Analysis of the molecular mechanism of Hsp70 chaperones

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The 70 kDa heat shock proteins (Hsp70s) are involved in a wide variety of cellular protein folding processes. The remarkable versatility of Hsp70s is based on the transient interaction of their substrate binding domain (SBD) with a short degenerative motif within the substrate polypeptide. This interaction is regulated by an allosteric control mechanism between their nucleotide binding domain (NBD) and SBD. Thereby, Hsp70·ATP has a low affinity to substrates but high substrate association and dissociation rates and Hsp70·ADP has a high affinity for substrates with low substrate exchange rates. The intrinsic ATP hydrolysis rate is very low but is stimulated substantially by synergistic action of the protein substrate itself and a cochaperone of the family of J-domain proteins (JDP). Nucleotide exchange factors accelerate ADP release and, at physiological ATP concentrations, conversion to the low affinity state. They thus regulate the life-time of chaperone-substrate complexes.In the past funding period we were able to contribute substantially to a better understanding of the Hsp70 chaperone machine. Our investigations of the mechanics of substrate binding revealed that the SBD has a different conformation when protein substrates are bound as compared to the conformation with bound peptides, changing how we envision the interaction of Hsp70s with folded proteins, aggregates and amyloids. We were able to solve the crystal structure of Hsp70 in the ATP bound open conformation. A mutagenesis study base on the crystal structure revealed fundamental aspects of the allosteric regulatory mechanism and allowed us to propose a mechanical model of allostery in Hsp70s. We also discovered a synergistic action of JDPs of different classes in Hsp70-mediated disaggregation of protein aggregates and we characterized a Hsp70 machinery that efficiently dissolves alpha-synuclein aggregates.In the coming funding period we will address four issues of central importance to the understanding of the Hsp70 system: (i) based on the recent crystal structure we want to analyze the kinetics of conformational changes induced by ATP binding to nucleotide-free Hsp70 and by substrate binding to Hsp70·ATP and how they are modulated by substrates and cochaperones. These investigations should not only yield a dynamic model of Hsp70 action but also information on energy transfer to substrate proteins. (ii) To understand how JDP stimulate the ATPase activity of Hsp70s we will try to define the interaction site of the J-domain on the NBD of Hsp70s and J-domain induced conformational changes in the NBD. (iii) We will investigate the JDP-substrate interaction to clarify how JDPs of different classes bind to substrates, whether and how they change the conformation of substrates, and how they target Hsp70 to substrates. (iv) The cooperation of JDPs and nucleotide exchange factors will be studied to elucidate the synergistic action during the Hsp70 chaperone cycle.