A novel strategy for inhibition of a-amylases: yellow meal worm
a-amylase in complex with the Ragi bifunctional inhibitor at
2.5 Å resolution
Stefan Strobl1, Klaus Maskos2, Georg Wiegand2, Robert Huber2,
F Xavier Gomis-Rüth2,†*and Rudi Glockshuber1*
Background: α-Amylases catalyze the hydrolysis of α-D-(1,4)-glucan linkages
in starch and related compounds. There is a wide range of industrial and
medical applications for these enzymes and their inhibitors. The Ragi
bifunctional α-amylase/trypsin inhibitor (RBI) is the prototype of the cereal
inhibitor superfamily and is the only member of this family that inhibits both
trypsin and α-amylases. The mode of inhibition of α-amylases by these cereal
inhibitors has so far been unknown.
Results: The crystal structure of yellow meal worm α-amylase (TMA) in
complex with RBI was determined at 2.5 Å resolution. RBI almost completely
fills the substrate-binding site of TMA. Specifically, the free N terminus and the
first residue (Ser1) of RBI interact with all three acidic residues of the active site
of TMA (Asp185, Glu222 and Asp287). The complex is further stabilized by
extensive interactions between the enzyme and inhibitor. Although there is no
significant structural reorientation in TMA upon inhibitor binding, the N-terminal
segment of RBI, which is highly flexible in the free inhibitor, adopts a 310-helical
conformation in the complex. RBI’s trypsin-binding loop is located opposite the
α-amylase-binding site, allowing simultaneous binding of α-amylase and trypsin.
Conclusions: The binding of RBI to TMA constitutes a new inhibition
mechanism for α-amylases and should be general for all α-amylase inhibitors of
the cereal inhibitor superfamily. Because RBI inhibits two important digestive
enzymes of animals, it constitutes an efficient plant defense protein and may be
used to protect crop plants from predatory insects.
α-Amylases (α-1,4-glucan-4-glucanohydrolase, EC 18.104.22.168)
catalyze the hydrolysis of α-D-(1,4)-glucan linkages in
starch components, glycogen and various other related car-
bohydrates. They constitute a widely distributed family of
enzymes found in micro-organisms, plants and animals,
and are central in carbohydrate metabolism. Insect and
mammalian α-amylases show a high degree of homology
in their primary  and tertiary structures . Neverthe-
less, several inhibitors of insect α-amylases were reported
to inhibit pig pancreatic α-amylase (PPA) and other mam-
malian α-amylases only with low affinity or not at all
[3–10]. The structural basis for inhibition of α-amylases by
the widely occurring natural inhibitors is in most cases
unknown. Until now, almost all structural studies on the
interaction of α-amylases with substrate analogs [11,12],
carbohydrate inhibitors [13,14] and proteinaceous inhibitors
[15,16] have been performed with PPA. We have recently
determined the first three-dimensional structure of an
α-amylase from insects, namely the α-amylase (TMA)
from the yellow meal worm (larvae of Tenebrio molitor) to
1.64 Å resolution by X-ray crystallography . TMA is a
monomeric protein of 471 residues that consists of three
domains (A–C). The enzyme contains four disulfide bridges,
a structural calcium ion and a chloride ion, which allosteri-
cally activates the enzyme. The long substrate-binding cleft
of TMA may accommodate six saccharide units, with sub-
strate hydrolysis taking place between the third and fourth
pyranose [2,14]. Asp185, Glu222 and Asp287 are supposed
to be key residues for catalysis . TMA’s polypeptide fold
resembles that of PPA, but there are important differences
in loop segments next to the active-site region . Because
TMA is the only insect α-amylase for which a three-dimen-
sional structure is available, it constitutes the model enz-
yme for structural studies on the specific inhibition of insect
The bifunctional α-amylase/trypsin inhibitor (RBI) from
Ragi (Eleusine coracana Gaertn.; Indian finger millet) is the
prototype of the cereal inhibitor superfamily and is the only
member of this family for which independent inhibitory
activities against both trypsin and α-amylases have been
Addresses: 1Institut für Molekularbiologie und
Biophysik, Eidgenössische Technische Hochschule
Hönggerberg, CH-8093 Zürich, Switzerland.
2Max-Planck-Institut für Biochemie, D-82152
†Present address: Centre d’Investigacions i
Desenvolupament (C.S.I.C.), Carrer Jordi Girona,
18-26, 08034 Barcelona, Spain.
Key words: α-amylase, Ragi bifunctional inhibitor,
trypsin, yellow meal worm (Tenebrio molitor),
Received: 31 March 1998
Revisions requested: 30 April 1998
Revisions received: 19 May 1998
Accepted: 29 May 1998
Structure 15 July 1998, 6:911–921
© Current Biology Ltd ISSN 0969-2126
reported . Like other inhibitors of this family, RBI
inhibits α-amylases from various sources. The proteins’
exact mode of α-amylase inhibition was so far unknown,
however. RBI is a monomer of 122 amino acids with five
disulfide bonds. The three-dimensional structure of recom-
binant RBI in solution was solved by nuclear magnetic reso-
nance (NMR) spectroscopy. The globular fold of RBI
consists of four α helices with simple ‘up-and-down’ topo-
logy and a small antiparallel β sheet . The trypsin-bind-
ing loop of RBI adopts the ‘canonical’, substrate-like
conformation, which is highly similar among completely
unrelated serine proteinase inhibitor families . The
existence of a ternary complex between trypsin, RBI and
α-amylase suggested that RBI’s α-amylase-binding site is
located opposite the trypsin-binding loop . But neither
the known structures of RBI and various α-amylases, nor a
comparison of the primary structures of RBI and other
α-amylase inhibitors from the cereal inhibitor superfamily,
gave hints on the exact location of RBI’s α-amylase-binding
Here, we describe the three-dimensional structure of the
complex between RBI and TMA at 2.5 Å resolution. It
reveals a completely new inhibition mode for proteina-
ceous α-amylase inhibitors. The N-terminal residues of
RBI, which are unstructured in solution, fold into a helical
conformation upon binding to the enzyme, whereas the
free N terminus directly targets the catalytic residues of
Results and discussion
Structure of the complex
In the RBI–TMA complex the inhibitor binds to the
active site of the enzyme, which lies in a V-shaped depres-
sion at the interface of the domains A and B (Figure 1).
This is in agreement with biochemical experiments,
which proved a competitive inhibition mode for the inter-
action between RBI and the porcine enzyme, PPA .
RBI exclusively interacts with residues from domains A
and B of TMA, which wall the substrate-binding site.
Altogether, 26 residues of the inhibitor interact with 28
residues of the enzyme. The contacts are summarized in
Table 1. The overall contact area between enzyme and
inhibitor is 1201 Å2.
The substrate-binding cleft of TMA, like that of PPA,
provides at least six subsites for binding of carbohydrate
moieties, as the pseudo-hexasaccharide inhibitor V-1532
(a member of the trestatin family) can easily be modeled
into the cleft of TMA on the basis of the X-ray structure of
the PPA–V-1532 complex . In the RBI–TMA complex,
the analogous subsites 1–5 in TMA are completely blocked
by residues of RBI (Figures 2 and 3). In particular, two
functional segments can be identified in RBI that interact
with the α-amylase in a very specific manner (Figure 3).
Segment 1, comprising the N-terminal residues Ser1–Ala11
and residues Pro52–Cys55, protrudes like an arrow head
into TMA’s substrate-binding groove and directly targets
the active site of the enzyme. The five N-terminal RBI
residues form the tip of the arrow. Although residues 1–5
are flexible in the solution structure of free RBI, they
adopt a 310-helical conformation in the complex with TMA
and fill the saccharide-binding subsites 3 (Ser1), 2 (Val2),
and 4 (Thr4). Ile7 and Met10 sterically block the access to
subsite 5 (Figure 3). Ser1 is involved in numerous hydro-
gen bonds with residues in the active site of TMA (Table 1,
Figure 4). The N-terminal amino group of Ser1 interacts
with the carboxyl groups of two catalytic residues in TMA,
Asp185 Oδ1 and Glu222 Oε1 and Oε2. The hydroxyl group
of Ser1 forms hydrogen bonds to the third catalytic residue
Asp287 (Oδ2), to TMA’s chloride-binding residue Arg183
(Nη2) and to His286 Nε2, which is invariant in the active
sites of α-amylases. The carbonyl oxygen atom of Ser1 inter-
acts via water-mediated hydrogen bonds with the catalytic
residue Asp185 (Oδ2) and with Nε2 of His99, which is
also absolutely conserved in α-amylases. Thus, Ser1 com-
pletely fills the sugar-binding subsite 3 of the enzyme. In
addition, Val2 N and Ser5 Oγ of RBI interact by direct
and water-mediated hydrogen bonds, respectively, with
the carboxyl group of the third catalytic residue of TMA,
Asp287. Hydrophobic interactions between Val2 (RBI) and
Trp56, Trp57 and Tyr60 of TMA, which cover the saccha-
ride-binding subsites 2 and 3, additionally position Val2
Structure 1998, Vol 6 No 7
A ribbon diagram of the RBI–TMA complex. RBI is shown in gold. The
three TMA domains, A (residues 1–97 and 160–379), B (residues
98–159) and C (residues 380–471), are depicted in blue, green and
red, respectively. Disulfide bridges in RBI and TMA are shown in red
and yellow, respectively. This figure was made with SETOR .
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Research Article Insect a-amylase in complex with a plant inhibitor Strobl et al. 921