Expression, purification, and molecular characterization of Plasmodium falciparum FK506-binding protein 35 (PfFKBP35).
The immunosuppressive drug FK506 binds its targets FK506-binding protein (FKBP) family and modulates cellular processes. Recent studies demonstrated that FK506 shows anti-malaria effects. Newly identified FK506-binding protein 35 from Plasmodium falciparum (PfFKBP35) is assumed to be the molecular target of FK506 in the parasite. Currently, molecular and structural basis of growth inhibition of the parasite by FK506 remains unclear. In this study, to examine characteristics of PfFKBP35 and also understand its molecular mechanism of the inhibition by FK506, we have cloned, expressed, and purified the full-length PfFKBP35 and its FK506-binding domain (FKBD). We demonstrate that the full-length PfFKBP35 and the FKBD were properly folded, and suitable for biochemical and biophysical studies. PfFKBP35 showed a basal activity in inhibiting the phosphatase activity of calcineurin in the absence of FK506, but the presence of FK506 greatly enhanced its calcineurin-inhibitory activity. Our NMR data indicate that the FKBD binds FK506 with a high affinity.
Yoon
Hye Rim
HR
Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
Kang
Cong Bao
CB
Chia
Joel
J
Tang
Kai
K
Yoon
Ho Sup
HS
D000595:Amino Acid Sequence; D024322:Amino Acids, Aromatic / Q000737:chemistry; D000818:Animals; D019703:Calcineurin / Q000032:analysis; D065095:Calcineurin Inhibitors; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D016054:DNA, Protozoan; D030562:Databases, Protein; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D018503:Genome, Protozoan; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D022021:Tacrolimus Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014170:Transformation, Genetic
D024322:Amino Acids, Aromatic; D065095:Calcineurin Inhibitors; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D019703:Calcineurin; D022021:Tacrolimus Binding Proteins