Avidity for Polypeptide Binding by Nucleotide-Bound Hsp104 Structures

Weaver, Clarissa L.; Duran, Elizabeth C.; Mack, Korrie L.; Lin, Jeffrey; Jackrel, Meredith E.; Sweeny, Elizabeth A.; Shorter, James; Lucius, Aaron L.

doi:10.1021/acs.biochem.7b00225

Cited by 13 publications

(29 citation statements)

References 35 publications

(95 reference statements)

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“…Accelerating the ATPase cycle increases the power output of the Hsp104 machine. Diminishing the cycle time spent in the ADP-bound open state counters substrate protein loss (6,41,42). Different conditions can cause these two determinants, ATPase activity and substrate loss, to concur or conflict, which helps to explain some otherwise puzzling behavior.…”

Section: Significancementioning

confidence: 99%

Structural and kinetic basis for the regulation and potentiation of Hsp104 function

Lin

Mayne

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution.

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Section: Significancementioning

confidence: 99%

Structural and kinetic basis for the regulation and potentiation of Hsp104 function

Lin

Mayne

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…The use of the slowly hydrolyzable ATP analog, ATPγS (Deville et al 2017; Gates et al 2017), ATP (Puchades et al 2017), or the ADP-BeFx (Han et al 2017) transition-state mimic were likely critical for capturing the substrate-bound states by mimicking hydrolysis or allowing partial hydrolysis to occur. Indeed, high-affinity substrate binding for Hsp104 was identified only in the presence of ATPγS compared with ATP, AMPPNP, and ADP (Gates et al 2017; Weaver et al 2017). Furthermore, mixtures of ATP and ATPγS can activate Hsp104 disaggregase activity in the absence of the Hsp70 chaperone system (Doyle et al 2007; DeSantis et al 2012).…”

Section: Cryo-em Structures Reveal Fundamental Insight Into Substratementioning

confidence: 99%

Spiraling in Control: Structures and Mechanisms of the Hsp104 Disaggregase

Shorter

Southworth

2019

Cold Spring Harb Perspect Biol

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Hsp104 is a hexameric AAA+ ATPase and protein disaggregase found in yeast, which couples ATP hydrolysis to the dissolution of diverse polypeptides trapped in toxic preamyloid oligomers, phase-transitioned gels, disordered aggregates, amyloids, and prions. Hsp104 shows plasticity in disaggregating diverse substrates, but how its hexameric architecture operates as a molecular machine has remained unclear. Here, we highlight structural advances made via cryoelectron microscopy (cryo-EM) that enhance our mechanistic understanding of Hsp104 and other related AAA+ translocases. Hsp104 hexamers are dynamic and adopt open “lock–washer” spiral states and closed ring structures that envelope polypeptide substrate inside the axial channel. ATP hydrolysis-driven conformational changes at the spiral seam ratchet substrate deeper into the channel. Remarkably, this mode of polypeptide translocation is reminiscent of models for how hexameric helicases unwind DNA and RNA duplexes. Thus, Hsp104 likely adapts elements of a deeply rooted, ring-translocase mechanism to the specialized task of protein disaggregation.

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“…All of the Tyr loops are seen to exchange more slowly by almost 100-fold. This transition appears to reflect the tight substrate binding conformation that acts to translocate substrate protein into the central pore (3,11,12,(24)(25)(26). Fig.…”

Section: Significancementioning

confidence: 99%

Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

Lin

Mayne

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.Hsp104 | hydrogen exchange | mass spectrometry | HX MS | HDX MS W e report an initial hydrogen exchange study of Hsp104, a large homohexameric member of the AAA+ superfamily (6 × 908 amino acid residues), in its various functional states. In Saccharomyces cerevisiae Hsp104 controls the prionogenesis and dissolution of the Sup35 translation termination factor (1). Hsp104 is interesting more generally for its ability to rescue aggregated proteins and even stably structured amyloids by mobilizing the driving energy of favorable ATP binding and hydrolysis to forcefully unfold proteins by threading them into or through its narrow central channel (1-5).Earlier low-resolution structures of Hsp104 and its bacterial homolog ClpB obtained by negative staining or cryo-electron microscopy (cryo-EM) showed symmetric flat hexamers (6-10). Recent higher resolution cryo-EM studies reveal more detailed global and fine-scale structural features, their dependence on the adenosine nucleotide that is bound, and enable mechanistic inferences (11,12). Thus, we now know what Hsp104 looks like, but the fundamental mechanisms and principles that determine how it works have so far been out of reach. The same is true for a rapidly growing number of other large protein molecules.Many methods have been used to measure changes and their connecting structural paths in allosteric systems, but there has been no way to connect site-resolved changes with site-resolved energetics and thus establish the importance and role of...

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Avidity for Polypeptide Binding by Nucleotide-Bound Hsp104 Structures

Cited by 13 publications

References 35 publications

Structural and kinetic basis for the regulation and potentiation of Hsp104 function

Structural and kinetic basis for the regulation and potentiation of Hsp104 function

Spiraling in Control: Structures and Mechanisms of the Hsp104 Disaggregase

Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

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