(Received for publication, September 8, 1995)
From the
The 2.6-Å x-ray crystal structure of bovine -thrombin
in complex with rhodniin, a protein inhibitor isolated from the bug Rhodnius prolixus, has been solved and refined. The structure
has enabled us to trace the N-terminal part of the 49-residue A-chain
of bovine
-thrombin for the first time, which is fixed in a
U-shaped loop on the molecular surface opposite the active site canyon.
Model building shows that the 25 amino acid residues that link the
A-chain and F2 kringle cannot run through the fibrinogen recognition
exosite. This demonstrates that this fibrinogen recognition exosite is
available in prothrombin and meizothrombin.
Thrombin plays a central role in hemostasis and thrombosis,
performing both coagulatory and regulatory functions (see (1) ). Prothrombin circulates in the plasma as a 579-residue
(in man; 582 residues in bovine) 4-domain zymogen, consisting of an
N-terminal Gla (-carboxyglutamic acid) domain, two tandem kringle
domains, and the catalytic domain (see Fig. 1). Activation of
prothrombin via two cleavages at Arg-PT271 (human; Arg-PT274 in bovine)
and Arg-15 yields the two-chain
-thrombin (for thrombin, A-chain
residues from Cys-1 to Arg-15 and the B-chain are identified via
chymotrypsinogen numbers deduced from topological
equivalence(2, 3) ; residues preceding Cys-1 are
numbered according to human prothrombin and designated by the prefix PT
(see Table 1)). Depending on the reaction conditions, either bond
may be cleaved first(4, 5) . Under physiological
conditions (i.e. in the presence of Ca
and
negatively charged phospholipids), activation is effected largely via
the prothrombinase complex (factors Xa and Va). In this case, the first
cleavage occurs at Arg-15, resulting in the catalytically active
membrane-bound intermediate meizothrombin. Subsequent cleavage at
Arg-PT271 releases the mature two-chain procoagulant
-thrombin
into the blood vessel. In the absence of factor Va, factor Xa performs
these cleavages in reverse order at a much slower rate(4) . Due
to an additional autocatalytic cleavage at Arg-PT285-Thr-PT286,
the human
-thrombin A-chain is further truncated to 36 residues.
Figure 1:
Schematic representation of prothrombin
and some of its activation products. The factor Xa cleavage sites in
prothrombin at Arg-PT271 and Arg-15 are indicated by the arginine side
chains. In human -thrombin there is an additional thrombin
cleavage site within the A-chain at Arg-PT284. Gla denotes the
-carboxyglutamic acid domain, which is followed by the F1 and F2 kringle domains.
Crystallographic studies of -thrombin reveal a compact
molecule, with the B-chain and the central portion of the A-chain
forming a single globular domain(1, 2, 3) .
The
-thrombin molecule is characterized by a deep narrow
canyon-like active site cleft, together with two positively charged
surface patches, representing the ``fibrinogen recognition
exosite'' and the ``heparin binding site'' (3) .
These two sites are of great functional significance in
thrombin's interactions with macromolecular substrates and
inhibitors (1) . Recent crystal structures of
-thrombin in
complex with F2 kringle (6) reveal that this kringle domain
binds to the heparin binding site, largely via specific electrostatic
interactions. The structure of prethrombin 2 has also recently been
solved(7) , exhibiting an overall organization identical to
that of
-thrombin; the C-terminal part of the A-chain shows some
displacement due to the intact peptide bond between Arg-15 and Ile-16.
As in trypsinogen(8, 9) and
chymotrypsinogen(10) , the low proteolytic activity of zymogen
species can be attributed to the malformation of the oxyanion hole,
completed on salt bridge formation between Asp-194 and the new N
terminus of Ile-16. The above structures together with that of
prothrombin fragment F1 (Gla and F1 kringle domains) (11, 12) have given us a detailed understanding of the
structural biology of
-thrombin (1, 3) and a
model for the organization of prothrombin (13) .
In
contrast, the organization of the N-terminal portion of the A-chain has
not been determined, which forms the covalent link to the C terminus of
the F2 kringle in prothrombin, prethrombin 1, and meizothrombin.
Although meizothrombin displays comparable amidolytic activity to
-thrombin against low molecular weight substrates, its activity
toward fibrinogen, factor V, and platelets is reduced(14) , and
prothrombin and prethrombin 1 do not bind to fibrinogen(15) .
One possible explanation for this was thought to be the partial
obstruction of the fibrinogen recognition exosite by the linker
peptide. On the other hand, Liu et al. (16) have
reported that
-thrombin, prethrombin 2, and meizothrombin bind
hirugen, suggesting that conformational changes rather than steric
hindrance interfere with macromolecular binding at the fibrinogen
recognition exosite. Furthermore, Ni et al. (17) have
shown that the fibrinogen recognition exosite is at least partially
accessible in prothrombin. In the present paper, we describe the
experimentally determined structure of almost the complete A-chain of
bovine
-thrombin and the course of the modeled 25-amino acid
residue linker. The N-terminal residues of the A-chain are ordered in
such a manner that excludes occupancy of the fibrinogen recognition
exosite by the linker.
Bovine thrombin was cocrystallized with the inhibitor
rhodniin isolated from the bug Rhodnius prolixus(18) .
Orthorhombic crystals (space group
P22
2
, two complexes per asymmetric
unit) and monoclinic crystals (space group C2, two complexes per
asymmetric unit) were grown from 10% polyethylene glycol 4000, 300
mM phosphate buffer, pH 6.
The structure was solved by
Patterson search techniques with bovine thrombin as obtained in complex
with N-(2-naphthylsulfonylglycyl)-DL-p-amidinophenylalanylpiperidine (19) as a search model. For the monoclinic data with
reflections up to 2.6 Å the model was refined to a R value of 0.189 using X-PLOR (20) with the parameters of
Engh and Huber(21) . The final model showed r.m.s. (
)deviations from ideal values for bond lengths and angles
of 0.01 Å and 1.66°, respectively. The orthorhombic crystals
diffracted up to 3.1 Å, and the model was refined to a R value of 0.175. r.m.s. deviations of the final model are 0.01
Å and 1.62° for bond lengths and angles, respectively. Full
details of the refinement and the final model are given in (22) . The coordinates of both crystal forms have been
deposited with the Brookhaven Protein Data Bank.
The 25-amino acid residue linker was modeled along the surface of the catalytic module of human thrombin to connect the thrombin A-chain with the F2 kringle using the programs O(23) and MAIN (24) (coordinates of the complex of human thrombin and F2 kringle were kindly provided by Prof. A. Tulinsky). Special attention was paid to burial of hydrophobic residues, charge compensation, and the additional requirements of complementarity to factor Xa. Bond and angle energies of the modeled linker were minimized using X-PLOR.
In three of the four independent bovine thrombin molecules
examined here, the complete B-chain and the central and C-terminal part
of the A-chain (Cys-1 to Arg-15) can be traced in continuous electron
density; for one molecule no electron density is observed for a short
segment (Ala-149-Asp-149) in the autolysis loop. The average
r.m.s. deviation for 301 C-atoms (excluding residues
Asp-147-Asp149) is 0.36 Å, with a conformation virtually
identical to those observed previously for thrombin. In addition, we
observe for the first time well defined electron density for the 16
residues preceding Cys-1, Phe-PT277 to Asp-PT292 (Fig. 2). These
residues exhibit a virtually identical conformation in all four copies,
characterized by an average
-carbon r.m.s. deviation of 0.69
Å. Most of these side chains are well defined and exhibit little
variability; only those of Lys-PT284 and Gln-PT278 differ considerably
between the four thrombin molecules. The five most N-terminal residues
of the A-chain can be traced by continuous electron density only for
one molecule in the orthorhombic crystals. In the other three thrombin
molecules, discontinuous density is found, possibly reflecting slightly
different conformations.
Figure 2:
Stereo view of the electron density of the
extended N-terminal fragment of the A-chain. The phenylalanines PT281
and PT286 make a face-to-face contact, and Phe-PT280 makes a
face-to-edge contact with Phe-PT281. Furthermore, the side chain of
Lys-235 makes a hydrogen bond to the face of the phenyl group of
Phe-PT280. Contour surface is shown at 0.8 . This figure was
produced using the program MAIN.
The N-terminal fragment (Thr-PT272 to
Asp-PT292) of the A-chain forms a large U-shaped loop with multiple
turn structure (see Fig. 3). It is situated in an elongated
depression on the ``back'' surface of the catalytic module.
These 21 residues cover approximately 700 Å of a
surface patch extending between the C-terminal helix of the B-chain,
Arg-206, Arg-50, and Asp-125, enclosing also the basic residues
Lys-107, Lys-129B, Lys-235, and Arg-244; this is to be compared with
1700 Å
covered by the whole A-chain.
Figure 3: Connolly surface (calculated and displayed with MAIN) of the observed structure of bovine thrombin. The N-terminal residues PT272 to PT292 are shown in thick lines and the central and C-terminal part of the A-chain and the B-chain in thin lines. The surface of the N-terminal residues is not shown. The view is onto the reverse side of thrombin, rotated 180° about the y axis compared with the standard orientation. The active site therefore runs from right to left along the back of the figure, with the fibrinogen recognition exosite at the left.
This 21-residue fragment is stabilized through electrostatic and hydrophobic interactions to the underlying B-chain module. Seven carbonyl groups and the negatively charged side chains of Asp-PT292 and Glu-PT290 are directed toward a positively charged patch of the B-chain surface. Only one carbonyl oxygen (Ala-PT288) is engaged in an inter-main chain hydrogen bond with an amide nitrogen (Asp-49), whereas five of the carbonyl groups are hydrogen bonded to side chain groups. Asp-PT292 is the only residue to be involved in a direct ionic contact with the catalytic module (Lys-9).
The majority of hydrophobic contacts is mediated through the 4 phenylalanine residues Phe-PT286, Phe-PT281, Phe-PT280, and Phe-PT277 (see Fig. 3). Their bulky aromatic side chains are almost completely buried in the interface between the A- and the B-chain, with residues Phe-PT286 and Phe-PT281 in the center of a large loop pointing toward a hydrophobic surface patch formed by Ile-47, Trp-51, Leu-53, Leu-105, Ile-242, Ile-238, and Leu-123. Three of these phenylalanine residues (Phe-PT286, Phe-PT281, and Phe-PT280) are clustered in an aromatic triad, with Phe-PT281 making face-to-face contacts with Phe-PT286 and face-to-edge contacts with Phe-PT280. Furthermore, Lys-235 points toward the electron-rich face of Phe-PT286 to form a 3.5-Å hydrogen bond (see Fig. 2). The benzene ring of the fourth phenylalanine, Phe-PT277, is oriented parallel to the guanidino group of Arg-206. These four phenylalanyl residues certainly represent a central element for the stabilization of this N-terminal A-chain fragment. This is further underscored by the fact that amino acids PT280 to PT287 are highly conserved in all six prothrombin sequences known (see Table 1); residues Gly-PT287, Phe-PT286, Thr-PT285, Phe-PT281, and Phe-PT280 (i.e. including three of the phenylalanines) are identical, and the three intervening amino acids, PT282-PT284, exhibit only two different types. The supporting amino acids are also highly conserved; five of the seven hydrophobic residues are identical in all 11 thrombin sequences known(25) , and in the other positions only hydrophobic residues are found.
In addition to these A-B interchain contacts, the loop is also stabilized through intrachain interactions. An important contribution is made by the side chain of Glu-PT290, which extends toward the loop center and connects its two peripheral ends by hydrogen bonds to main chain amide nitrogens. This loop is further stabilized by a few inter-main chain hydrogen bonds. Intermolecular crystal contacts, in contrast, do not seem to affect the loop structure to a large extent. In only two of the four structures examined a weak ionic contact between the partially disordered side chain of Lys-PT284 and a symmetry-related rhodniin molecule is observed; the side chain is disordered in the other cases.
Inspection of the electron densities
for some other bovine thrombin structures in different space groups
determined in our laboratory ()suggest a similarly folded
A-chain segment, PT277-PT292. It might be interesting to note
that the homologous PT285-PT292 segment of human thrombin is
found with a similar conformation in the D-Phe-Pro-Arg-chloromethylketone-thrombin complex, albeit in
much weaker electron density(3) . The congruence in the
conformation of segment PT277-PT292 observed in the bovine
thrombin structures leads us to assume that this conformation and
location are inherently preferred and also maintained in the uncleaved
proforms.
In human thrombin, Lys-PT284 is replaced by an arginine
residue, and its peptide bond becomes autocatalytically cleaved leading
to the N-terminally truncated A-chain that starts with Thr-PT285. In
the bovine thrombin crystal structures presented here the six amino
acids flanking this residue on both sides form one turn of an
approximate -helix (see Fig. 3). Despite this P1-arginine
and the preceding P2-proline residue (replacing the bovine thrombin
glutamic acid) this segment presumably adopts a similar conformation in
the intermediate elongated human (pro)thrombin forms. Residues
P4-P4` would presumably require considerable rearrangements to
facilitate binding into the active site of an attacking human thrombin
molecule and to adopt a canonical conformation for
cleavage(26) . Only human and rat prothrombin exhibit an
arginine at position P1, and only human prothrombin possesses a proline
at position P2 that accurately matches thrombin cleavage site
specificity.
In the intact form of bovine prothrombin, a 25-residue linker peptide connects the last cysteine of F2 kringle (Cys-PT249) with Thr-PT272 of the A-chain (see Table 1). The corresponding linkers in human, rat, mouse, and chicken species are shorter by two, seven, and eight residues, respectively. Assuming the conformation observed in our four bovine thrombin molecules from Phe-PT277 onward, we have tried to model an appropriate peptide along the surface of human thrombin linking Phe-PT277 with the C terminus of the F2 kringle (see Fig. 4). This linking peptide was positioned to cover a hydrophobic patch centered around Ile-PT268 formed by Leu-14G, Tyr-14J, Tyr-134, Pro-204, and Phe-204A. Ile-PT268 is conserved in the prothrombin sequences of human, bovine, rat, mouse, and chicken species, and even in hagfish a leucine is found at that position. Tyr-14J and Pro-204 of the supporting surface patch are also strictly conserved, and the residues found at the other three positions are mainly hydrophobic(25) .
Figure 4:
Stereo view of the prethrombin 1 model in
the same orientation as Fig. 3. A ribbon representation shows
secondary structure elements for human thrombin and F2
kringle(6) . The C trace for residues
PT249-PT292 with the observed N-terminal fragment of the A-chain
(residues PT277-PT292) is shown in black, and the 28
residues of the modeled linker peptide are shown in white.
This figure was drawn using the program
MOLSCRIPT(28) .
In a factor Xa-prothrombin enzyme-substrate encounter complex the position of Ile-PT268 and the overall apolar nature of this region would nicely match the extended hydrophobic patch west of the S1 pocket in factor Xa(27) . Furthermore, the charged patches on the module surface around the cleavage site between linker and A-chain are complementary to an attacking factor Xa molecule. The modeled linker peptide also covers part of the heparin binding site not occupied by F2 kringle, with the five glutamate residues in the N-terminal part of the linker contacting the basic residues Arg-165, Lys-169, and Arg-175. The highly negatively charged linking peptide together with at least two uncompensated negatively charged side chains of F2 kringle (i.e. Asp-PT173 and Asp-PT207) might mediate binding to factor Xa, whose large positively charged heparin binding site would otherwise be repulsed by that of thrombin. The proposed conformation would also allow this linking part to follow the same course in rat and mouse prothrombin, where the linker is seven residues shorter. In our model for prethrombin 1, the two factor Xa cleavage sites are only approximately 15 Å apart. Thus both sites would be accessible to the same attacking factor Xa molecule in the prothrombinase complex without large rearrangements, in agreement with experimental results.
Although the linker chain could take several other adjacent courses, a course through the active site cleft and/or through the fibrinogen recognition exosite would require a linker peptide of at least 115-Å length. Such a trace would not be possible for the chains of rat, mouse, chicken, or hagfish, and even for human and bovine prothrombin the linker chain would have to be extremely extended. Therefore, a direct influence of the linking peptide on this important fibrinogen recognition exosite can probably be excluded.