Isolation and characterization of novel cyclotides from Viola hederaceae: solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide.
Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 (vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional (1)H NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded beta-sheet, a short 3(10) helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and approximately 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC(50) value of 0.87 microm.
Chen
Bin
B
Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia.
Colgrave
Michelle L
ML
Daly
Norelle L
NL
Rosengren
K Johan
KJ
Gustafson
Kirk R
KR
Craik
David J
DJ
D000163:Acquired Immunodeficiency Syndrome / Q000188:drug therapy; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D047169:Cyclotides / Q000737:chemistry* / Q000494:pharmacology*; D003545:Cysteine / Q000737:chemistry; D047168:Cystine Knot Motifs; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000494:pharmacology*; D018515:Plant Leaves / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry; D013997:Time Factors; D032413:Viola / Q000378:metabolism*
D019380:Anti-HIV Agents; D047169:Cyclotides; D004220:Disulfides; D010936:Plant Extracts; D012997:Solvents; C502455:VHL-1 protein, Viola hederaceae; D003545:Cysteine