Prev. Article Next Article Table of Contents

Article

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^†^,^‡

Supporting Info

Maria Sunnerhagen,*^§ Glenn A. Olah, Johan Stenflo,^# Sture Forsén,^§ Torbjörn Drakenberg,^§ and Jill Trewhella*

Physical Chemistry 2, Chemical Center, Lund University, P.O. Box 124, S-221 00 Lund, Sweden, Chemical Science and Technology Division, Mail Stop G758, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, and Clinical Chemistry, Lund University, Wallenberg Laboratory, Malm General Hospital, S-214 01 Malm, Sweden

Biochemistry, 1996, 35 (36), pp 11547–11559

DOI: 10.1021/bi960633j

Publication Date (Web): September 10, 1996

†

This work was supported by grants from the Swedish Board of Technical Development and the Swedish Medical Research Council. The NMR spectrometer was purchased with grants from the Knut and Alice Wallenberg Foundation and the Swedish Council for Planning and Coordination of Research. Support for J.T. and G.A.O. was provided by National Institutes of Health Award GM40528 and the Department of Energy's Office of Health and Environmental Research project HA-02-02-03. M.S. acknowledges the Fredrika Bremer Society and Fysiografiska Sällskapet for generous travel grants.

‡

The coordinates for the apo and 1 Ca forms have been deposited in the Brookhaven Protein Data Bank (access no. 1WHE and 1WHF, respectively).

To whom correspondence should be addressed.

Chemical Center, Lund University.

Current address: Molecular Biophysics, Karolinska Institute, Doktorsringen 6A, S-171 77 Stockholm, Sweden.

Los Alamos National Laboratory.

Clinical Chemistry, Lund University.

Abstract

Coagulation factor X is a serine protease containing three noncatalytic domains: an N-terminal γ-carboxyglutamic acid (Gla)¹ domain followed by two epidermal growth factor (EGF)-like domains. The isolated N-terminal EGF domain binds Ca²⁺ with a K_d 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).