Leishmania Major HSP90, N-terminal domain, in complex with ATP mimetic

Hsp90 (heat shock protein 90) is a molecular chaperone whose functions include assisting in protein folding, in both stress responses and regular cell signaling. Despite being up regulated under stress conditions, Hsp90 accounts for at 1-2 % of the total cell proteins under normal conditions. It is found in almost all organisms with the notable exception of Archaebacteria. Its functions are essential in all eukaryotes but dispensable under normal growth conditions in Eubacteria. Hsp90 is named after the apparent molecular weight of its encoded protein, which is composed of three distinct domains, an ATP binding N-terminal domain joined by a charged linker to the middle and C-terminal domains involved in substrate binding and dimerization. The Hsp90 activities are dependent on ATP hydrolysis and coupled to its concomitant conformational change, involving a transient association of the N-terminal domains. The mechanism linking this ATPase and chaperone activities is not well understood but what is known is that interfering with the ATPase activity either by mutation or pharmacological means, its chaperone capabilities of signaling clients is severely compromised.


Hsp90, despite being a molecular chaperone of a panoply of clients involved in cell signaling, is actively targeted in research of anticancer therapeutics mainly due to the little toxicity reported for the inhibitors that have reached the clinical stage. This specific inhibition of a rather promiscuous protein is poorly characterized but is known to be linked to the largest structural changes that are involved in the chaperone cycle. All the known inhibitors of Hsp90 target the ATP binding site in the N-terminal domain, highlighting the unique structural properties of this protein and highlighting the paradoxical behavior of this target. In infectious diseases, the role of Hsp90 has been dual; on one hand several viruses have co-opted this host chaperone to assist the folding of their own proteins hence inhibiting the host enzyme having a deleterious effect on the virus. On the other hand, Hsp90's ubiquitousness among the eukaryotes coupled with its peculiar involvement in both the normal homeostatic control of cell mechanisms and the stress responses make them a very attractive drug target in the case of parasitic protozoan. Additionally, the development of specific molecular probes specific for protozoan parasite's Hsp90 will help elucidate the functions of this enzyme.


In the case of Leishmania parasites, their genomes contain multicopy gene clusters of Hsp90 and it is the most abundant heat shock protein, ~ 2.5% of its entire proteome. A fundamental function associated with Hsp90 in Leishmania donovani is sensing the thermal shift during the transition from the sand-fly vector to the mammalian host, triggering the morphological differentiation between the promastigote to the amastigote forms. This transition could be mimed by the inactivation of the parasite chaperone, with compounds like geldanamycin or radicicol. Geldanamycin also induces a cell growth arrest therefore derivative compounds could be useful as therapeutic agents.


Here we report the structure of the N-terminal domain of L. major Hsp90 (LmjF33.0312 :M1-K213) in complex with AMPPNP ( 5'-adenylylimidodiphosphate) to 2.0 Å resolution. This is the first structure of an N-terminal domain with a non-hydrolizable ATP analogue. The structure shows the nucleotide binding site with the so-called lid loop in an open conformation which is consistent with the domain being a monomer. This lid closes as the Hsp90 dimerizes and links the ATPase activity to its conformational changes.