C. parvum HSP70 (cgd2_20), ATP binding domain, in complex with ADP and inorganic phosphate

Heat shock proteins are ubiquitous and highly conserved proteins, occurring in all cellular organisms. Their main role is to act as molecular chaperones which bind to unfolded proteins, either nascent polypeptides or denatured ones, facilitating their refolding to the native state. Heat shock protein 70 (Hsp70) forms one of the major heat shock protein families. For the most part, Hsp70 proteins are induced in response to stress, although some variants are constitutively expressed in cells. Besides their role in refolding proteins, the Hsp70s also participate in a multitude of cellular processes such as the assembly or disassembly of multiprotein complexes, protein translocation, protein degradation and signal transduction.


The Hsp70 protein consists of two domains joined by a short linker. It has an N-terminal nucleotide binding domain (NBD) responsible for the ATPase activity and a C-terminal substrate binding domain. The Hsp70 chaperone activity is linked to its ATPase activity, with substrate affinity varying according to the nucleotide present in the NBD. The catalytic cycle begins when an ADP-bound Hsp70 binds to the peptide substrate, allowing it to refold. The ADP-bound state shows higher affinity for the peptide substrate than the ATP-bound state. Releasing of ADP and Pi allows the subsequent binding of ATP and the bound substrate is consequently released. It also re-establishes the starting point of the chaperone cycle. Under physiological ATP concentrations, nucleotide dissociation is the rate limiting step for substrate release. Nucleotide dissociation is thus a crucial step in the cycle, therefore this step is highly regulated and subject to strong evolutionary variation. The dissociation of bound nucleotides requires an opening of the nucleotide binding cleft. The equilibrium between the closed and the opened states is probably the determining factor for the intrinsic rates of nucleotide dissociation. Some Hsp70s show a low dissociation rate, though faster than ATP hydrolysis in the absence of substrates, but the addition of the latter enhances the hydrolysis rate making the nucleotide release the limiting step. In vivo a group of partner proteins called nucleotide exchange factors (NEFs) are associated with Hsp70s function and regulation, facilitating the transition between open and closed states.


Since Hsp70s participate in a wide variety of cellular processes, they are an active drug target for cancer research. In our case, we are also interested in its connection to the host-parasite relationship and its therapeutic potential. Hsp70s and Hsp90s are main players in the stress response therefore modulation of their chaperone activity could have great impact on the parasite's viability. An intracellular parasite like Cryptosporidium parvum encounters multiple conditions during its life cycle, including immune reactions from the host, therefore it heavily relies on the stress response to maintain its cellular homeostasis. To gain further understanding in the function of this parasitic chaperone we have determined several crystallographic structures of the N-terminal domain in complex with ADP + Pi (3L4I), AMPPNP (3KVG) and an apo form (3L6Q). This ensemble gives us a detailed picture of the role of the different residues in the binding and hydrolysis of the ATP. Interestingly, in the apo structure two sulfate ions, from the crystallization solution, were bound to the nucleotide binding site. Their positions did not overlap with either γ or β phosphates from the nucleotide. The sulfate ions positions coincide with that of the inorganic phosphate present in the ADP structure and the second position is most commonly occupied by the Mg2+ ion in other Hsp70. The location of these ions highlights putative differences between the C. parvum and other eukaryotic Hsp70s chaperones.