©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure of the Metal-free -Carboxyglutamic Acid-rich Membrane Binding Region of Factor IX by Two-dimensional NMR Spectroscopy (*)

(Received for publication, November 14, 1994; and in revised form, February 2, 1995)

Steven J. Freedman (1) (2) (3) Barbara C. Furie (1) (2) (3) Bruce Furie (1) (2) (3) James D. Baleja (3)(§)

From the  (1)Center for Hemostasis and Thrombosis Research, Division of Hematology-Oncology, New England Medical Center and the Departments of (2)Medicine and (3)Department of Biochemistry, Tufts University School of Medicine and Sackler School of Biomedical Sciences, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The -carboxyglutamic acid-rich domain of blood coagulation Factor IX is required for the binding of the protein to phospholipid membranes. To investigate the three-dimensional structure of this domain, a synthetic peptide corresponding to residues 1-47 of Factor IX was studied by ^1H NMR spectroscopy. In the absence of metal ions, the proton chemical shift dispersion in the one-dimensional NMR spectrum indicated that the peptide contains regular structural elements. Upon the addition of Ca(II) or Mg(II), large chemical shift changes were observed in the amide proton and methyl proton regions of the spectrum, consistent with the conformational transitions that metal ions are known to induce in native Factor IX. The apopeptide was studied by two-dimensional NMR spectroscopy at 500 MHz to determine its solution structure. Protons were assigned using total correlation spectroscopy, nuclear Overhauser effect spectroscopy, and double quantum-filtered correlation spectroscopy experiments. Intensities of cross-peaks in the nuclear Overhauser effect spectrum were used to generate a set of interproton distance restraints. The structure of the apopeptide was then calculated using distance geometry methods. There are three structural elements in the apopeptide that are linked by a flexible polypeptide backbone. These elements include a short amino-terminal tetrapeptide loop (amino acids 6-9), the disulfide-containing hexapeptide loop (amino acids 18-23), and a carboxyl-terminal alpha helix (amino acids 37-46). Amide hydrogen exchange kinetics indicate that the majority of the peptide is solvent accessible, except in the carboxyl-terminal element. The structured regions in the apopeptide are insufficient to support phospholipid binding, indicating the importance of additional structural features in the Ca(II)-stabilized conformer.


INTRODUCTION

Factor IX is a member of a family of vitamin K-dependent plasma zymogens involved in blood coagulation(1) . These proteins are synthesized as precursor proteins with a propeptide and an amino-terminal domain rich in glutamic acid residues. Prior to cleavage, the propeptide, which contains the recognition unit for a vitamin K-dependent carboxylase, directs post-translational -carboxylation of the adjacent glutamic acid residues in this amino-terminal domain(2) . This modification is critical for the biological function of Factor IX. Factor IX contains 12 -carboxyglutamic acid (Gla) (^1)residues. The Gla residues are metal ion ligands(3) . Upon interaction with Ca(II), Factor IX binds to acidic phospholipid membranes(4, 5) . Metal ions induce two sequential conformational transitions; the first is metal ion-nonspecific, and the second is Ca(II)-specific(5) . Only the second conformational change results in a structure competent to bind phospholipids. Both conformational changes in Factor IX can be detected by quenching of tryptophan fluorescence (6) or by metal-dependent antibody recognition of newly exposed epitopes(5) . Therefore, -carboxylation is a primary structural modification during biosynthesis that is a prerequisite to the Ca(II)-induced structural transition.

The region containing the Gla residues has been designated the Gla domain in vitamin K-dependent factors. The Gla domain is interposed between a propeptide and an aromatic amino acid stack domain in the prozymogens(1) . In Factor IX, the propeptide and Gla domain (residues -18 to 38) and the aromatic amino acid stack domain (residues 39 to 46) are encoded by exon II and exon III, respectively(7, 8) . The Gla and aromatic amino acid stack domains are highly homologous among the vitamin K-dependent factors. For instance, human Factor IX and bovine prothrombin are 54% identical in this region.

The crystal structure of Ca(II)-bound bovine prothrombin fragment I reveals a Gla domain structure that coordinates seven calcium ions(9) . The carboxyl-terminal three quarters of the Gla domain consists primarily of 9-10 helical turns spread over three different alpha helices. The carboxyl-terminal two helices are separated by a reverse turn, and a single alpha helical turn precedes the disulfide loop. The remaining amino-terminal segment forms a large loop structure. The calcium ions line an internal carboxylate groove in the amino-terminal half of the domain. In the absence of calcium ions, there is insufficient electron density to define the polypeptide backbone of the first 35 residues(19, 20) . The Gla domain is apparently disordered, except for an alpha helix at the carboxyl terminus (residues 36-47). This alpha helix is common to both the apo- and Ca(II)-bound fragment I structures.

We have previously demonstrated that the Gla domain and the adjacent aromatic amino acid stack domain (residues 1-47) form the unit responsible for binding phospholipid membranes in Factor IX(10) . This was demonstrated by studying the properties of a chemically synthesized peptide, Factor IX (1-47), that contains 12 Gla residues. This peptide binds Mg(II) and Ca(II) ions and undergoes the metal ion-induced structural transitions, as monitored by intrinsic tryptophan fluorescence quenching and recognition of metal-induced epitopes by conformation-specific antibodies. In the presence of Ca(II), but not Mg(II) or a metal-free environment, Factor IX (1-47) bound specifically to acidic phospholipid vesicles, with a K of 0.64 µM. Thus, Factor IX (1-47) is an excellent model for examining the structural properties of Factor IX at an atomic level, since it is sufficiently small for two-dimensional NMR studies, and retains the functional properties of the native protein. We report here the three-dimensional structure of apoFactor IX (1-47) as determined by two-dimensional NMR spectroscopy.


MATERIALS AND METHODS

Preparation of Factor IX (1-47)

Factor IX (1-47) was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl)/NMP chemistry on an Applied Biosystems model 430A peptide synthesizer as described previously(10) . The two cysteines of the crude, deprotected peptide were allowed to spontaneously oxidize in a solution of 50 mM ammonium bicarbonate, pH 8.0, by stirring at 4 °C for 18 h. Subsequently, the peptide sample was lyophilized and then purified by HPLC over a preparative reverse phase C18 column (Vydac) using a linear gradient of 30-40% acetonitrile, 0.1% trifluoroacetic acid. Purification was performed on a Beckman System Gold HPLC with a flow rate of 4 ml/min. For NMR spectroscopy, the purified peptide was dissolved in HPLC-grade H(2)O, and the concentration was quantitated by absorption at 280 nm ( = 8,370 M cm). The samples were treated with Chelex 100 to remove trace metal ions, and the solution was adjusted to pH 5.2 with HCl and NaOH. For samples containing metal ions, CaCl(2) and MgCl(2) were titrated into peptide solutions, and then the pH was readjusted to 5.7 and 5.8, respectively. For the sample containing CaCl(2), 1 M NaCl was included since it was necessary for solubility of the peptide.

NMR Spectroscopy

For a 3 mM sample in D(2)O, peptide was lyophilized and then redissolved in 99.96% D(2)O. Samples in H(2)O were prepared by dissolving the peptide in H(2)O containing 10% D(2)O as the deuterium lock signal. Spectra were collected on a Bruker AMX-500 spectrometer with a proton frequency of 500.14 MHz. The carrier frequency was set on the water resonance, which was suppressed using presaturation. One-dimensional NMR spectra were collected for samples, which ranged in concentration between 0.8 mM and 3 mM peptide depending on the presence or absence of metal ions. Temperatures between 5 and 35 °C were used to evaluate the stability of the sample and resonance line widths. The data were acquired with 1024 real points and spectral widths of approximately 7000 Hz. One-dimensional spectra were processed on a Sun SPARCstation 2 using the program Felix (Biosym Technologies, San Diego, CA). The solvent peak was removed with a sine bell convolution function. The spectra were processed using sine bell window functions shifted by 45 °C and zero filled to 4096 real points. Two-dimensional NOESY spectra were recorded with mixing times of 75 and 200 ms for a 2.1 mM peptide sample at 25 °C, and with a mixing time of 200 ms for the sample at 15 °C. A total of 2048 real data points was acquired in t(2), 512 time-proportional phase incrementation increments in t(1), a spectral width of 6024.1 Hz in the F2 dimension, 144 summed scans, and a relaxation delay of 1.3 s between scans. Spectra were multiplied with sine bell window functions shifted by 36 °C in t(2) (applied over 1024 points) and 45 °C in t(1) (applied over all 512 points) and zero filled to a 2048 by 1024 (real) matrix using the Bruker NMR processing program. NOESY cross-peak intensities were converted into distances and calibrated using published methods(11, 12) . A TOCSY spectrum was recorded and processed using identical parameters as the NOESY, except with a mixing time of 38.6 ms, 64 summed scans, and using an MLEV-17 mixing sequence(13) . A DQF-COSY spectrum was recorded with 2048 real t(2) points and with a spectral width of 6024.1 Hz, 96 summed scans, and 768 time-proportional phase incrementation increments. Spectra were multiplied by a sine bell window function shifted by 22.5 °C in t(2) and 30 °C in t(1) and zero filled to a 2048 by 1024 (real) matrix.

Sequence-specific resonance assignments were made in two steps: 1) identification of intraresidue spin systems using the ^1H-^1H through-bond connectivities found in TOCSY and DQF-COSY spectra and 2) sequentially assigning residues on the basis of sequential d, d, and d NOE connectivities(14) . NOESY spectra were obtained at two different temperatures (25 and 15 °C) to separate resonances that may be overlapping or to shift resonances bleached out by saturation of the water resonance under one set of conditions but not the other. Finally, a NOESY spectrum collected on a sample in D(2)O was used to distinguish aromatic from amide protons, and expose alpha protons that were near the presaturated water resonance in H(2)O spectra. Short-range and medium-range NOE interactions could then be determined using a complete set of proton resonance assignments.

The vicinal spin-spin coupling constants ^3J were used to calculate torsion angles(14) . The coupling constants were measured from the splitting of amide cross-peaks in a NOESY spectrum that was resolution-enhanced by multiplying with a squared sine bell window function shifted by 30 °C and applied over 2048 (real) points in t(2). For some residues, torsion angle constraints were confined to the negative angle region by referring to both the size of the coupling constant and the intensity of NOE cross-peaks for intraresidue d(i,i), and sequential d(i,i+1) and d(i,i+1)(14) . Only residues with ^3J < 6 Hz were used for structure determination.

Structure determination used a set of 600 distance restraints (intraresidue, 222; sequential, 236; short and medium range, 142) and 21 torsion angles that were entered into the DGII program of InsightII (Biosym Technologies, San Diego, CA). A combination of distance geometry and simulated annealing methods (15) generated 15 structures, all of which converged. Using the alpha carbon trace of the well defined residues, the structures were superimposed with the lowest energy structure. Average root mean square (r.m.s.) deviation values of the backbone atoms in different regions of the peptide reflected the quality of the structures determined.

Amide hydrogen exchange kinetics were performed by following the time-dependent decay of amide proton resonance intensity after dissolving Factor IX (1-47) lyophilized from H(2)O into D(2)O solution at pH 5.0 and 25 °C. One-dimensional spectra were acquired at various time points over a 35-min period. Intensities of specific amide proton resonances at each time point were measured from the peak heights. The initial intensities of each amide proton observed were normalized to 100%. The data points were fitted to a first order exponential decay equation, and corresponding exchange rate constants were derived from each decay curve.


RESULTS

Factor IX (1-47) peptide contains the Gla and aromatic amino acid stack domains of Factor IX. This peptide approximates the minimal region of Factor IX required for phospholipid binding. As a first step to the determination of the phospholipid binding structure of Factor IX, we analyzed ^1H NMR spectra of Factor IX (1-47) under the following three different conditions: metal-free (apo)Factor IX (1-47), Ca(II)-bound Factor IX (1-47), and Mg(II)-bound Factor IX (1-47). At temperatures between 5 and 35 °C and pH values between 4.5 and 7.5, we obtained one-dimensional spectra (Fig. 1A) for the apoFactor IX (1-47), which were similar and insensitive to changes in ionic strength. The proton resonances gave spectrally disperse peaks, especially in the amide proton region, where many peaks were at least 0.15 ppm from the random coil value of about 8.4 ppm(14) . This indicates that apoFactor IX (1-47) is at least partially structured.


Figure 1: One-dimensional ^1H NMR spectra of Factor IX (1-47). PanelA, Factor IX (1-47) in the absence of metal ions. The spectrum shows some dispersion of amide resonances indicating regions of structure. The sample contains Chelex-treated peptide (2.1 mM) in H(2)O at pH 5.2, 25 °C. The spectrum was acquired with 128 scans. PanelB, Factor IX (1-47) in the presence of calcium ions. The sample contains 0.8 mM peptide, 11.2 mM CaCl(2), and 1 M NaCl at pH 5.7, 25 °C. The spectrum was acquired with 256 scans. PanelC, Factor IX (1-47) in the presence of magnesium ions. The sample contains 1.5 mM peptide and 15 mM MgCl(2) at pH 5.8, 25 °C. The spectrum was acquired with eight scans.



Upon the addition of Ca(II), the proton spectrum revealed increased dispersion of resonances in all regions of the spectrum (>0.4 ppm from the random coil values) (Fig. 1B). Specifically, there were downfield-shifted amide proton resonances and upfield-shifted methyl proton resonances. In addition, the metal-free and Ca(II)-bound forms of the peptide were in slow exchange on an NMR time scale. For example, the tryptophan indole amide protons from apoFactor IX (1-47) and the Factor IX (1-47):Ca(II) binary complex could be observed simultaneously at 10.25 and 10.19 ppm, respectively. These results suggest that a new structure is adopted following the addition of calcium ions that is different from and likely more structured than the metal-free form.

The Factor IX (1-47):Mg(II) binary complex (Fig. 1C) yielded a spectrum that differed from that of apoFactor IX (1-47) and Factor IX (1-47):Ca(II). Increased dispersion of resonances was observed relative to the apoFactor IX (1-47), and the spectrum differed from that of the Factor IX (1-47):Ca(II) complex, particularly in the amide region. ApoFactor IX (1-47) was also in slow exchange with the Mg(II)-bound form of Factor IX (1-47). Sharper resonance line widths could be achieved by reducing the temperature to 5 °C, thus suggesting exchange broadening(14) .

To understand the differences between the structures of the Gla domain of the vitamin K-dependent proteins in the absence of calcium ions and in the presence of calcium ions, we determined the structure of apoFactor IX (1-47) by two-dimensional NMR spectroscopy. We collected two-dimensional NOESY, TOCSY, and DQF-COSY spectra of apoFactor IX (1-47) in H(2)O at pH 5.2 and 25 °C and a NOESY spectrum at 15 °C. Additionally, a NOESY spectrum was obtained in D(2)O to simplify aromatic side chain proton assignments and to uncover alpha protons buried underneath the water resonance. We assigned all of the proton resonances by determining intraresidue spin systems and making sequential connectivities of alpha and beta protons to backbone amide protons of neighboring residues ( Table 1and Fig. 2). In the amide-amide proton region of the NOESY spectrum (Fig. 3), we observed sequential NH-NH contacts, which indicated alpha helical structure in the carboxyl terminus of the peptide(14) . Following the assignment of all proton resonances, short range and medium range interactions were defined from the NOESY spectrum. The absence of any long range interactions indicated the lack of a compact tertiary structure or the presence of different conformers rapidly interconverting on an NMR time scale. Coupling constants between vicinal alpha and amide protons were measured from the splitting of amide proton cross-peaks of a resolution-enhanced NOESY spectrum. From the coupling constants, corresponding torsion angles were measured that were then used to define the backbone conformation of the peptide.




Figure 2: Two-dimensional ^1H NMR spectra of apoFactor IX (1-47) in the alpha-NH proton region. PanelA, a two-dimensional DQF-COSY spectrum shows intraresidue alpha-NH cross-peaks identified by one-letter symbols and residue numbers. PanelB, the same region of a two-dimensional NOESY spectrum. For simplicity of illustration, only line connectivities and residue numbers of the intraresidue and sequential alpha-NH cross-peaks of the carboxyl-terminal alpha helix are shown. The NOE mixing time is 200 ms. Gla residues are represented by the symbol X. Note that the W42alpha-Q43NH cross-peak shown in parentheses overlaps with the 3 side-chain proton of Trp-42 (W423). The experimental conditions are the same as for Fig. 1A.




Figure 3: Two-dimensional ^1H NMR spectrum of apoFactor IX (1-47) in the amide-amide proton region. For simplicity of illustration, only cross-peaks involving sequential amide protons for the carboxyl-terminal alpha helix are labeled by residue number. The NOE mixing time is 200 ms. The experimental conditions are the same as those for Fig. 1A.



Although short range and medium range interactions were observed throughout the peptide, most of these interactions were concentrated in three regions. First, the side-chain and backbone protons of Leu-6 and Phe-9 made extensive interresidue contacts. Second, the covalent connectivity of a disulfide bond defines a loop between Cys-18 and Cys-23, and contacts were observed throughout this hexapeptide sequence. Third, as suggested by the coupling constant data, amide-amide proton contacts, and medium range interactions of residue i with residue i+3, the peptide sequence from Arg-37 to Val-46 appeared alpha helical (14) (Fig. 4). In this segment, we found coupling constants below 6.0 Hz, and sequential d(i,i+1), d(i,i+3), and d(i,i+3) in a sequence of 3 or more residues.


Figure 4: Summary of sequential and medium-range NOEs. The amino acid sequence of residue numbers 35-47, which contains the carboxyl-terminal alpha helix, is shown. The amino acid type is shown at the top of the figure. The boxes below each residue are black for ^3J< 6 Hz, open for ^3Jgeq 8 Hz, and shaded for ^3Jbetween 6 and 8 Hz.J< 6 Hz are found in alpha helical structures. The first threelinesbelow the sequence show (i,i+1) connectivities that were analyzed from a NOESY spectrum. A thinline indicates a weak NOE, and a thickline indicates a strong one. Medium-range NOEs typical of alpha helical structures are shown in the lower fourlines.



To determine the structure of apoFactor IX (1-47), NOESY cross-peaks were converted into a set of distance restraints. This was achieved by using the number of contours of a cross-peak to establish upper and lower limit distances after calibrating against internal standards of NH-alpha distances(11, 12) . A set of 600 distance restraints, of which 222 were intraresidue, 236 were sequential, and 142 were short and medium range, and 21 torsion angles were used to generate 15 final structures by a combination of simulated annealing and distance geometry methods. A pairwise superposition of backbone atoms of all structures with the lowest energy structure showed large root mean square deviations. This is consistent with the absence of long range interactions in the NOESY spectra and in contrast to the tertiary interactions in the Ca(II)-bound Gla domain of prothrombin (9) . Three structured regions were found by comparing all calculated structures of apoFactor IX (1-47). Leu-6 to Phe-9 is a tetrapeptide loop with a r.m.s. deviation of 0.9 Å (Fig. 5C). Cys-18 to Cys-23 is a disulfide-containing hexapeptide loop with an r.m.s. deviation of 1.3 Å (Fig. 5B). Arg-37 to Val-46 is an alpha helix of 2.5 turns with an r.m.s. deviation of 0.8 Å (Fig. 5A). These three structured segments exist autonomously in that they appear to have no interactions with each other or with other portions of the molecule (Fig. 6). In total, they include 20 residues within the 47-residue peptide.


Figure 5: Stereoview overlay of the 15 calculated structures for different segments of apoFactor IX (1-47). All segments are shown superimposed with the alpha carbon backbone of the lowest energy structure. PanelA, residues 36-47 is the carboxyl-terminal alpha helix, which is only well defined for residues 37-46 (average r.m.s. deviation is 0.7 and 0.8 Å for the alpha carbons and backbone atoms, respectively). PanelB, residues 18-23 is the hexapeptide, disulfide loop (average r.m.s. deviation is 1.1 and 1.3 Å for the alpha carbons and backbone atoms, respectively). PanelC, residues 6-9 of Factor IX (1-47) is a tetrapeptide loop (average r.m.s. deviation is 0.5 and 0.9 Å for the alpha carbons and backbone atoms, respectively).




Figure 6: Positions of the three structured regions of apoFactor IX (1-47). The well defined portions of the molecule shown in Fig. 5are indicated by ribbons within the context of the lowest energy structure of apoFactor IX (1-47). The structured regions are independent, noninteracting structural motifs.



Amide hydrogen exchange kinetics were used to partially characterize the dynamics of the apoFactor IX (1-47) structure. The protonated peptide was dissolved in D(2)O, and the disappearance of amide protons was observed over time. Only eight amide protons were observed at 7 min, and none remained at 35 min (data not shown). The 8 amides that were relatively stable to exchange were assigned to the carboxyl-terminal portion of the peptide in the region that had alpha helical structure (residues 35, 38, 39, and 41-45). The amide intensities of these residues measured at various time points between 7 and 35 min were fitted to a first-order exponential decay equation in order to derive corresponding rate constants. The exchange rate constants ranged between 0.24 min (Trp-42) and 0.10 min (Tyr-45). These rates are approximately 50 times slower than the intrinsic exchange rates measured for solvent accessible amide protons in polypeptides at 25 °C and pH 5.0(14) . All other residues had exchange rates that were greater than 0.24 min.

The three segmental structures calculated for apoFactor IX (1-47) were compared with analogous portions of the Ca(II)-bound prothrombin fragment I crystal structure. We used the lowest energy apoFactor IX (1-47) structure for superposition with Ca(II)-bound prothrombin. A superposition of the entire 1-47 sequence showed that the prothrombin structure was more compact than apoFactor IX (1-47) with no obvious similarities between the two structures. However, the three substructures from each molecule had root mean square deviation values of approximately 1 Å and were therefore very similar to the analogous substructures of prothrombin (Fig. 7, A-C).


Figure 7: Superposition of the three structured segments of apoFactor IX (1-47) with the analogous portions of the prothrombin fragment I crystal structure. PanelA, the 6-9 loop; panelB, the 18-23 disulfide loop; panelC, the 37-46 alpha helix. ApoFactor IX (1-47), black; prothrombin, gray.




DISCUSSION

Factor IX is a key component in the blood coagulation cascade. Factor IX is converted to its enzymatically active form, Factor IXa, by Factor XIa or the Factor VIIa:tissue factor complex. In turn, Factor IXa and its cofactor, Factor VIIIa, activate Factor X(1) . The biological role of Factor IX and Factor IXa as a substrate and enzyme in these processes is critically dependent on their calcium-dependent membrane binding function. First, Factor IXa and Factor VIIIa assemble on membrane surfaces in a calcium-dependent interaction to form the tenase complex. Membrane surfaces substantially enhance the rate of Factor X activation by the Factor IXa:Factor VIIIa complex (16) . Second, Factor IX is activated in the presence of Ca(II) by the Factor VIIa:tissue factor complex(17) ; this reaction requires phospholipids. Therefore, the phospholipid binding activity of Factor IX is integral to its biological role in blood coagulation.

The phospholipid binding site on Factor IX has been localized to the Gla/aromatic amino acid stack domains at the amino terminus of the protein(10) . The unique malonate structure of Gla is essential for function since des--carboxyl Factor IX does not bind phospholipids (5) . Occupancy of calcium-binding sites in the Gla domain induces a conformational transition that exposes a membrane binding surface in this region of Factor IX(5) . Conversely, removal of calcium ions with EDTA reverses the structural transition and phospholipid binding. Determination of the structures of the two conformational states is critical in understanding the nature of this reversible calcium-mediated transition, which leads to phospholipid binding. Presently, there are no Factor IX structures that describe this phenomenon at an atomic level. Furthermore, there are no NMR- or x-ray-derived structures for full-length metal-free Gla domains of vitamin K-dependent proteins.

In this report, we have described the NMR-derived structure of apoFactor IX (1-47), a peptide that undergoes the calcium-mediated conformational transition and contains the calcium-dependent phospholipid binding properties of Factor IX(10) . The addition of Ca(II) or Mg(II) resulted in changes in the one-dimensional NMR spectra that are consistent with the conformational transitions previously observed biochemically. The structure of apoFactor IX (1-47) determined by two-dimensional NMR revealed three well defined structural regions of the peptide backbone: two loops, Leu-6-Phe-9 and Cys-18-Cys-23, and an alpha helix, Arg-37-Val-46, separated by poorly defined regions in the polypeptide backbone.

There is evidence to suggest that Gla domains of native proteins only form compact structures in the presence of metal ions. The Gla domain of apoprothrombin fragment I is disordered in the crystal structure (19, 20) , suggesting an unfolded or highly mobile structure. Additionally, full-length Factor IX crystallized in the absence of metal ions lacks electron density for the Gla domain. (^2)In contrast, in the presence of calcium ions, the Gla domain of prothrombin fragment I has a compact structure(9, 21) . Vysotchin et al.(22) showed that the Factor IX Gla domain, either isolated as a 6-kDa fragment, or in the context of a 25-kDa fragment containing both EGF domains, undergoes a lower amplitude and 5-10 °C lower melting transition in the absence of metal ions compared with the presence of calcium ions(22) . They interpreted the results as a calcium-induced folding transition to a more compact state. In sum, these results suggest that the conformational equilibrium mediated by metal ions is between a relatively flexible apostructure and a compact metal-bound structure rather than between two different well ordered structures. The results presented here are consistent with this model since apoFactor IX (1-47) contains unstructured regions as well as three well ordered regions.

Although we have no direct experimental evidence for a spatially diffuse structure, there are several arguments that suggest that this is the case. First, the dispersion of ^1H resonances in the apoFactor IX (1-47) spectrum is much less than the metal-containing spectra. Second, the absence of long range interactions between protons in the NOESY spectrum precludes the definition of any tertiary structure in the molecule. Particularly noteworthy is the absence of interaction between the amino-terminal Tyr-1 and Gla-17, Gla-21, and Gla-27, a distinctive feature of the prothrombin:Ca(II) complex(9) . Third, there is undefinable electron density in the Gla region of crystallized apoprothrombin fragment I (19, 20) . Fourth, following exchange of apoFactor IX (1-47) into D(2)O solution at pH 5.0, the majority of amide proton resonances disappear within 7 min. This suggests that large regions of peptide are exposed to solvent by comparison with the rapid exchange rates of unstructured peptides(14) . Thus, the NMR data, consistent with the calorimetric data, provide no evidence for a rigid compact apoFactor IX (1-47) structure.

In the absence of calcium ions, the Gla/aromatic amino acid stack domains of crystallized prothrombin fragment I has a single defined structural element, a carboxyl-terminal alpha helix (36-47)(9) . This same alpha helix is found in the Ca(II)-bound form. This finding is consistent with the results of Vysotchin et al.(22) , who demonstrated by circular dichroism that a proteolytically-derived, 6-kDa Gla module from human Factor IX includes alpha helical character(22) . We have shown that in the absence of metal ions, this residual helical structure in Factor IX is found from amino acids 37-46 and includes 2.5 helical turns. Thus, Factor IX and prothrombin share this structural element in the absence of metal ions. In further support of the secondary structure of this region, a hydrogen/deuterium exchange experiment indicated that backbone amide protons of the carboxyl-terminal portion of the peptide were resistant to exchange. The likely basis for this finding is that the amide protons are stabilized by hydrogen bonding. This helical segment is clearly important for Gla domain structure and function, since truncation fragments lacking most or all of this sequence do not exhibit normal phospholipid binding(10, 23) . Despite conservation of only 4 out of the 10 amino acids between human Factor IX and bovine prothrombin in the alpha helical segment identified, the backbone structures are very similar (Fig. 7C). Therefore, the conserved alpha helical structure is perhaps important for orienting the conserved aromatic amino acid side chains of Phe-41, Trp-42, and Tyr-45 in all Gla domains.

The structured tetrapeptide loop Leu-Gla-Gla-Phe, representing residues 6-9, is a highly conserved sequence in the vitamin K-dependent proteins. In apoFactor IX (1-47) this sequence forms a loop that is primarily defined by extensive interactions between the Leu-6 side chain and the aromatic ring of Phe-9 (Leu-6 Halpha, Hbeta, H, and H to Phe-9 H, H, and H). Moreover, this loop is conserved in structure between apoFactor IX (1-47) and Ca(II)-bound prothrombin fragment I (Fig. 7A). Presently, Leu-6, Gla-7, and Gla-8 have been recognized as playing important roles in Gla domain structure and phospholipid binding for vitamin K-dependent proteins in the presence of calcium ions(24, 25, 26, 27, 28) . These conclusions are based primarily on mutagenesis studies. The conservation of this tetrapeptide loop sequence in the vitamin K-dependent proteins, its preserved structure in Factor IX (1-47) in the absence of metal ions, and its structural similarity to the homologous region in prothrombin fragment I in the presence of Ca(II) implicate this loop as a vital part of the Gla domain structure in vitamin K-dependent factors.

The disulfide-containing hexapeptide loop sequence, 18-23, in apoFactor IX (1-47) is also structured. This is due to the disulfide bond formed between Cys-18 and Cys-23 that is conserved in all of the vitamin K-dependent factors. By comparison to the prothrombin fragment I crystal structure, the two loops are structurally similar (Fig. 7B). The disulfide bond is an integral part of Gla domain structures, since disruption of this bond by a distal Cys Ser mutation in protein C or double Cys Ser mutations in prothrombin dramatically reduces the anticoagulant and coagulant activities, respectively(29) . (^3)This loop contains conserved Gla residues at positions 20 and 21. Gla-20 in prothrombin and Gla-21 in protein C play important roles since mutagenesis of either residue affects activity(25, 26) . From the crystal structure of prothrombin fragment I(9) , Gla 20 plays a central role in maintaining the fragment I structure since it interacts with calcium ions and Arg-55. The aromatic amino acid stack residues (Phe-41, Trp-42, and Tyr-45) interact with the Cys-18-Cys-23 loop in the prothrombin:Ca(II) complex. However, this interaction is not observed in the absence of Ca(II) for apoFactor IX (1-47).

ApoFactor IX (1-47) lacks some of the structural elements of Ca(II)-bound prothrombin fragment I(9) . The missing secondary structures include the large amino-terminal -like loop and two additional segments of alpha helices. In addition, apoFactor IX (1-47) does not have organized and interacting secondary structures as does Ca(II)-bound prothrombin fragment I. Specifically, the carboxyl-terminal alpha helix and the disulfide loop are independent structural units. Thus, the inability of apoFactor IX (1-47) to bind phospholipid membranes is a consequence of both these factors.

We believe the apoFactor IX (1-47) structure determined here reflects the structure of this fragment in the context of the intact Factor IX molecule. Intact Factor IX has been characterized by two conformational transitions from apo- to metal-bound forms: the apo- to Mg(II)-bound transition and the apo- to Ca(II)-bound transition(5) . In recent work, we have shown these transitions to be structurally and functionally mimicked by the Factor IX (1-47) peptide based on fluorescence quenching, conformational antibody recognition, and phospholipid binding(10) . Using these criteria, we infer that the metal-free structure is an adequate model for the Gla and aromatic amino acid stack domains of intact apoFactor IX. The structural findings presented for apoFactor IX (1-47) are also supported by the x-ray structures of apoFactor IX^2 and apoprothrombin fragment I(9) , itself a portion of the intact prothrombin species, but having extensive sequence carboxyl-terminal to the Gla and aromatic amino acid stack domains. These structures also suggest a relatively disordered Gla domain. Although there are known limitations of x-ray crystallography in defining regions of extreme mobility, our data are consistent with a disordered structure since NMR has the advantage of defining regions of structure that are mobile in solution. In this regard, our work adds two structured subregions not previously described for apoGla domains by x-ray crystallography, the 6-9 and 18-23 loops.

While it remains possible that crystallization of Ca(II):prothrombin fragment I has frozen out structural elements that do not exist in solution, we have evidence to the contrary. From preliminary NMR work with the Factor IX (1-47) peptide bound to Ca(II), we find NH-NH main-chain contacts in the NOESY spectrum for 95% of the amino acids, some of which are strong, and some of which are weak; the strong contacts have been tentatively assigned to alpha helical regions, which are exactly those predicted by the prothrombin x-ray structure. We also observe a number of long range interactions between methyl groups and aromatic side-chains. All long range NOEs are consistent with the short interresidue distances observed in the Ca(II):prothrombin fragment I structure obtained by x-ray crystallography(30) . This contrasts with the apoFactor IX (1-47) NOESY spectrum where no long range interactions are identified and where NH-NH contacts are confined to the limited regions of defined structure. This suggests the Factor IX (1-47):Ca(II) structure is more ordered, with extensive alpha helical and loop character and tertiary interactions, compared with the apoFactor IX (1-47) structure. However, an extensive comparison between the apo- and Ca(II)-bound Factor IX (1-47) structures is not yet available due to technical difficulties pertaining to solubility of the Ca(II):peptide complex, especially under conditions of low ionic strength (i.e. solubility of the Ca(II):peptide is partially improved in a 1 M NaCl solution).

The three portions of Factor IX (1-47) structured in the absence of metal ions, including 20 of 47 residues or about 40% of the peptide, may be particularly critical as part of the Ca(II):Gla domain structure. This is strongly suggested by the occurrence of homologous substructures in the prothrombin fragment I:Ca(II) crystal structure. It is likely that in the presence of Ca(II) these preformed secondary structural elements associate with each other and additional regions in the molecule to form the compact folded structure observed for prothrombin. In prothrombin, calcium ions facilitate the formation of intramolecular, noncovalent interactions that define the structure of the Gla domain. We speculate that the motifs described within are strategically structured so that they rapidly associate when Ca(II) is present, leading to the expression of the phospholipid binding site.


FOOTNOTES

*
This work was supported by Grant HL42443 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6872; Fax: 617-636-6409.

(^1)
The abbreviations used are: X or Gla, -carboxyglutamic acid; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; DQF-COSY, double quantum-filtered correlation spectroscopy; r.m.s., root mean square; HPLC, high performance liquid chromatography.

(^2)
W. Bode, personal communication.

(^3)
J. V. Ratcliffe, B. Furie, and B. C. Furie, unpublished observations.


ACKNOWLEDGEMENTS

We thank Margaret Jacobs for synthesizing the Factor IX (1-47) peptide.


REFERENCES

  1. Furie, B., and Furie, B. C. (1988) Cell 53, 505-518 [Medline] [Order article via Infotrieve]
  2. Jorgensen, M., Cantor, A., Furie, B. C., Brown, C. L., Shoemaker, C. B., and Furie, B. (1987) Cell 48, 185-191 [Medline] [Order article via Infotrieve]
  3. Sperling, R., Furie, B. C., Blumenstein, M., Keyt, B., and Furie, B. (1978) J. Biol. Chem. 253, 3898-3906 [Medline] [Order article via Infotrieve]
  4. Jones, M. E., Griffith, M. J., Monroe, D. M., Roberts, H. R., and Lentz, B. R. (1985) Biochemistry 24, 8064-8069 [Medline] [Order article via Infotrieve]
  5. Liebman, H. A., Furie, B. C., and Furie, B. (1987) J. Biol. Chem. 262, 7605-7612 [Abstract/Free Full Text]
  6. Morita, T., Isaacs, B. S., Esmon, C. T., and Johnson, A. E. (1984) J. Biol. Chem. 259, 5698-5704 [Abstract/Free Full Text]
  7. Kurachi, K., and Davie, E. W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6461-6464 [Abstract]
  8. Choo, K. H., Gould, K. G., Rees, D. J. G., and Brownlee, G. G. (1982) Nature 299, 178-180 [Medline] [Order article via Infotrieve]
  9. Soriano-Garcia, M., Padmanabhan, K., de Vos, A. M., and Tulinsky, A. (1992) Biochemistry 31, 2554-2566 [Medline] [Order article via Infotrieve]
  10. Jacobs, M., Freedman, S. J., Furie, B. C., and Furie, B. (1994) J. Biol. Chem. 269, 25494-25501 [Abstract/Free Full Text]
  11. Detlefsen, D. J., Thanabal, V., Pecoraro, V. L., and Wagner, G. (1991) Biochemistry 30, 9040-9046 [Medline] [Order article via Infotrieve]
  12. Hyberts, S. G., Goldberg, M. S., Havel, T. F., and Wagner, G. (1992) Protein Sci. 1, 736-751 [Abstract/Free Full Text]
  13. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 65, 355-360
  14. W ü thrich, K. (1986) NMR of Proteins and Nucleic Acids , John Wiley & Sons, Inc., New York
  15. Havel, T. F. (1991) Prog. Biophys. Mol. Biol. 56, 43-78 [CrossRef][Medline] [Order article via Infotrieve]
  16. van Dieigen, G., Tans, G., Rosing, J., and Hemker, H. C. (1981) J. Biol. Chem. 256, 3433-3441 [Abstract/Free Full Text]
  17. Osterud, B., and Rapaport, S. I. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5260-5264 [Abstract]
  18. Deleted in proof
  19. Park, C. H., and Tulinsky, A. (1986) Biochemistry 25, 3977-3982 [Medline] [Order article via Infotrieve]
  20. Harlos, K., Boys, C. W. G., Holland, S. K., Esnouf, M. P., and Blake, C. C. F. (1987) FEBS Lett. 224, 97-103 [CrossRef][Medline] [Order article via Infotrieve]
  21. Soriano-Garcia, M., Park, C. H., Tulinsky, A., Ravichandran, K. G., and Skrzypczak-Jankun, E. (1989) Biochemistry 28, 6805-6810 [Medline] [Order article via Infotrieve]
  22. Vysotchin, A., Medved, L. V., and Ingham, K. C. (1993) J. Biol. Chem. 268, 8436-8446 [Abstract/Free Full Text]
  23. Schwalbe, R. A., Ryan, J., Stern, D. M., Kisiel, W., Dahlbäck, B., and Nelsestuen, G. L. (1989) J. Biol. Chem. 264, 20288-20296 [Abstract/Free Full Text]
  24. Zapata, G. A., Berkowitz, P., Noyes, C. M., and Hiskey, R. G. (1987) Fed. Proc. 46, 2230
  25. Ratcliffe, J. V., Furie, B., and Furie, B. C. (1993) J. Biol. Chem. 268, 24339-24345 [Abstract/Free Full Text]
  26. Zhang, L., Jhingan, A., and Castellino, F. J. (1992) Blood 80, 942-952 [Abstract]
  27. Zhang, L., and Castellino, F. J. (1990) Biochemistry 29, 10828-10834 [Medline] [Order article via Infotrieve]
  28. Zhang, L., and Castellino, F. J. (1994) J. Biol. Chem. 269, 3590-3595 [Abstract/Free Full Text]
  29. Zhang, L., and Castellino, F. J. (1991) Biochemistry 30, 6696-6704 [Medline] [Order article via Infotrieve]
  30. Freedman, S. J., Jacobs, M., Furie, B. C., Baleja, J. D., and Furie, B. (1994) Blood 84, 531 (abstr.)

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