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Next: Biosynthesis Up: SFTI Previous: Bowman-Birk Inhibitors and the
Sequence and Structure
SFTI-1 contains 14 amino acids, two of which are Cys residues that are joined in a disulfide bond. As shown in Figure 1.11, this disulfide bond separates SFTI-1 into two loops corresponding to a reactive loop, which possesses strong sequence identity to the BBI active site, and a secondary loop which completes the cyclic backbone of the molecule. An inspection of the reactive loop sequence of SFTI-1 shows that it clearly belongs to the BBI family of trypsin inhibitors (Figure 1.11). About forty different members of the BBI family have been identified and a comparison of the trypsin reactive loops from a range of BBIs to SFTI-1 shows similarity in both inter-cystine length and sequence.
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With a K of 0.1-0.5 nM [8],
SFTI-1 is the most potent trypsin inhibitor yet discovered and studies
utilising minimised peptides mimicking the reactive loop of BBIs
suggest that SFTI's remarkable efficiency is, at least partially,
related to its reactive loop sequence. By screening a range of
minimal synthetic variants of the BBI reactive loop the residues
crucial for activity have been identified. Using the nomenclature of
Schechter and Berger [86] in which the primary scissile
bond is between P1 and P1', significant activity loss against trypsin
is seen in the BBI reactive loop at positions P
, P
, P
' and
P
' [87]. The optimal residue for a number of these
positions has also been determined using the same methodology, and in
each case SFTI-1 appears to possess this optimal residue. As the
primary contact with the protease, changes in P
are correlated
with broad changes in specificity
[89,90,88,85],
and in this position SFTI contains a Lys which is specific for trypsin
inhibition [89]. In P
and P
'
SFTI-1 contains Thr and Ile respectively. These two residues have
been found to be optimal for their respective positions
[92,91], although Thr
in P
was measured in chymotrypsin assays. An optimal residue for
P
' has not been determined, but an alanine scan of a BBI reactive
loop mimetic showed an
2000
reduction in activity
against trypsin when this Pro was substituted [87].
SFTI-1 contains a Pro in this position and a Pro is absolutely
conserved in this position across the BBI family, further highlighting
its importance for activity. This remarkable convergence of optimal
active site residues in SFTI-1, and its potent inhibitory effect,
underscores the concept of SFTI-1 as a natural peptide mimetic of the
BBI trypsin reactive loop.
Both the crystal structure [9], in complex with
bovine -trypsin, and the solution structure
[8], by
H NMR, have been solved
for SFTI-1. In both cases SFTI-1 was shown to consist of two
anti-parallel
-strands that are connected at both ends by turns
-- resulting in a cyclic peptide. Remarkably, the complexed
structure and the solution structure are very similar (mean RMSD for
the backbone atoms of 0.25
) indicating that SFTI-1 must possess
a highly rigid structure that does not undergo major conformational
change when complexed with trypsin. Contributing to this rigidity is
the disulfide bridge that separates the molecule into two loops,
however, as revealed in the crystal structure, an extensive network of
hydrogen bonds further stabilises the molecule [9].
As summarised in Figure 1.12, SFTI-1 possesses three
intramolecular main-chain hydrogen bonds -- between Gly1 HN-Phe12 O
and Phe12 HN-Arg2 O in the secondary loop and between Thr4 HN-Ile10
O in the reactive loop. The reactive loop is further stabilised by a
bifurcated hydrogen bond between the hydroxyl group of Thr4 and both
the main-chain amide of Ile10 and the sidechain hydroxyl of Ser6. The
hydroxyl group from Ser6 is also bonded to the main-chain carbonyl
group of Pro8. All of these hydrogen bonds were confirmed in the
solution structure by measurement of slowly exchanging amide protons
[8], however a hydrogen bond between
the sidechain of Asp14 and the main-chain amide of Gly1 that was
predicted by Luckett et al. [9], based on the
crystal structure, could not be confirmed in the latter study. SFTI-1
contains three Pro residues and the bond between Ile7 and Pro8 has
been shown to be in the cis conformation in both the crystal
and solution structure
[8,9]. Along with the
disulfide bond and hydrogen bond network this cis-peptide bond
acts to maintain the reactive site loop of SFTI in the shape of a
-hairpin.
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The structures of several BBI inhibitors have been solved using X-ray
crystallography, both alone
[94,80,93]
and in complex with trypsin
[96,95,97].
Additionally, the solution structure of a soyabean BBI has been solved
using H NMR [98]. In each case the
trypsin reactive loop forms a two stranded anti-parallel
-sheet
joined with a type VIb
-hairpin. The reactive loops are
stabilised by an absolutely conserved disulfide bridge and hydrogen
bonds [99]. In dicot BBIs a conserved
cis-Pro further stabilises the reactive loop
[100]. The similarities between key
elements of the structure of SFTI-1 and the structures of the reactive
loops of the BBIs clearly indicate that they would share a similar
fold and a superimposition of the structures of SFTI and the reactive
loop of a selection of BBIs highlights this structural similarity
(Figure 1.13). The conserved elements -- a disulfide
bond, a cis-Pro in position P
', and intramolecular hydrogen
bonding -- would therefore appear to be the three principle
restraining features important for the inhibitory effect of these
proteins.
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As suggested by their structural similarities the interactions between SFTI and trypsin are similar to the interactions between the protease and the trypsin reactive loops of the BBIs. The crystal structure of SFTI-1 in complex with trypsin [9] shows that the interactions centre around Lys5, in position P1, which extends into the S1 pocket of the enzyme and makes both direct and water mediated contact with the specificity-determining Asp189, the hydroxyl group of Ser190 and the mainchain carbonyl of Gly219. An extensive array of hydrogen bonds and ion pairs forms between the enzyme and the inhibitor and these are summarised in Figure 1.14. These hydrogen bonds include interactions between the mainchain carbonyl of Lys5, which is within hydrogen bonding distance of Ser195 and His57 of the trypsin catalytic triad.
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The majority of the contact points between SFTI-1 and trypsin are made
on one side of the reactive loop between the residues from Cys3 to
Ile7 and the distant side of the reactive loop, between Pro8 and
Cys11, appears to play an important stabilising role despite making no
direct contacts with the enzyme [9]. It has been
suggested that the highly stabilised reactive loop of SFTI prevents
hydrolysis of the peptide by preventing the structural change that
would normally accompany hydrolysis
[8,9]. The importance of
Thr in position P for inhibitory activity, at least in
chymotrypsin, appears to support this conjecture, as its removal, and
the subsequent loss of two hydrogen bonding interactions, could
destabilise the loop leading to a loss of inhibitory activity. The
secondary loop, which has no direct role in enzyme interactions, may
aid binding efficiency by stabilising the entire structure through
additional hydrogen bonding. This is supported by the exceptional
similarity between the solution and crystal structure of SFTI-1 which
suggests that a lack of binding-induced conformational change
contributes to the low K
as SFTI-1 can bind with minimal entropy
costs.




Next: Biosynthesis Up: SFTI Previous: Bowman-Birk Inhibitors and the Jason Mulvenna
2005-04-24