show that N-CoR exploits its multivalency to form a multi-site complex that diplays an equilibrium between different conformational states. This conformational equilibrium is modulated by cognate ligands, RAR point mutation and RXR H12 deletion. Now, we can state that, in addition to NR conformation and ligand-induced allosteric changes, intrinsic disorder is substantially embedded in the synergetic regulation of RXR/RAR activity and its resulting abilities to communicate with the intracellular components.

biophysics↗

bioRxiv2018

DNA-segment-capture model for loop extrusion by structural maintenance of chromosome (SMC) protein complexes

Cells possess remarkable control of the folding and entanglement topology of long and flexible chromosomal DNA molecules. It is thought that structural maintenance of chromosome (SMC) protein complexes play a crucial role in this, by organizing long DNAs into series of loops. Recent experimental data suggest that SMC complexes are able to translocate on DNA, as well as pull out lengths of DNA via a \"loop extrusion\" process. We describe a Brownian loop-capture-ratchet model for translocation and loop extrusion based on known structural, catalytic, and DNA-binding properties of the Bacillus subtilis SMC complex. Our model provides an example of a new class of molecular motor where large conformational fluctuations of the motor track - in this case DNA - are involved in the basic translocation process. Quantitative analysis of our model leads to a series of predictions for the motor properties of SMC complexes, most strikingly a strong dependence of SMC translocation velocity on tension in the DNA track that it is moving along, with \"stalling\" occuring at subpiconewton tensions. A closely related hallmark of the loop-capture mechanism is a strong dependence of translocation step size on DNA tension. We also discuss how the same mechanism might be used by other structurally related SMC complexes (E. coli MukBEF and eukaryote condensin, cohesin and SMC5/6) to organize genomic DNA.

biophysics↗

bioRxiv2017

A catch-bond drives stator mechanosensitivity in the Bacterial Flagellar Motor

The bacterial flagellar motor (BFM) is the rotary motor which powers the swimming and swarming of many motile bacteria. The torque is provided by stator units, ion motive force powered ion channels known to assemble and disassemble dynamically in the BFM. This turnover is mechano-sensitive, with the number of engaged units dependent upon the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechano-sensitivity is unknown. Here we directly measure the kinetics of arrival and departure of the stator units in individual wild-type motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. Obtaining the real-time stator stoichiometry before and after periods of forced motor stall, we measure both the number of active stator units at steady-state as a function of the load and the kinetic association and dissociation rates, by fitting the data to a reversible random sequential adsorption model. Our measurements indicate that BFM mechano-sensing relies on the dissociation rate of the stator units, which decreases with increasing load, while their association rate remains constant. This implies that the lifetime of an active stator unit assembled within the BFM increases when a higher force is applied to its anchoring point in the cell wall, providing strong evidence that a catch-bond mechanism can explain the mechano-sensitivity of the BFM.

biophysics↗

bioRxiv2017

Molecular chaperones inject energy from ATP hydrolysis into the non-equilibrium stabilisation of native proteins

Protein homeostasis, namely the ensemble of cellular mechanisms collectively controlling the activity, stability and conformational states of proteins, depends on energy-consuming processes. De novo protein synthesis requires ATP hydrolysis for peptide bond formation. Controlled degradation by the chaperone-gated proteases requires ATP hydrolysis to unfold target proteins and render their peptide bonds accessible to hydrolysis. During and following translation, different classes of molecular chaperones require ATP hydrolysis to control the conformational state of proteins, favor their folding into their active conformation and avoid, under stress, their conversion into potentially harmful aggregates. Furthermore, specific ATP-fueled unfolding chaperones can dynamically revert aggregation itself. We used here various biochemical assays and physical modeling to show that both bacterial chaperones GroEL (HSP60) and DnaK (HSP70) can use the energy liberated by ATP hydrolysis to maintain proteins in their active state even under conditions that do not favor, thermodynamically, the native state. The energy from ATP hydrolysis is thus injected by the chaperones in the system and converted into an enhanced, non-equilibrium steady-state stabilization of the native state of their substrates. Upon ATP consumption, the chaperone substrates spontaneously revert to their equilibrium non-native state.

biochemistry↗