Biology subjects

Oliveira, R. J. d.

Publications and source records attributed to Oliveira, R. J. d..

3 recordsLinked to original sources

bioRxiv2023

Uncovering the association mechanism between two intrinsically flexible proteins

The understanding of protein-protein interaction mechanisms is key to the atomistic description of cell signalling pathways and for the development of new drugs. In this context, the mechanism of intrinsically disordered proteins folding upon binding has attracted attention. The VirB9 C-terminal domain (VirB9Ct) and the VirB7 N-terminal motif (VirB7Nt) associate with VirB10 to form the outer membrane core complex of the Type IV Secretion System injectisome. Despite forming a stable and rigid complex, VirB7Nt behaves as a random coil while VirB9Ct is intrinsically dynamic in the free state. Here we combined NMR, stopped-flow fluorescence and computer simulations using structure-based models to characterize the VirB9Ct-VirB7Nt coupled folding and binding mechanism. Our data indicated that VirB9Ct binds to VirB7Nt by way of a conformational selection mechanism. However, at higher temperatures energy barriers between different VirB9Ct conformations are more easily surpassed. Under these conditions the formation of non-native initial encounter complexes may not be neglected, providing alternative pathways towards the native complex conformation. These observations highlight the intimate relationship between folding and binding, calling attention to the fact that the two molecular partners must search for the most favored intramolecular and intermolecular interactions on a rugged and funnelled conformational energy landscape, along which multiple intermediates may lead to the final native state.

biophysics↗

bioRxiv2022

Ratchet, swivel, tilt and roll: A complete description of subunit rotation in the ribosome

Protein synthesis by the ribosome involves large-scale rearrangements of the "small" subunit (SSU; [~]1 MDa), which include inter- and intra-subunit rotational motions. With more than 1000 structures of ribosomes and ribosomal subunits now publicly available, it is becoming increasingly difficult to design precise experiments that are based on a comprehensive analysis of all known rotation states. To overcome this limitation, we present the Ribosome Angle Decomposition (RAD) method, where the orientation of each small subunit head and body is described in terms of three angular coordinates (rotation, tilt and tilt direction) and a single translation. To demonstrate the utility of the accompanying software (RADtool) we applied it to all published ribosome and mitoribosome structures. This identified and analyzed 1077 fully-assembled ribosome complexes, as well as 280 isolated small subunits from 48 organisms. The RAD approach quantitatively distinguishes between previously described qualitative rotational features, determines when rotation-only descriptions are insufficient, and shows that tilt-like rearrangements of the SSU head and body are pervasive in both prokaryotic and eukaryotic ribosomes. Together, the presented database and technique provide a robust platform for systematically analyzing, visualizing, and comparing subunit orientations of ribosomes from all kingdoms of life. Accordingly, the RAD resource establishes a common foundation with which structural, simulation, single-molecule and biochemical efforts can precisely interrogate the dynamics of this prototypical molecular machine.

molecular biology↗

bioRxiv2021

The dynamics of subunit rotation in a eukaryotic ribosome

Protein synthesis by the ribosome is coordinated by an intricate series of large-scale conformational rearrangements. Structural studies can provide information about long-lived states, however biological kinetics are controlled by the intervening free-energy barriers. While there has been progress describing the energy landscapes of bacterial ribosomes, very little is known about the energetics of large-scale rearrangements in eukaryotic systems. To address this topic, we constructed an all-atom model with simplified energetics and performed simulations of subunit rotation in the yeast ribosome. In these simulations, the small subunit (SSU; ~1MDa) undergoes spontaneous and reversible rotations (~ 8{degrees}). By enabling the simulation of this rearrangement under equilibrium conditions, these calculations provide initial insights into the molecular factors that control dynamics in eukaryotic ribosomes. Through this, we are able to identify specific inter-subunit interactions that have a pronounced influence on the rate-limiting free-energy barrier. We also show that, as a result of changes in molecular flexibility, the thermodynamic balance between the rotated and unrotated states is temperature-dependent. This effect may be interpreted in terms of differential molecular flexibility within the rotated and unrotated states. Together, these calculations provide a foundation, upon which the field may begin to dissect the energetics of these complex molecular machines.

biophysics↗