Crystal Structure of Thrombin in Complex with S-Variegin: Insights of a Novel Mechanism of Inhibition and Design of Tunable Thrombin Inhibitors Page: 4
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A Novel Mechanism of Thrombin Inhibition
genix, Milano, Italy) as described previously [16,17]. Values for
concentration of peptide needed for 50% inhibition (ICso) and
inhibition constant (K) were calculated from data obtained were
fitted using Origin software (MicroCal, Northampton, MA, USA).
A detailed account for the selection and use of equations to fit the
data is available in Materials and Methods S1.
Zebrafish larvae venous thrombosis model
Zebrafish and the larvae were maintained as previously described
. Intravenous microinjections of peptides were performed using
Nanoject II (Drummond, Broomall, PA, USA) with glass injection
needles. Ten nanolitres of peptides or phosphate buffered saline
(PBS) were injected into 4 days post-fertilization larvae through the
posterior cardinal vein. Laser ablation of larval veins were
performed with a pulsed nitrogen laser light pumped through
coumarin 440 dye (445 nm) (MicroPoint Laser system, Photonic
Instrument, St Charles, IL, USA) at 10 pulses/s with laser intensity
setting at 10. Laser ablation of each larvae was carried out 20 min
after microinjection of the peptide or PBS. The laser beam was
aimed at the caudal vein around five somites towards the caudal end
from the anal pore and triggered for 3 s. Thrombus formation
following vein injury, due to laser ablation, was monitored and the
time taken for complete occlusion of the injured vein was recorded.
The crystal structure of the thrombin:s-variegin complex was
determined at 2.4 A resolution (Table 1 and Figure 1C-D). The
electron density of the complex structure is well defined except for
termini residues of chain A [T(1HTFGSGElC) and TArgl5]. The
structure of thrombin in the complex superimposes well with other
Only 17 out of the 32 residues (VHis 12 to VLeu28) of s-variegin
have well-defined density (Figure 1C). The first seven N-terminal
residues do not make direct contact with thrombin  and s-
variegin is cleaved by thrombin between VLys 10 and V'Met 1 [ 16].
It is likely that the N-terminal fragment V(1SDQGDVAEPKIo) has
dissociated from thrombin after cleavage before crystallization. In
contrast, the C-terminal fragment MH22 V(ll'MHKTAPPFD-
FEAIPEEYLDDES32) remains bound to thrombin after cleavage
. The N-terminal VMetll1 and the last five residues
v(28LDDES32) of the fragment are not observed, reflecting
disorder in the termini.
The C-termini of hirulog-1/-3, hirugen and hirudin have the
following sequence DFEEIPEEYL(Q), with the Gln only present
in hirudin. s-Variegin has an almost identical sequence
V(19DFEAIPEEYLDDES32), with four extra residues in the C-
terminus. Despite the identity, there are large differences between
their conformations. The C-terminus is disordered in the hirulog-
1/bivalirudin structure (PDB code: 2HGT) , forming a 310 helix
in hirulog-3 (PDB code: 1ABI)  and hirugen (PDB code:
1HGT ) and forming a full i-helical turn in sulfo-hirudin (PDB
code: 2PW8) . In s-variegin, these residues remain in an
extended conformation until the last observed residues (VLeu28).
The extra residues in C-terminus, although not observed in the
present structure may cause the peptide to adopt the fully
extended conformation (Figure S1).
Interactions with thrombin catalytic residues
The active site of thrombin in the crystal structure was
compared to the published data for the thrombin:hirugen
structure (unoccupied active site) (Figure 2). Of the three catalytic
residues, the most striking differences are with the Oy atom of
TSer195 and the orientation of the imidazole ring of THis57. In
the thrombin:s-variegin structure, TSerl95 Oy is displaced by
1.19 A, pointing towards s-variegin. Distance between TSer195
Oy and the side chain Ns of VHisl2 is 3.35 A, possibly forming
hydrogen bond (Table S 1). At the same time, the distance between
Ns of THis57 and Oy of TSerl95 increases to 3.60 A from 2.79 A,
breaking the crucial strong hydrogen bond needed to form the
catalytic charge relay system. Without stabilization by the strong
hydrogen bond between THis57 and TSer195, the imidazole ring
of THis57 is now rotated slightly and leads to a displacement of its
Ns by 0.56 A (Figure 2A). The newly formed hydrogen bond
between TSer 195 and VHis 12 delocalize the electrons of TSer 195
Oy, making TSer195 a weak nucleophile and incapable of
efficiently attacking the backbone C of the substrate. This explains
the observed classical non-competitive inhibition for MH22 .
Interactions with prime subsites
In addition to the new hydrogen bond, the following
interactions anchor s-variegin P2' to P5' residues v(12HKTA15)
to the thrombin prime subsites (Figure 3A). Besides the catalytic
residues, TLeu41, TCys42, TCys58, TTrp60D, TLys60F and
TGlu192 are also in contact with VHisl2. Two hydrogen bonds
can be formed between VHis 12 with TGlu39 and TGlu192 (Table
S1). The P3' ('Lysl3) interacts with TArg35, TGlu39, TTrp60D,
TLys60F, TAsn143, TThr147, and TGlu192. The P4' (VThrl4)
side chain is directed towards the base of the highly flexible
autolysis-loop. The side chain occupies a surface lined by TLeu40,
TTrpl41, TGly142, TAsn143, TGlnl51 and TGly193. Interactions
within this P3' subsite are strengthened by two hydrogen bonds
between VThrl4 with TAsn143 and TGlnl51 (Table Sl). The P5'
'Ala is surrounded by TGln38, TGlu39, TArg73 and TGlnl51.
Thus there are extensive interactions between the variegin peptide
and thrombin prime subsites.
Interactions with exosite-I
s-Variegin fits firmly into the canyon-like cleft extending from
the thrombin active site to exosite-I. The walls of this hydrophobic
cleft are formed by the 60- and autolysis- loops near the active site,
and 34- and 70- loops at exosite-I, while many apolar residues in
these loops line the bottom [2,3]. s-Variegin is in close contact with
multiple residues in exosite-I as depicted in Figure 3B. All but four
residues of s-variegin (VPhe 1l8, 'Asp 1l9, VAla22 and VGlu26) have
their side chains buried in the interfaces with thrombin (Figure 3A).
Interestingly, the high identity between C-terminus of s-
variegin, hirulog-1/-3, hirugen and hirudin are not reflected in
their respective salt bridges formation with exosite-I of thrombin.
Despite the presence of multiple anionic residues in the s-variegin
C-terminus and highly cationic exosite-I, only one strong salt
bridge is formed (VGlu26:TArg77A). This salt bridge (3.84 A) is
not observed in hirulog-1/-3, hirugen and hirudin structures as
TArg77A adopts a different rotamer that points away from the
inhibitor (Figure 4A and S2A). In addition, a weak salt bridge is
also likely between VGlu21 and TArg75 (4.64 A). In hirulog-1/-3,
hirugen and hirudin structures the analogous Glu makes an ion
pair with TArg75 of a 2 fold symmetry-related thrombin, although
this interaction is suggested to occur within the same thrombi-
n:inhibitor pair in solution [4,22,32]. In our structure, the TArg75
side chain is rotated by 80.5 about C3, compared to the
thrombin:hirulog-3 complex (Figure 4A and S2A) facilitating this
interation. In hirulog-1/-3 and hirugen structures, an ion pair
between TArg73 and the Asp, analogous to VAspl9, can be
observed. However, formation of this ion pair in the thrombin:s-
variegin complex is not possible as the VAsp 19 side chain points in
an opposite direction into solvent. This difference is most likely
October 2011 1 Volume 6 1 Issue 10 1 e26367
.: PLoS ONE I www.plosone.org
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Koh, Cho Yeow; Kumar, Sundramurthy; Kazimirova, Maria; Nuttall, Patricia A.; Radhakrishnan, Uvaraj P.; Kim, Seongcheol et al. Crystal Structure of Thrombin in Complex with S-Variegin: Insights of a Novel Mechanism of Inhibition and Design of Tunable Thrombin Inhibitors, article, October 2011; [San Francisco, California]. (digital.library.unt.edu/ark:/67531/metadc287041/m1/4/: accessed November 18, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT College of Arts and Sciences.