(Received for publication, November 14, 1994; and in revised form, February 2, 1995)
From the
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
H 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
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.
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) (
)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 helices.
The carboxyl-terminal two helices are separated by a reverse turn, and
a single
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
helix at the carboxyl terminus
(residues 36-47). This
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.
Sequence-specific resonance assignments were made in two steps: 1)
identification of intraresidue spin systems using the H-
H 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
O was
used to distinguish aromatic from amide protons, and expose
protons that were near the presaturated water resonance in
H
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 J
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
. 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
J
< 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
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 HO into
D
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.
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 H 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 H 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
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
, 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
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 HO at pH 5.2 and 25 °C and a NOESY
spectrum at 15 °C. Additionally, a NOESY spectrum was obtained in
D
O to simplify aromatic side chain proton assignments and
to uncover
protons buried underneath the water resonance. We
assigned all of the proton resonances by determining intraresidue spin
systems and making sequential connectivities of
and
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
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
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 H NMR spectra
of apoFactor IX (1-47) in the
-NH proton region. PanelA, a two-dimensional DQF-COSY spectrum shows intraresidue
-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
-NH cross-peaks of the carboxyl-terminal
helix
are shown. The NOE mixing time is 200 ms. Gla residues are represented
by the symbol X. Note that the W42
-Q43NH cross-peak shown in parentheses overlaps with the
3 side-chain proton of
Trp-42 (W42
3). The experimental conditions are the same as for Fig. 1A.
Figure 3:
Two-dimensional H 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
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
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 helix, is shown. The amino acid
type is shown at the top of the figure. The boxes below each residue
are black for
J
< 6 Hz, open for
J
8 Hz, and
shaded for
J
between 6 and 8 Hz.
J
< 6 Hz are found in
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
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-
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
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 carbon backbone of the
lowest energy structure. PanelA, residues
36-47 is the carboxyl-terminal
helix, which is only well
defined for residues 37-46 (average r.m.s. deviation is 0.7 and
0.8 Å for the
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
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
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 DO, 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
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
helix. ApoFactor IX (1-47), black; prothrombin, gray.
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
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. ()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 H 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
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
helix (36-47)(9) . This same
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
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
helical segment identified, the backbone
structures are very similar (Fig. 7C). Therefore, the
conserved
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 H, H
, 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) . (
)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
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
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 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 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
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.