title: Molecular analysis of Plasmodium falciparum co-chaperone Aha1 supports its interaction with and regulation of Hsp90 in the malaria parasite.
abstract: The recent recognition of Plasmodium falciparum Hsp90 (PfHsp90) as a promising anti-malaria drug target has sparked interest in identifying factors that regulate its function and drug-interaction. Co-chaperones are well-known regulators of Hsp90's chaperone function, and certain members have been implicated in conferring protection against lethal cellular effects of Hsp90-specific inhibitors. In this context, studies on PfHsp90's co-chaperones are imperative to gain insight into the regulation of the chaperone in the malaria parasite. In this study, a putative co-chaperone P. falciparum Aha1 (PfAha1) was identified and investigated for its interaction and regulation of PfHsp90. A previous genome-wide yeast two-hybrid study failed to identify PfAha1's association with PfHsp90, which prompted us to use a directed assay to investigate their interaction. PfAha1 was shown to interact with PfHsp90 via the in vivo split-ubiquitin assay and the association was confirmed in vitro by GST pull-down experiments. The GST pull-down assay further revealed PfAha1's interaction with PfHsp90 to be dependent on MgCl(2) and ATP, and was competed by co-chaperone Pfp23 that binds PfHsp90 under the same condition. In addition, the PfHsp90-PfAha1 complex was found to be sensitive to disruption by high salt, indicating a polar interaction between them. Using bio-computational modelling coupled with site-directed mutagenesis, the polar residue N108 in PfAha1 was found to be strategically located and essential for PfHsp90 interaction. The functional significance of PfAha1's interaction was clearly that of exerting a stimulatory effect on the ATPase activity of PfHsp90, likely to be essential for promoting the activation of PfHsp90's client proteins.
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Chun Song
authors.initials: CS
authors.lastname: Chua
authors.forename: Huiyu
authors.initials: H
authors.lastname: Low
authors.forename: Norbert
authors.initials: N
authors.lastname: Lehming
authors.forename: T S
authors.initials: TS
authors.lastname: Sim
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D018503:Genome, Protozoan; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D000251:Adenosine Triphosphatases