Thrombin functions through its RGD sequence in a non-canonical conformation.
Previous studies have suggested that thrombin interacts with integrins in endothelial cells through its RGD (Arg-187, Gly-188, Asp-189) sequence. All existing crystal structures of thrombin show that most of this sequence is buried under the 220-loop and therefore interaction via RGD implies either partial unfolding of the enzyme or its proteolytic digestion. Here, we demonstrate that surface-absorbed thrombin promotes attachment and migration of endothelial cells through interaction with alpha(v)beta(3) and alpha(5)beta(1) integrins. Using site-directed mutants of thrombin we prove that this effect is mediated by the RGD sequence and does not require catalytic activity. The effect is abrogated when residues of the RGD sequence are mutated to Ala and is not observed with proteases like trypsin and tissue-type plasminogen activator, unless the RGD sequence is introduced at position 187-189. The potent inhibitor hirudin does not abrogate the effect, suggesting that thrombin functions through its RGD sequence in a non-canonical conformation. A 1.9-Angstroms resolution crystal structure of free thrombin grown in the presence of high salt (400 mm KCl) shows two molecules in the asymmetric unit, one of which assumes an unprecedented conformation with the autolysis loop shifted 20 Angstroms away from its canonical position, the 220-loop entirely disordered, and the RGD sequence exposed to the solvent.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Matthew E
ME
Papaconstantinou
Christopher J
CJ
Carrell
Agustin O
AO
Pineda
Kevin M
KM
Bobofchak
F Scott
FS
Mathews
Christodoulos S
CS
Flordellis
Michael E
ME
Maragoudakis
Nikos E
NE
Tsopanoglou
Enrico
E
Di Cera
D002465:Cell Movement; D042783:Endothelial Cells / Q000502:physiology; D006801:Humans; D009842:Oligopeptides / Q000502:physiology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000737:chemistry* / Q000502:physiology*
D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D013917:Thrombin