Hsp70 chaperones are central hubs of the protein quality control network and collaborate with co-chaperones having a J-domain (an ∼70-residue–long helical hairpin with a flexible loop and a conserved ...His–Pro–Asp motif required for ATP hydrolysis by Hsp70s) and also with nucleotide exchange factors to facilitate many protein-folding processes that (re)establish protein homeostasis. The Hsp70s are highly dynamic nanomachines that modulate the conformation of their substrate polypeptides by transiently binding to short, mostly hydrophobic stretches. This interaction is regulated by an intricate allosteric mechanism. The J-domain co-chaperones target Hsp70 to their polypeptide substrates, and the nucleotide exchange factors regulate the lifetime of the Hsp70–substrate complexes. Significant advances in recent years are beginning to unravel the molecular mechanism of this chaperone machine and how they treat their substrate proteins.
Significance Heat shock protein 70 (Hsp70) molecular chaperones help maintain protein homeostasis. Hsp70 functions require regulated promiscuous binding and release of a wide range of protein ...substrates. ATP binding to the Hsp70 nucleotide-binding domain (NBD) regulates the affinity and kinetics of substrate binding to their substrate-binding domain (SBD). Our work sought deeper understanding of the role of conformational dynamics for allosteric signaling in Hsp70s: The SBD undergoes a seesaw-like conformational change from a high substrate affinity state to one with lower substrate affinity, and we show that this conformational change results in drastic changes in conformational flexibility for the SBD that are essential for efficient substrate binding and release. These insights will help efforts to use Hsp70s as therapeutic targets.
Binding of ATP to the N-terminal nucleotide-binding domain (NBD) of heat shock protein 70 (Hsp70) molecular chaperones reduces the affinity of their C-terminal substrate-binding domain (SBD) for unfolded protein substrates. ATP binding to the NBD leads to docking between NBD and βSBD and releasing of the α-helical lid that covers the substrate-binding cleft in the SBD. However, these structural changes alone do not fully account for the allosteric mechanism of modulation of substrate affinity and binding kinetics. Through a multipronged study of the Escherichia coli Hsp70 DnaK, we found that changes in conformational dynamics within the βSBD play a central role in interdomain allosteric communication in the Hsp70 DnaK. ATP-mediated NBD conformational changes favor formation of NBD contacts with lynchpin sites on the βSBD and force disengagement of SBD strand β8 from strand β7, which leads to repacking of a βSBD hydrophobic cluster and disruption of the hydrophobic arch over the substrate-binding cleft. In turn, these structural rearrangements drastically enhance conformational dynamics throughout the entire βSBD and particularly around the substrate-binding site. This negative, entropically driven allostery between two functional sites of the βSBD–the NBD binding interface and the substrate-binding site–confers upon the SBD the plasticity needed to bind to a wide range of chaperone clients without compromising precise control of thermodynamics and kinetics of chaperone–client interactions.
Sending Signals Dynamically Smock, Robert G; Gierasch, Lila M
Science (American Association for the Advancement of Science),
04/2009, Volume:
324, Issue:
5924
Journal Article
Peer reviewed
Open access
Proteins mediate transmission of signals along intercellular and intracellular pathways and between the exterior and the interior of a cell. The dynamic properties of signaling proteins are crucial ...to their functions. We discuss emerging paradigms for the role of protein dynamics in signaling. A central tenet is that proteins fluctuate among many states on evolutionarily selected energy landscapes. Upstream signals remodel this landscape, causing signaling proteins to transmit information to downstream partners. New methods provide insight into the dynamic properties of signaling proteins at the atomic scale. The next stages in the signaling hierarchy--how multiple signals are integrated and how cellular signaling pathways are organized in space and time--present exciting challenges for the future, requiring bold multidisciplinary approaches.
The interior of cells is highly crowded with macromolecules, which impacts all physiological processes. To explore how macromolecular crowding may influence cellular protein folding, we interrogated ...the folding landscape of a model β-rich protein, cellular retinoic acid-binding protein I (CRABP I), in the presence of an inert crowding agent (Ficoll 70). Urea titrations revealed a crowding-induced change in the water-accessible polar amide surface of its denatured state, based on an observed ca. 15% decrease in the change in unfolding free energy with respect to urea concentration (the m-value), and the effect of crowding on the equilibrium stability of CRABP I was less than our experimental error (i.e., ≤1.2 kcal/mol). Consequently, we directly probed the effect of crowding on the denatured state of CRABP I by measuring side-chain accessibility using iodide quenching of tryptophan fluorescence and chemical modification of cysteines. We observed that the urea-denatured state is more compact under crowded conditions, and the observed extent of reduction of the m-value by crowding agent is fully consistent with the extent of reduction of the accessibility of the Trp and Cys probes, suggesting a random and nonspecific compaction of the unfolded state. The thermodynamic consequences of crowding-induced compaction are discussed. In addition, over a wide range of Ficoll concentration, crowding significantly retarded the unfolding kinetics of CRABP I without influencing the urea dependence of the unfolding rate, arguing for no appreciable change in the nature of the transition state. Our results demonstrate how macromolecular crowding may influence protein folding by effects on both the unfolded state ensemble and unfolding kinetics.
It is hard to imagine a more extreme contrast than that between the dilute solutions used for
in vitro studies of protein folding and the crowded, compartmentalized, sticky, spatially inhomogeneous ...interior of a cell. This review highlights recent research exploring protein folding in the cell with a focus on issues that are generally not relevant to
in vitro studies of protein folding, such as macromolecular crowding, hindered diffusion, cotranslational folding, molecular chaperones, and evolutionary pressures. The technical obstacles that must be overcome to characterize protein folding in the cell are driving methodological advances, and we draw attention to several examples, such as fluorescence imaging of folding in cells and genetic screens for in-cell stability.
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