title: Uptake of the antifungal cationic peptide Histatin 5 by Candida albicans Ssa2p requires binding to non-conventional sites within the ATPase domain.
abstract: Candida albicans Hsp70 Ssa1/2 proteins have been identified as cell wall binding partners for the antifungal cationic peptide Histatin 5 (Hst 5) in vivo. C. albicans Ssa2p plays a major role in binding and translocation of Hst 5 into fungal cells, as demonstrated by defective peptide uptake and killing in C. albicans SSA2 null mutants. Candidal Hsp70 proteins are classical chaperone proteins with two discrete functional domains consisting of peptide binding and ATP binding regions. Pull-down assays with full-length and truncated Ssa2 proteins found that the ATPase domain was required for Hst 5 binding. Further mapping of Ssa2p by limited digestion and peptide array analyses identified two discrete Hst 5-binding epitopes within the ATPase region. Expression of Ssa2p in C. albicans cells carrying mutations in the first epitope identified by thermolysin digestion (Ssa2128-132A3) significantly reduced intracellular transport and fungicidal activity of Hst 5, confirming its importance as a binding site for Hst 5 function in vivo. Since this Hst 5 binding site lies within the Ssa2p ATPase domain near the ATP-binding cleft, it is possible that ATP modulates Hst 5 binding to Ssa2p. Indeed, gel filtration assays demonstrated that although nucleotides are not required for Hst 5 binding, their presence improved binding affinity by 10-fold. Thus, C. albicans Ssa2p binds Hst 5 at a surface-localized epitope in a subunit of the ATPase domain; and this region is required for intracellular translocation and killing functions of Hst 5.
authors.affiliation: Department of Oral Biology, School of Dental Medicine, Public Health and Health Professions and Biomedical Sciences, University at Buffalo, Buffalo, NY 14214, USA.
authors.forename: Jianing N
authors.initials: JN
authors.lastname: Sun
authors.forename: Wansheng
authors.initials: W
authors.lastname: Li
authors.forename: Woong Sik
authors.initials: WS
authors.lastname: Jang
authors.forename: Namrata
authors.initials: N
authors.lastname: Nayyar
authors.forename: Mark D
authors.initials: MD
authors.lastname: Sutton
authors.forename: Mira
authors.initials: M
authors.lastname: Edgerton
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002176:Candida albicans / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics; D018604:Epitope Mapping; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D054808:Histatins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C068075:HTN3 protein, human; D054808:Histatins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases