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

Abstract

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.

Exploring the effect of mechanical anisotropy of protein structures in the unfoldase mechanism of AAA+ molecular machines

“…This approach mimics the set-up from LOT experiments and simulation studies for other AAA+ machines. We note that more detailed models, such as the approach based on targeted transitions between spastin motor’s configurations [63] while acting on its substrate, which we employed for the ClpY simulations, are prohibitive due to the large size of the substrate (the MT lattice). The first event observed during these simulations in both set-ups was the unfolding of the C-terminal region of the pulled β -tubulin corresponding to the unraveling of its H11, H11’, and H12 helices and the E10-strand.…”

“…Often, the rate-limiting step of the remodeling action of AAA+ machines corresponds to the process of SP unfolding, which is modulated by the protein structure, either through the topology near the terminal engaged by the machine or through internal features. In stringent cases, protein degradation may stall altogether due to strong mechanical resistance associated with complex structure, such as in knotted proteins, or internal structure that can act as a stop signal [31][32][33][34]. More broadly, mechanical resistance near the engaged SP terminal can be overcome by repetitive force application, however, unfolding rates are strongly dependent on the type of secondary structure present and the strength and extent of its connectivity with the SP core (such as van der Waals vs. hydrogen bonding, buried vs. solvent-exposed structure).…”

“…Native contacts in this region persist over long times with limited contribution of non-native contacts.Diverging dynamic contribution of native and non-native contacts of the WT-DHFR and CP variants highlights the importance of kinetic aspects of the unfolding and translocation processes. Quantitatively, these kinetic aspects can be characterized by determining the waiting time per residue during the translocation process that provides a fingerprint of the mechanical resistance of the polypeptide chain (see Methods)[34,70,71]. As shown in Figure10, in the high-energy barrier pathways, in both N-and C-terminal pulling of the WT-DHFR, large dwell times reflect the strong mechanical resistance near the engaged terminal.…”

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

“…Often, the rate-limiting step of the remodeling action of AAA+ machines corresponds to the process of SP unfolding, which is modulated by the protein structure, either through the structure near the terminal engaged by the machine or through internal features. In stringent cases, protein degradation may stall altogether due to strong mechanical resistance associated with complex structure, such as in knotted proteins, or internal structure that can act as a stop signal [ 31 , 32 , 33 , 34 ]. More broadly, mechanical resistance near the engaged SP terminal can be overcome by repetitive force application; however, unfolding rates are strongly dependent on the type of secondary structure present and the strength and extent of its connectivity with the SP core (such as van der Waals vs. hydrogen bonding, buried vs. solvent-exposed structure).…”

“…Given the extensive experimental and computational research on the mechanisms of similar AAA+ machines, such as Clp ATPases and the Rpt1-6 motor of the proteasome, our modeling is informed by the results obtained in these prior studies. The computationally prohibitive long time scales involved in SP remodeling mediated by AAA+ motors have largely resulted in two-pronged computational approaches, as in our studies that separately probed the details of conformational states of microtubule-severing machines, katanin and spastin, and the protein disaggregation machine ClpB during ATP-driven transitions [ 43 ] and the protein unfolding and translocation mechanisms associated with SP threading through the ClpY or proteasome pores [ 34 , 41 , 44 , 45 , 46 , 47 , 48 ]. To address these stringent constraints, our spastin model includes a non-allosteric pore description and an external pulling force that accelerates observation of unfolding and translocation events, as done in our recent simulations of ClpY-mediated threading of I27 and proteasome-mediated threading of the green fluorescent protein [ 47 , 48 ].…”