The Relative Orientation of Gla and EGF Domains in Coagulation Factor X Is Altered by Ca²⁺ Binding to the First EGF Domain. A Combined NMR−Small Angle X-ray Scattering Study^,

Abstract

Coagulation factor X is a serine protease containing three noncatalytic domains: an N-terminal gamma-carboxyglutamic acid (Gla)1 domain followed by two epidermal growth factor (EGF)-like domains. The isolated N-terminal EGF domain binds Ca2+ with a Kd of 10(-3) M. When linked to the Gla domain, however, its Ca2+ affinity is increased 10-fold. In this paper, we present the NMR solution structure of the factor X Gla-EGF domain pair with Ca2+ bound to the EGF domain, as well as small angle X-ray scattering (SAXS) data on the Gla-EGF domain pair with and without Ca2+. Our results show that Ca2+ binding to the EGF domain makes the Gla and EGF domains fold toward each other using the Ca2+ site as a hinge. Presumably, a similar mechanism may be responsible for alterations in the relative orientation of protein domains in many other extracellular proteins containing EGF domains with the consensus for Ca2+ binding. The results of the NMR and SAXS measurements reported in this paper confirm our previous result that the Gla domain is folded also in its apo state when linked to the EGF domain [Sunnerhagen, M., et al. (1995) Nat. Struct. Biol. 2, 504-509]. Finally, our study clearly demonstrates the powerful combination of NMR and SAXS in the study of modular proteins, since this enables reliable evaluation of both short-range (NMR) and long-range interactions (SAXS).

Deletion of Epidermal Growth Factor-like Domains Converts Mammalian Tolloid into a Chordinase and Effective Procollagen C-proteinase

“…These observations are in agreement with a number of studies where it was demonstrated that conformational changes in structure can be associated with calcium ion binding, e.g. troponin C (41), fibrillin-1 (42,43), and the clotting factors IX and X, as well as the complement component C1r, where there is a change in orientation or alignment of domains (44,45). Furthermore, the conserved carboxylate residues contained in the EGF domain that are responsible for binding calcium ions with high affinity (D/N)X(D/N)(E/Q)(X) m (D/N)*(X) n (Y/F) (see Ref.…”

“…Furthermore, we propose that a hairpin conformation of mTld holds the CUB4 and -5 domains in locations that exclude substrates cleavable by BMP-1 or limit their access to the active site. Indeed, binding of Ca 2ϩ by an isolated EGF domain has been shown to result in little effect on its conformation; instead changes appear to have longer range effects involving neighboring domains (44,45). Taken together, our data imply a role of the two C-terminal CUB in regulating the enzymatic activity of mTld by a substrate-exclusion mechanism.…”

Localization of Phosphatidylserine Binding Sites to Structural Domains of Factor Xa

“…A Ca 2ϩ -dependent interaction between the EGF-like and Gla modules appears to enhance the affinity of the site on the EGF-like module to the point that it is tighter (17, 18) (k d ϳ 120 M) than the catalytic domain site. Consistent with this, nuclear magnetic resonance shows that Ca 2ϩ binding tightens the fold of the isolated EGF N domain and bends Gla and EGF N domains toward each other around a hinge located in the Gla domain, referred to as a helical or hydrophobic stack (19).…”

“…In terms of secondary structure, this translated into a large increase in ⌰ 222 /⌰ 208 and a nearly insignificant decrease in ␣-helical content (Tables I and II). It is known from NMR studies that Gla-EGF N undergoes a significant structural reorganization in which the EGF and Gla domains fold onto each other upon binding Ca 2ϩ (19). C6PS induced a structural change in Gla-EGF N in the absence of Ca 2ϩ (⌰ 222 /⌰ 208 increased by 333% and helicity increased by 2.3%; Fig.…”

“…This is not surprising, because the solution structure of bovine factor X a EGF N is reported to consist almost entirely of anti-parallel ␤-sheets and turns (39) and Ca 2ϩ binding to Gla-EGF N seems not to alter its fold (19). This is another example of how the ⌰ 222 /⌰ 208 ratio reflects more than the ␣-helical content of a protein, and, in this case, must reflect Ca 2ϩ -induced structural rearrangements that alter the electronic symmetry of EGF N .…”