title: Characterization of the binding between a 70-kDa heat shock protein, HspA1A, and phosphoinositides.
abstract: HspA1A, a seventy-kilodalton heat shock protein, binds to specific anionic lipids and this interaction regulates important physiological phenomena like apoptosis, tumor growth, and lysosomal rescue. However, whether HspA1A binds to phosphoinositides has yet to be established and quantified. Therefore, in this study, we determined the binding affinity of HspA1A to several phosphoinositides and characterized five aspects of their molecular interaction. First, we established that HspA1A binds phosphatidylinositol monophosphates with higher affinity than di- and triphosphorylated inositides. Second, using high concentrations of potassium we found that HSPA1A embeds within the lipid bilayer of all phosphoinositides tested. However, the effects of the high salt concentrations were significantly different between the different phosphoinositides. Third, using calcium and reaction buffers equilibrated at different pH values we found that these differentially affected HspA1A-phosphoinositide binding, revealing a lipid-specific pattern of binding. Fourth, by assessing the binding properties of the two HspA1A domains, the nucleotide-binding domain and the substrate-binding domain, we determined that in most cases the full-length protein is necessary for binding to phosphoinositides. Fifth, by including in the reactions nucleotides and protein substrates we determined that they minimally and differentially affected phosphoinositide-binding. Collectively, these findings strongly suggest that the HspA1A-phosphoinositide binding is complex yet specific, is mediated by both electrostatic and hydrophobic interactions, is not related to the lipid-head charge, and depends on the physicochemical properties of the lipid.
authors.affiliation: Department of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USA.
authors.forename: Chelsea
authors.initials: C
authors.lastname: McCallister
authors.affiliation: Department of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USA.
authors.forename: Brianna
authors.initials: B
authors.lastname: Kdeiss
authors.affiliation: Department of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Oliverio
authors.affiliation: Department of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USA. Electronic address: nnikolaidis@fullerton.edu.
authors.forename: Nikolas
authors.initials: N
authors.lastname: Nikolaidis
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D051379:Mice; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
chemical_list: D018840:HSP70 Heat-Shock Proteins; C498550:Hsp70.3 protein, mouse; D008051:Lipid Bilayers; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D002118:Calcium
keywords: Heat-shock proteins; Lipid-binding; Liposomes; Membranes; Stress