Structural analysis of a biologically active echistatin analogue des(46-49)-[Ala8,37]-echistatin gamma with three disulfide bonds by 2D-NMR and computer graphics.
An echistatin analogue, designated as des(46-49)-[Ala8,37]-echistatin gamma, was synthesized chemically by solid-phase peptide synthesis. The analogue was made by replacing Cys8 and Cys37 residues with two alanines and the deletion of C-terminal peptide 46-49 of echistatin gamma, resulting in an artificial polypeptide of 45 amino acids with three disulfide bonds. In the platelet aggregation assay, the analogue exhibits almost the same activity as echistatin gamma, indicating that the linear sequence of des(46-49)-[Ala8,37]-echistatin gamma contains all of the primary-structure information that is required for proper folding of this synthetic polypeptide. The tertiary structure of the analogue, as determined from high-resolution nuclear magnetic resonance (NMR) coupled with dynamic simulated annealing, is very similar to that of echistatin alpha1 which differs from echistatin gamma by 8 residues. In particular the two important sites of the Arg-Gly-Asp (RGD) loop and the C-terminal Lys45, both of which show some degree of disorder, are maintained in similar spatial orientation and proximity as those in echistatin alpha 1 even without the constraint provided by the disulfide bond of the (Cys8-Cys37) pair. These results provide new insights in further defining distinct structural features of echistatin gamma, which are involved in supporting the active polypeptide conformation to achieve biological activity in the absence of one pair of disulfide bonds.
Chuang
L C
LC
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Chen
P Y
PY
Chen
C
C
Huang
T H
TH
Wang
K T
KT
Chiou
S H
SH
Wu
S H
SH
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003196:Computer Graphics; D004220:Disulfides / Q000737:chemistry*; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry*; D017434:Protein Structure, Tertiary
D004220:Disulfides; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; C057986:echistatin