Kinetic effects in directional proteasomal degradation of the green fluorescent protein.

The 26S eukaryotic proteasome is an ATP-dependent degradation machine at the center of the ubiquitin-proteasome system that maintains cell viability through unfolding and degradation of ubiquitinated proteins. Its 19S regulatory particle uses a powerful heterohexameric AAA+ ATPase motor that unfolds substrate proteins and threads them through the narrow central pore for degradation within the associated 20S peptidase. In this study, we probe unfolding and translocation mechanisms of the ATPase motor by performing coarse-grained simulations of mechanical pulling of the green fluorescent protein substrate through the pore. To discern factors controlling the N-C or C-N directional processing of the substrate protein, we use three distinct models involving continuous pulling, at constant velocity or constant force, or discontinuous pulling with repetitive forces. Our results reveal asymmetric unfolding requirements in N- and C-terminal pulling upon continuous application of force in accord with the softer mechanical interface near the N-terminal and restraints imposed by the heterogeneous pore surface. By contrast, repetitive force application that mimics variable gripping by the AAA+ motor results in slower unfolding kinetics when the force is applied at the softer N-terminal. This behavior can be attributed to the dynamic competition between, on the one hand, refolding and, on the other, rotational flexibility and translocation of the unfolded N-terminal α-helix. These results highlight the interplay between mechanical, thermodynamic, and kinetic effects in directional degradation by the proteasome.

Residue-Specific Impact of EDTA and Methionine on Protein Oxidation in Biotherapeutics Formulations Using an Integrated Biotherapeutics Drug Product Development Workflow.

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing ∼200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.

Exploring the Effect of Mechanical Anisotropy of Protein Structures in the Unfoldase Mechanism of AAA+ Molecular Machines.

Essential cellular processes of microtubule disassembly and protein degradation, which span lengths from tens of µm to nm, are mediated by specialized molecular machines with similar hexameric structure and function. Our molecular simulations at atomistic and coarse-grained scales show that both the microtubule-severing protein spastin and the caseinolytic protease ClpY, accomplish spectacular unfolding of their diverse substrates, a microtubule lattice and dihydrofolate reductase (DHFR), by taking advantage of mechanical anisotropy in these proteins. Unfolding of wild-type DHFR requires disruption of mechanically strong ß-sheet interfaces near each terminal, which yields branched pathways associated with unzipping along soft directions and shearing along strong directions. By contrast, unfolding of circular permutant DHFR variants involves single pathways due to softer mechanical interfaces near terminals, but translocation hindrance can arise from mechanical resistance of partially unfolded intermediates stabilized by ß-sheets. For spastin, optimal severing action initiated by pulling on a tubulin subunit is achieved through specific orientation of the machine versus the substrate (microtubule lattice). Moreover, changes in the strength of the interactions between spastin and a microtubule filament, which can be driven by the tubulin code, lead to drastically different outcomes for the integrity of the hexameric structure of the machine.

Unfolding and Translocation of Knotted Proteins by Clp Biological Nanomachines: Synergistic Contribution of Primary Sequence and Topology Revealed by Molecular Dynamics Simulations.

We use Langevin dynamics simulations to model, at an atomistic resolution, how various natively knotted proteins are unfolded in repeated allosteric translocating cycles of the ClpY ATPase. We consider proteins representative of different topologies, from the simplest knot (trefoil 31), to the three-twist 52 knot, to the most complex stevedore, 61, knot. We harness the atomistic detail of the simulations to address aspects that have so far remained largely unexplored, such as sequence-dependent effects on the ruggedness of the landscape traversed during knot sliding. Our simulations reveal the combined effect on translocation of the knotted protein structure, i.e., backbone topology and geometry, and primary sequence, i.e., side chain size and interactions, and show that the latter can dominate translocation hindrance. In addition, we observe that due to the interplay between the knotted topology and intramolecular contacts the transmission of tension along the polypeptide chain occurs very differently from that of homopolymers. Finally, by considering native and non-native interactions, we examine how the disruption or formation of such contacts can affect the translocation processivity and concomitantly create multiple unfolding pathways with very different activation barriers.