Structure of the propeptide of prothrombin containing the gamma-carboxylation recognition site determined by two-dimensional NMR spectroscopy.
The propeptides of the vitamin K dependent blood clotting and regulatory proteins contain a gamma-carboxylation recognition site that directs precursor forms of these proteins for posttranslational gamma-carboxylation. Peptides corresponding to the propeptide of prothrombin were synthesized and examined by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). CD spectra indicate that these peptides have little or no secondary structure in aqueous solutions but that the addition of trifluoroethanol induces or stabilizes a structure containing alpha-helical character. The maximum helical content occurs at 35-40% trifluoroethanol. This trifluoroethanol-stabilized structure was solved by two-dimensional NMR spectroscopy. The NMR results demonstrate that residues -13 to -3 form an amphipathic alpha-helix. NMR spectra indicate that a similar structure is present at 5 degrees C, in the absence of trifluoroethanol. Of the residues previously implicated in defining the gamma-carboxylation recognition site, four residues (-18, -17, -16, and -15) are adjacent to the helical region and one residue (-10) is located within the helix. The potential role of the amphipathic alpha-helix in the gamma-carboxylation recognition site is discussed.
Sanford
D G
DG
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111.
Kanagy
C
C
Sudmeier
J L
JL
Furie
B C
BC
Furie
B
B
Bachovchin
W W
WW
D000595:Amino Acid Sequence; D001665:Binding Sites; D019735:Carbon-Carbon Ligases; D002942:Circular Dichroism; D003080:Cold Temperature; D004795:Enzyme Stability / Q000187:drug effects; D008025:Ligases / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry*; D011516:Prothrombin / Q000737:chemistry*; D014270:Trifluoroethanol / Q000494:pharmacology
D010446:Peptide Fragments; D011498:Protein Precursors; D014270:Trifluoroethanol; D011516:Prothrombin; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase