The effect of aspartate hydroxylation on calcium binding to epidermal growth factor-like modules in coagulation factors IX and X.
Hydroxylation of aspartic acid to erythro-beta-aspartic acid (Hya) occurs in epidermal growth factor (EGF)-like modules in numerous extracellular proteins with diverse functions. Several EGF-like modules with the consensus sequence for hydroxylation bind Ca2+, and it has therefore been suggested that the hydroxyl group is essential for Ca2+ binding. To determine directly the influence of beta-hydroxylation on calcium binding in the EGF-like modules from coagulation factors IX and X, we have now measured calcium binding to both the fully beta-hydroxylated and the non-beta-hydroxylated modules of the two proteins. At low ionic strength, the Hya-containing module of factor X has a slightly higher Ca2+ affinity, but at physiological salt concentrations this difference is no longer significant for either factor IX or X. Analysis of the 1H NMR chemical shift differences between the hydroxylated and nonhydroxylated factor X modules show that hydroxylation has no effect on the domain fold. Furthermore, measurements on factor IX show that hydroxylation has no effect on the Ca2+/Mg2+ specificity of the ion binding site. We conclude that the hydroxyl group is not a direct ligand for the calcium ion in these EGF-like modules, nor is it essential for high-affinity Ca2+ binding.
Sunnerhagen
M S
MS
Department of Physical Chemistry, Lund University, Sweden.
Persson
E
E
Dahlqvist
I
I
Drakenberg
T
T
Stenflo
J
J
Mayhew
M
M
Robin
M
M
Handford
P
P
Tilley
J W
JW
Campbell
I D
ID
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D004815:Epidermal Growth Factor / Q000737:chemistry; D005164:Factor IX / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry
D002135:Calcium-Binding Proteins; D001224:Aspartic Acid; D014443:Tyrosine; D004815:Epidermal Growth Factor; D005164:Factor IX; D005170:Factor X; D002118:Calcium