The multiple facets of the Hsp90 machine

International audienceThe Ninth International Conference on the Hsp90 Chaperone Machine concluded in October 2018, in Leysin, Switzerland. The program highlighted findings in various areas, including integrated insight into molecular mechanism of Hsp90, cochaperones, and clients’ structure and function.Heat shock protein-90 (Hsp90) is a molecular chaperone critical for the folding, stability, and activity of client proteins 1. Hsp90 and its orthologs, including bacterial HtpG, mitochondrial TRAP1 and endoplasmic reticulum Grp94, exist as dimers, hydrolyze ATP, and cycle between distinct conformational states. Hsp90 preferentially binds proteins in near native states facilitating their remodeling for protein interactions and signaling. At the 9th International Conference on the Hsp90 Chaperone Machine approximately one-third of the attendees shared their data on Hsp90 structure and function through short talks (Figure 1). Here, we distill and summarize their finding

Comprehensive fitness maps of Hsp90 show widespread environmental dependence

Gene-environment interactions have long been theorized to influence molecular evolution. However, the environmental dependence of most mutations remains unknown. Using deep mutational scanning, we engineered yeast with all 44,604 single codon changes encoding 14,160 amino acid variants in Hsp90 and quantified growth effects under standard conditions and under five stress conditions. To our knowledge, these are the largest determined comprehensive fitness maps of point mutants. The growth of many variants differed between conditions, indicating that environment can have a large impact on Hsp90 evolution. Multiple variants provided growth advantages under individual conditions; however, these variants tended to exhibit growth defects in other environments. The diversity of Hsp90 sequences observed in extant eukaryotes preferentially contains variants that supported robust growth under all tested conditions. Rather than favoring substitutions in individual conditions, the long-term selective pressure on Hsp90 may have been that of fluctuating environments, leading to robustness under a variety of conditions

A structural perspective on the dynamics of kinesin motors

Despite significant fluctuation under thermal noise, biological machines in cells perform their tasks with exquisite precision. Using molecular simulation of a coarse-grained model and theoretical arguments we envisaged how kinesin, a prototype of biological machines, generates force and regulates its dynamics to sustain persistent motor action. A structure based model, which can be versatile in adapting its structure to external stresses while maintaining its native fold, was employed to account for several features of kinesin dynamics along the biochemical cycle. This analysis complements our current understandings of kinesin dynamics and connections to experiments. We propose a thermodynamic cycle for kinesin that emphasizes the mechanical and regulatory role of the neck-linker and clarify issues related the motor directionality, and the difference between the external stalling force and the internal tension responsible for the head-head coordination. The comparison between the thermodynamic cycle of kinesin and macroscopic heat engines highlights the importance of structural change as the source of work production in biomolecular machines.Comment: 35 pages, 8 figures, 1 Tabl

The Origin of Minus-end Directionality and Mechanochemistry of Ncd Motors

Adaptation of molecular structure to the ligand chemistry and interaction with the cytoskeletal filament are key to understanding the mechanochemistry of molecular motors. Despite the striking structural similarity with kinesin-1, which moves towards plus-end, Ncd motors exhibit minus-end directionality on microtubules (MTs). Here, by employing a structure-based model of protein folding, we show that a simple repositioning of the neck-helix makes the dynamics of Ncd non-processive and minus-end directed as opposed to kinesin-1. Our computational model shows that Ncd in solution can have both symmetric and asymmetric conformations with disparate ADP binding affinity, also revealing that there is a strong correlation between distortion of motor head and decrease in ADP binding affinity in the asymmetric state. The nucleotide (NT) free-ADP (?-ADP) state bound to MTs favors the symmetric conformation whose coiled-coil stalk points to the plus-end. Upon ATP binding, an enhanced flexibility near the head-neck junction region, which we have identified as the important structural element for directional motility, leads to reorienting the coiled-coil stalk towards the minus-end by stabilizing the asymmetric conformation. The minus-end directionality of the Ncd motor is a remarkable example that demonstrates how motor proteins in the kinesin superfamily diversify their functions by simply rearranging the structural elements peripheral to the catalytic motor head domain

Unfolding and translocation by mechanical pulling with a constant force through a non-allosteric ClpY pore.

<p>Probability density map of the fraction of native contacts and radius of gyration for simulations of mechanical pulling through a non-allosteric pore with constant force <i>F</i> = 125 pN.</p

Coarse-Grained Simulations of Topology-Dependent Mechanisms of Protein Unfolding and Translocation Mediated by ClpY ATPase Nanomachines

<div><p>Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an <i>α</i>/<i>β</i> model protein. We contrast this mechanism with our previous studies which used an all-<i>α</i> SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation.</p></div

Structural details of <i>α</i>/<i>β</i> proteins.

<p>(A.) Model protein utilized in this study. (B.) Protein L. The <i>β</i>-strands are shown in green, <i>α</i>-helix in blue, and loop regions in magenta. The C-terminus is indicated by a red sphere and the N-terminus by a blue sphere. (C.) BLN-model sequence (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004675#sec012" target="_blank">Methods</a>) of the <i>α</i>/<i>β</i> model SP and variants with contiguous stretches of non-attractive amino acids (red).</p

Unfolding and translocation pathway of the <i>α</i>/<i>β</i> protein by allosteric ClpY.

<p>(A) The <i>α</i>/<i>β</i> model protein (magenta) fused at the C-terminus with the SsrA degradation tag (yellow) binds to the central channel loops (blue) of ClpY (green). The dimensions of ClpY and pore size are indicated. For clarity, two ClpY subunits are not shown and two are shown in gray. (B) The central channel loops engage the substrate protein. Iterative pulling results in unfolding of the substrate to <i>Q</i>_<i>N</i> ≃ 0.7. (C) Following several allosteric cycles, in the absence of the ClpQ peptidase co-factor, the substrate is partially translocated (<i>Q</i>_<i>N</i> ≃ 0.5). (D) The harmonic restraints in the distal region (<math><mrow><mi>z</mi><mo>-</mo><msub><mi>z</mi><mo>¯</mo><mrow><mi>l</mi><mi>o</mi><mi>o</mi><mi>p</mi><mi>s</mi></mrow></msub><mo>></mo><mn>14</mn><mo>.</mo><mn>5</mn><mo>Å</mo></mrow></math>) are added to account for the ClpQ assistance. The allosteric motions of ClpYQ result in complete translocation and refolding of the tagged substrate protein. Molecular images in this paper are created using Visual Molecular Dynamics [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004675#pcbi.1004675.ref058" target="_blank">58</a>].</p

Unfolding and translocation pathways effected by repetitive forces exerted by the ClpY channel loops onto the SP.

<p>Time series of the fraction of native contacts, <i>Q</i>_<i>N</i>, the radius of gyration <i>R</i>_<i>g</i> of the SP, and forces along the pore axis <i>F</i>_<i>z</i> in individual trajectories that result in (A) simultaneous unfolding and translocation or (B) unfolding prior to translocation. Reversibility of initial unfolding is observed in (B).</p