title: Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.
abstract: Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA 22908, USA. Electronic address: asl2c@virginia.edu.
authors.forename: Alicia S
authors.initials: AS
authors.lastname: Linford
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia Health System, Charlottesville, VA 22908, USA.
authors.forename: Nona M
authors.initials: NM
authors.lastname: Jiang
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA; Emerald Bio, Bainbridge Island, WA 98110, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Edwards
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Sherman
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA; Department of Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Wesley C
authors.initials: WC
authors.lastname: Van Voorhis
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA; Emerald Bio, Bainbridge Island, WA 98110, USA.
authors.forename: Lance J
authors.initials: LJ
authors.lastname: Stewart
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA; Seattle Biomedical Research Institute, Seattle, WA 98109, USA; Departments of Global Health and Medical Education & Biomedical Informatics, University of Washington, Seattle, WA 98195, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Myler
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA; Emerald Bio, Bainbridge Island, WA 98110, USA.
authors.forename: Bart L
authors.initials: BL
authors.lastname: Staker
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia Health System, Charlottesville, VA 22908, USA; Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA; Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA. Electronic address: wap3g@virginia.edu.
authors.forename: William A
authors.initials: WA
authors.lastname: Petri
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004748:Entamoeba histolytica / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D017027:Protein Tyrosine Phosphatases
keywords: Entamoeba histolytica; LMW-PTP crystal structure; LMW-PTP putative substrate identification; Low molecular weight protein tyrosine phosphatase (LMW-PTP); Substrate-trapping