1. health and fitness
2. disease
3. cancer

Eur. J. Biochem. 267, 1975±1984 (2000) q FEBS 2000
Characterization of potato proteinase inhibitor II reactive site mutants
Jules Beekwilder, Bert Schipper, Petra Bakker, Dirk Bosch and Maarten Jongsma
Department of Molecular Biology, Center for Plant Breeding and Reproduction Research (CPRO), Wageningen, the Netherlands
Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains.
We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression
host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating
each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly
inhibits both trypsin (Ki ˆ 0.4 nm) and chymotrypsin (Ki ˆ 0.9 nm), and is quite robust towards mutations at
positions P2 or P1 0 . In contrast, the first reactive site inhibits only chymotrypsin (Ki ˆ 2 nm), and this activity is
very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain
II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is
discussed.
Keywords: mutational analysis; Pichia pastoris; serine proteinase inhibitor; Solanum tuberosum.
Protease inhibitors are abundant proteins in the storage organs
and seeds of plants [1]. In addition, their synthesis is induced to
high levels in response to stress, infection and wounding [2].
Potato expresses many different proteinase inhibitors belonging
to a wide range of inhibitor families. Members of the potato
proteinase inhibitor II (PI-2) family have been shown to inhibit
serine proteases, such as trypsin, chymotrypsin, subtilisin,
oryzin and elastase [3,4]. Until now, they have been found only
among the Solanaceae. Proteins and messenger RNAs have
been identified in potato tubers [5,6], wounded tomato and
tobacco leaves [4,7], eggplant fruits [8], green tomatoes [9] and
ornamental tobacco flower stigma [10]. There is medical
interest in the properties of PI-2. It has been shown to have
anticarcinogenic properties, protecting mouse embryo fibroblasts from radiation-induced transformation [11]. Although the
mechanism underlying this effect is poorly understood, it is
correlated to the capacity of PI-2 to inhibit chymotrypsin-like
enzymes [12].
Plant protease inhibitors such as PI-2 have been proposed to
function as part of the plant defense system [13]. The plant
defense role is deduced from observations that proteinase
inhibitors in leaves are synthesized in response to wounding
[2,14,15] or viral infection [2,16]. In addition, direct evidence
for a protective role has been obtained; it was shown that
transgenic tobacco plants over-expressing potato PI-2 gained
resistance against the tobacco hornworm Manduca sexta [17].
Also, when the PI-2 gene was introduced into rice under its own
wound-inducible promoter, plants were protected from the
Correspondence to J. Beekwilder, CPRO, PO Box 16, 6700 AA
Wageningen, the Netherlands. Fax: 1 31 31 741 8094,
Tel.: 1 31 31 747 7109, E-mail: m.j.beekwilder@plant.wag-ur.nl
Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; cv, cultivar;
OMTKY3, turkey ovomucoid third domain; PCI, polypeptide chymotrypsin
inhibitor; P1, primary specificity site of protease inhibitor;
Ph-CO-Arg-NH-Np, Na-benzoyl-l-Arg-p-nitroanilide; PI-2, potato
proteinase inhibitor II; S1, pocket in the protease protein complementary to
the primary specificity site of a protease inhibitor or substrate; SAAPLpNA,
N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide.
(Received 10 November 1999, revised 1 February 2000, accepted
2 February 2000)
major lepidopteran rice pest, Sesamia inferens [18]. PI-2 probably acts by inhibiting digestive proteases from the insects,
thereby restricting the availability of amino acids [13].
The anti-nutrient activity of PI-2 has been shown to be
overcome by pest insects like Spodoptera exigua [19]. When
these larvae are reared on tobacco leaves expressing potato
PI-2, the spectrum of digestive proteases of the caterpillar
changes towards enzymes that can not be inhibited by PI-2. It
was our aim to reinforce the defense of crops to pest insects
such as S. exigua, by complementing the plant's inhibitor
arsenal. We looked for complementary proteinase inhibitors by
screening collections of proteinase inhibitors from a wide range
of sources [20], or by altering the specificity of plants own
inhibitors, by phage display [21]. PI-2-like proteins may constitute a convenient protein framework for the introduction of
insecticidal inhibitors into plants. Naturally occurring PI-2
genes encode two, three or six repeats of a 54-amino-acid
polypeptide domain, in which each of the domains may carry a
different specificity. If needed, this allows for the introduction
of several inhibitor specificities in one gene.
The cDNA and protein isolated from potato tubers have a
two-domain organization [5,6,22]. Remarkably, the region that
corresponds to a single domain protein found in tubers lies
across the two repeats of PI-2, rather than within one of the
repeats. This implies that the N-terminal and C-terminal
portions of the repeat are redundant for this otherwise fully
functional protein. Recently, it has been shown that both
termini are connected by disulfides and together make up a fold
similar to the naturally occurring single domain, although built
from two discontinuous parts of the primary sequence [23].
The specificity of a serine-protease inhibitor is governed
mostly by a few amino acids in what is called the reactive site
loop [24]. For PI-2, these have been located by studying the
crystal structure of PCI, one of the monomeric forms from
potato PI-2, in complex with a chymotrypsin-like enzyme [22].
In this complex, 73 of 103 contacts between the inhibitor and
protease involve an array of five amino acids. The two outer
residues of these five are cysteines, which are invariable in all
natural PI-2 variants. The central three amino acids vary
considerably in identity (Fig. 1), and have been shown to be
extremely flexible in solution [25]. In this study we substituted
1976 J. Beekwilder et al. (Eur. J. Biochem. 267)
q FEBS 2000
pH 6.0, 1.34% yeast nitrogen base, 1% glycerol) and BMM
medium (100 mm K3PO4, pH 6.0, 1.34% yeast nitrogen base,
0.5% methanol) were prepared according to the instructions in
the Pichia Expression Kit manual. Bovine pancreatic b-trypsin
type III (EC 3.4.21.4) and bovine pancreatic a-chymotrypsin
type II (EC 3.4.21.1) were purchased from Sigma Chemical Co.
(St. Louis, USA). Substrates Na-benzoyl-l-Arg-p-nitroanilide
(Ph-CO-Arg-NH-Np) and N-succinyl-Ala-Ala-Pro-Leu-pnitroanilide (SAAPLpNA) were from Sigma and Bachem
Feinchemikalien AG (Bubensdorf ), respectively.
The following oligonucleotides (Isogen, Amsterdam) were
used for PCR
1 5 0 -CCCCAAGCTTGCCCCCGAAATTGTGGTAATCTTGGGTTTG-3 0
2 5 0 -CGGAGCGGCCGCCTACATAGCGGGGTACATA-3 0
3 5 0 -CCGGCCATGGCTAAGGCTTGCCCCCGAAATTGCGATCCACATATTGCCTAC-3 0
5 0 -TCAAAATGCCCACGTTCAGAAGG-3 0
4 5 0 -GTATCTCTCGAGAAAAGAGAGGCTGAAGCTAAGGCTTGC-3 0
Restriction sites are underlined.
Construction of variants
Fig. 1. Partial alignment of naturally occurring PI-2-like proteins from
potato (Solanum tuberosum), ornamental tobacco (Nicotiana alata),
tobacco (N. tabacum), tomato (Lycopersicon esculentum), sweet pepper
(Capsicum annuum) and egg plant (S. melongata), and mutants of
potato PI-2 used in this study. Only parts that contribute to the first
reactive-site loop (first part) and to the second, third, etc. reactive-site loop
(second part) are depicted. Residues P1, P2 and P1 0 are indicated in bold.
The numbers in the last column refer to accession numbers in the Swissprot database.
the three central residues of the two reactive sites of potato PI-2
for alanines, in order to assess the role of the individual
domains in the inhibition of trypsin and chymotrypsin.
M AT E R I A L S A N D M E T H O D S
Strains, materials and media
All DNA manipulations were carried out with Escherichia coli
strain XL-1 blue, grown in Luria±Bertani medium supplied
with 100 mg´mL21 ampicillin. The Pichia pastoris strain
GS115 and the pPIC9 plasmid were purchased from Invitrogen
(San Diego, CA, USA), as part of the Pichia Expression Kit.
YPD medium (1% yeast extract, 2% peptone, 2% glucose),
MGY medium [1.34% yeast nitrogen base (Difco), 1%
glycerol, 0.4 mg´L21 biotin], BMG medium (100 mm K3PO4,
The construction of plasmids pB301 (encoding PI-2 variant
AAA-AAA), pB302 (TLE-PRN), pB303 (TRE-PRN), pB304
(AAA-PRN), pB305 (TLE-AAA), pB306 (TRE-AAA) [26],
R23 (AAA-HRS), R25 (AAA-VRS), psp2 (AAA-KRS), psp3
(AAA-SRH) and psp4 (AAA-RRS) was as described previously
[21]. The capital letters refer to the amino acids at positions
4±6 and 61±63 of PI-2 (Fig. 1). Plasmid R4 (encoding variant
AAA-ARA) was selected from a prevoulsy undescribed phage
display library (M. J. Beekwilder, unpublished results). Plasmid
pB309 (variant PRN-AAA) was created by amplifying the part
coding for mature PI-2 from pB301 [26] with primers 1 and 2
and Pwo polymerase (Boehringer, Mannheim). The obtained
385-bp DNA fragment was cleaved with restriction enzymes
NotI and HindIII and exchanged with the 371 bp HindIII±NotI
fragment of pB301, yielding plasmid pB309. Plasmid pB310
(variant PRN*-AAA) was made by amplifying the same fragment with primers 3 and 2. This 394 bp fragment was cleaved
with restriction enzymes NcoI and NotI, and exchanged for the
380-bp NcoI±NotI fragment in pB301, yielding plasmid pB310.
Constructs were tested by sequencing the entire ORF.
Expression constructs for PI-2 in Pichia pastoris
XhoI and NotI restriction sites were added to the PI-2 variants
by PCR on plasmids pB301, pB302, pB303, pB304, pB305,
pB306, pB309, pB310, R4, R23, R25, psp2, psp3 and psp4
using primers 4 and 2. After digestion with both enzymes, the
fragments were ligated into XhoI±NotI digested pPIC9 and
transformed to E. coli. This creates a translational fusion
between the leader sequence of the Saccharomyces mating
factor a and PI-2 as depicted in Fig. 2. Constructs were tested
by sequencing the entire cistron.
Yeast transformation and expression of recombinant PI-2
A 2-mg sample of each of the plasmids was isolated and
linearized with restriction enzyme Sal I. Digested plasmids were
introduced into P. pastoris GS115 by electroporation. Transformants were selected on MGY medium plates. The GS115
strain carries a mutation in the his4 gene, and therefore can not
survive on the minimal MGY plates. When the introduced
q FEBS 2000
PI-2 mutational analysis (Eur. J. Biochem. 267) 1977
Fig. 2. Structure of the PI-2 gene construct introduced into P. pastoris. (A) AOX1 promoter, the yeast a factor leader, the mature PI-2 gene with two
reactive sites, the stop codon and the XhoI and NotI restriction sites used for cloning. (B) DNA and protein sequence around the signal cleavage sites of the
yeast a factor for KEX2 and STE13 enzymes in yeast are shown, and around the stop codon of PI-2. Characters in bold indicate the mature PI-2 sequence.
plasmid has been integrated into the his4 locus, the mutated
part is replaced by the wild-type HIS4 sequences on pPIC9,
resulting in complementation of the histidine auxotrophy.
For each of the constructs, six individual single colonies of
transformants were transferred to 10 mL BMG medium, and
grown overnight at 28 8C and 300 r.p.m. The BMG medium
was removed by centrifugation at 1000 g for 5 min, and cells
were resuspended in 10 mL BMM medium to D600 ˆ 1.0.
Cells were grown for another 96 h at 28 8C and 300 rpm,
during which an additional 50 mL of methanol were added
daily in order to induce the AOX1 promoter, which drives the
synthesis of the PI-2 variants and secretion into the culture
medium. After removal of the cells by centrifugation at 1000 g
for 10 min, 25 mL of the supernatant from each of the cultures
was tested for trypsin inhibition using a radial diffusion assay,
as described previously [27]. For constructs AAA-AAA, TLEPRN, TRE-PRN, TLE-AAA, TRE-AAA, PRN-AAA and
PRN*-AAA, the best inhibitor-expressing clones were selected
and grown in 50 mL BMG medium at 28 8C. For the other
constructs, the best expressing 10 mL culture was used for
further purification. Cells were spun down and resuspended in
200 mL BMM medium to an D600 of 1.0 in 1-L flasks. Cultures
were grown for 96 h, during which 1 mL of methanol was
added daily. Cells were removed by centrifugation at 7000 g
followed by filtering through Acrocap 2 mm filters (Gelman
Sciences, Ann Arbor, MI, USA).
Protein purification
To purify the produced protein from the medium, the filtrate
was diluted three times in 50 mm acetic acid, the pH was set to
4.5 by addition of HCl, and the diluted medium was applied to
a 1-mL HiTrap SP column (Pharmacia). Bound material was
eluted by a salt gradient from 50 mm HAc to 50 mm HAc/1 m
NaCl. Fractions were collected, and 25 mL from each fraction
was assayed for trypsin inhibition in radial diffusion assay [27].
For a number of fractions, samples were taken and run on a
15% SDS/PAGE gel. For further analysis, fractions containing
the dominant 16 kDa band were dialyzed against a 50 mm
Tris/HCl/50 mm NaCl pH 7.6 buffer. The protein concentration was determined using the Bio-Rad Protein Assay. The
concentrations of PI-2 measured in that way were corrected by
a factor 0.73 (TLE-PRN) or 0.67 (AAA-AAA), which were
determined by comparing the Bio-Rad values with the absorption at 276 nm in 6 m guanidine hydrocloride and using an
extinction coefficient for PI-2 of 13 050 m21´cm21.
N-terminal sequencing
Purified samples for protein sequencing were size-separated by
SDS/PAGE (15% polyacrylamide) and transferred to Immobilon PSQ membrane (Millipore) with a Mini Trans-Blot
apparatus (Bio-Rad) using CAPS electroblotting buffer (10 mm
3[cyclohexylamino]-1-propanesulfonic acid in 10% MeOH/
1.5 mm Tris, pH 11). The filters containing the immobilized
proteins were washed thoroughly with deionized water and
subsequently saturated with 100% methanol. Blots were stained
for less than 1 min with 0.1% Coomassie Brilliant Blue R-250
in 40% methanol/1% acetic acid. After destaining with 50%
methanol and washing with deionized water, bands of interest
were excised (250 pmol per sample) and administered to an
Applied Biosystems Model 476A amino-acid sequencer. The
machine was run for 5±10 cycles.
Ki determination
The concentrations of active enzyme molecules in trypsin and
chymotrypsin solutions were determined by active site titration
[28]. In general, trypsin and chymotrypsin solutions were
40±50% active. The apparent equilibrium dissociation constants (Ki,app) of PI-2 variants towards trypsin and chymotrypsin
were determined according to the method of Green & Work
[29], modified according to Empie & Laskowski [30]. To
increasing concentrations of inhibitor in 100 mm Tris/HCl
pH ˆ 7.8 with 10 mm CaCl2 and 0.1 mg´mL21 BSA was added
protease to a concentration of 180, 60 or 15 nm in a total
volume of 150 mL. The concentration of enzyme was chosen so
that the ratio between the concentration of the enzyme ([E]) and
the observed affinity of the inhibitor (Ki) was minimally 100, to
enable accurate determination of Ki [31]. The equilibrium was
allowed to establish for 30 min at room temperature. Subsequently, 50 mL substrate was added (Ph-CO-Arg-NH-Np for
trypsin, SAAPLpNA for chymotrypsin) to a final concentration
1978 J. Beekwilder et al. (Eur. J. Biochem. 267)
of 1 mm. The residual protease activity was monitored by
measuring the change in extinction at 405 nm.
The equilibrium constant (Ki) and concentration of the
inhibitor ([I]stock) were determined by using the formula
described by Bieth [31].
…1 ÿ a†
‡ ‰EŠ …1 ÿ a†
a
where [I] is the total inhibitor concentration, a is the fraction of
total enzyme not bound to the inhibitor (measured as the ratio
of the substrate breakdown in the presence and absence of the
inhibitor), and [E] is the total enzyme concentration. This
formula was applied to different values of [I], by adding
different volumes (v) of the inhibitor stock solution (with
concentration [I]stock) into the total volume (V ). The result of
the experiments is that we have measured for a number of
volumes v the relative enzyme activity a. These data were
analyzed by non-linear regression with the program sigmaplot
5 using the formula:
‰IŠ ˆ K i v
…1 ÿ a†
‰IŠstock ˆ K i ‡ ‰EŠ …1 ÿ a†
V
a
In this way, for every inhibitor a Ki and [I]stock was determined.
The Ki values were further corrected according to [31] by the
formula
‰S0 Š
K i …apparent† ˆ K i 1 ‡
Km
where Km is the Michaelis constant of the substrate (0.77 mm
for Ph-CO-Arg-NH-Np on trypsin, 0.44 for SAAPLpNA on
q FEBS 2000
chymotrypsin), [S0] is the concentration of added substrate, and
Ki(apparent) is the observed Ki.
This mode of determination implies that it is not taken into
account that some inhibitors have two binding sites for the
enzyme tested. In our hands, more complex calculations
describing a system with four equilibria, as provided by
Bosterling & Quast [32] do not lead to consistent Ki values.
Therefore we report only Ki values that hold for both reactive
sites, without knowing their relative contribution.
R E S U LT S
Cloning and recombinant expression of PI-2-variants in
P. pastoris
The mutational study of PI-2 was focused on the central three
residues of both reactive sites, located at positions 4±6 (domain
1) and 61±64 (domain 2) (Fig. 1). Mutants of PI-2 studied here
are listed in Fig. 1, in which they are aligned to variants
isolated from solanaceous plants. We started from the part of
the cDNA which encodes the mature potato PI-2 [6,26]. Here
the protein encoded by this piece of DNA is called variant
TLE-PRN, indicating the nature of the residues in both reactive
sites. Either of these sites, or both, were substituted by three
alanine residues, resulting in variant TLE-AAA, AAA-PRN
and AAA-AAA. Also variants TRE-PRN, TRE-AAA and
PRN-AAA were created, to study the effect of substituting the
first reactive site with residues from the second. As an attempt
to graft the second reactive site loop onto the first domain,
mutant PRN*-AAA was created. As a second set, variants of
Fig. 3. Purification of PI-2 variant
TRE-PRN. (A) HiTrap SP cation-exchange
chromatography of 0.5 L P. pastoris culture
medium from a strain producing PI-2 variant
TRE-PRN. The protein content of fractions
eluted by a NaCl gradient (interrupted line) was
measured by absorbance at 280 nm (continuous
line). (B) Fifteen percent polyacrylamide/SDS
gel analysis of marker proteins (lane M), the
crude medium (lane S), the diluted medium
(lane D), the flow-through of the column
(lane F) and from a number of fractions
recovered from the column as described for (A)
(lanes 1±44). Protein was detected using
Coomassie Brilliant Blue. (C) Qualitative assay
of trypsin inhibitor activities of the same
fractions analyzed in (B), as indicated by clear
zones in a colored background.
q FEBS 2000
PI-2 mutational analysis (Eur. J. Biochem. 267) 1979
Table 1. PI-2 production in yeast. The yield of protein after purification
for the variants produced in P. pastoris are given in mg inhibitor protein per
litre culture medium.
Variant
Yield (mg´L21)
AAA-AAA
TLE-PRN
TRE-PRN
AAA-PRN
TLE-AAA
TRE-AAA
PRN-AAA
PRN*-AAA
AAA-ARA
AAA-HRS
AAA-VRS
AAA-KRS
AAA-SRH
AAA-RRS
1Š.10
11Š.32
2Š.45
3Š.69
0Š.67
0Š.43
1Š.95
0Š.85
1Š.01
1Š.20
1Š.15
0Š.21
2Š.11
0Š.20
PI-2 isolated by selection on trypsin of a phage display library
of PI-2 were involved, being AAA-HRS, AAA-KRS, AAARRS, AAA-VRH and AAA-VRS [21]. In addition a mutant
isolated previously, AAA-ARA, was involved. These mutants
were included to focus in more detail on the second reactive
site.
The PI-2 gene and its variants were subcloned in the
P. pastoris vector pPIC9 (Fig. 2), and the variant proteins were
expressed to be secreted into the culture medium of P. pastoris.
As judged from an SDS/PAGE gel stained with Coomassie
Brilliant Blue, the major supernatant protein is PI-2 (Fig. 3B,
lane S). The supernatant was further purified by FPLC on a
HiTrap SP column (Pharmacia). A typical elution profile is
shown in Fig. 3A. The fractions containing protein were tested
for the presence of inhibitory activity towards trypsin and
chymotrypsin by a radial diffusion assay (Fig. 3C). In general,
the protein profile corresponds to the protease-inhibiting
activity and the incidence of a band on the SDS/PAGE gel, as
shown in Fig. 3B, lanes 1±44. The quantity of the purified
protein is shown in Table 1, and ranges from 0.2 to 11 mg´L21
culture, depending on the variant.
Fig. 4. The protein content of purified
fractions of a number of inhibitors used, as
analyzed by electrophoresis in a 15%
polyacrylamide/SDS gel and staining with
Coomassie Brilliant Blue. The variants analyzed
in lanes A±J are indicated above the lanes. Lanes
D and J represent two different fractions of the
AAA-PRN variant.
Primary sequence of the expressed PI-2 variants
Purified fractions of each variant were analyzed on an
SDS/PAGE gel, as shown in Fig. 4. Some variants occur as two
bands, which do not separate completely on FPLC (e.g. Fig. 4,
lane A). In all cases, the most active fraction, according to the
radial diffusion assay, is dominated by the largest band, which
has a size corresponding to 16 kDa. For most variants, molecules of this size can be separated accurately from the smaller
molecules (compare variant AAA-PRN in Fig. 4, lanes D and
J). Fractions in which the smaller band is dominant (e.g.
Figure 4, lane J) are also active.
N-terminal sequencing was performed on a fraction of
variant TLE-PRN representing the larger species (Fig. 4, lane
B), and on a fraction of AAA-PRN, representing the smaller
species (Fig. 4, lane J). The major amino-acid signal of the
TLE-PRN sample was EAEAKA#TLE, where the # indicates
an empty signal. KA#TLE corresponds to the predicted
N-terminus of the native PI-2 protein found in potato, as shown
in Fig. 1 (the empty signal is interpreted to represent a cysteine
residue, which does not give a signal in Edman degradation).
The EAEA part corresponds to a glutamic acid-alanine repeat
which is normally efficiently removed by the STE13 protease
[33] (Fig. 2B).
The AAA-PRN sample of Fig. 4, lane J, which represents the
lower molecular mass species, produces two amino-acid
signals: EAKA#A and SEGSPE. The EAKA#A corresponds
to the expected N-terminus of the variant AAA-PRN,
N-terminally extended with one of the two glutamic acidalanine repeats. The SEGSPE sequence, which is the dominant
signal, corresponds to position 18 of the sequence of native PI-2
indicating that this fragment represents an N-terminally
truncated form of variant AAA-PRN, lacking part of the first
reactive site. A protein of similar size is present in all AAAAAA and PRN*-AAA samples (Fig. 4, lanes A and H). For
further analyses, fractions which were dominated by protein of
the size of intact PI-2 were used where possible (Fig. 4).
Inhibitory properties of alanine-substitution mutants
All synthesized variants of PI-2 were tested for inhibition of
bovine b trypsin and bovine a chymotrypsin, as described
above. A typical result is presented in Fig. 5, where the
1980 J. Beekwilder et al. (Eur. J. Biochem. 267)
q FEBS 2000
Table 2. Apparent equilibrium dissociation constants of PI-2 variants.
Ki values were determined by the method of Green & Work [29] in buffer
containing 0.1 m Tris, 10 mm CaCl2, 0.1 mg´mL21 BSA, pH 7.8.
Variant
Ki trypsin (nm)
Ki chymotrypsin (nm)
AAA-AAA
TLE-PRN
TRE-PRN
AAA-PRN
TLE-AAA
TRE-AAA
PRN-AAA
PRN*-AAA
AAA-ARA
AAA-HRS
AAA-VRS
AAA-KRS
AAA-SRH
AAA-RRS
. 300a
0.81 Š^ 0.074
0.79 Š^ 0.081
0.38 ^
Š 0.081
. 300a
18.5 Š^ 1.6
. 300a
. 300a
2.00 ^
Š 0.56
0.39 ^
Š 0.040
0.83 Š^ 0.12
0.50 Š^ 0.101
0.42 ^
Š 0.060
0.15 Š^ 0.015
72.4
0.56
1.65
0.93
2.09
122
217
156
4.2
1.0
0.24
1.4
0.55
4.1
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
5.7
0.054
0.48
0.073
0.41
31
94
13
1.0
0.25
0.070
0.28
0.11
0.74
a
The quantity of protease inhibitor available does not permit to accurately
determine Ki values . 300 nm.
enzymatic activity is plotted vs. the added volume of TRE-PRN
protein. The determined apparent equilibrium dissociation
constants are in Table 2. The wild-type PI-2 inhibits both
trypsin and chymotrypsin, with Ki ˆ 0.81 and 0.56 nm,
respectively. The contribution of the two individual reactive
sites to these activities is discussed below, on the basis of Ki
values obtained for relevant PI-2 variants.
The first reactive site does inhibit chymotrypsin
The first reactive site only inhibits chymotrypsin. When the
first domain is left intact and the second reactive site is
inactivated by alanine substitutions (variant TLE-AAA), the
affinity for chymotrypsin decreases 3.5-fold, from 0.6 to
2.1 nm. If, in addition, the first reactive site is knocked out,
chymotrypsin inhibition is reduced 35-fold to a Ki of 70 nm
(variant AAA-AAA).
The P1 leucine residue of the first reactive site is crucial. If it
is replaced by arginine, chymotrypsin inhibition is greatly
reduced. The Ki of variant TRE-AAA (122 nm) is 58 times
weaker than that of TLE-AAA (2.1 nm). Introducing PRN of
the second site (PRN-AAA), or the entire second reactive site
loop (PRN*-AAA), does not improve the affinity for chymotrypsin relative to the AAA-AAA variant.
The second reactive site inhibits trypsin
The central three residues of the second inhibitory loop are
crucial for the inhibition of trypsin. In most mutants in which
these are replaced by alanines (AAA-AAA, TLE-AAA, PRNAAA, PRN*-AAA), the Ki for trypsin decreases below the
detection level, meaning . 300-fold compared with the wildtype PI-2 TLE-PRN. This contrasts to what is observed when
the first reactive site is knocked out by alanine substitutions
(mutant AAA-PRN). This implies that the second domain must
be the site which inhibits trypsin.
When the effect of more subtle mutations is tested, the
second reactive site appears to be very robust. The affinity for
trypsin is hardly affected by mutations at the P2 or P1 0 position.
A mutant with alanine at both P2 and P1 0 (AAA-ARA;
Ki ˆ 2 nm) has only a 2.2±5-fold lower affinity than PI-2
variants with the intact second reactive site, like TLE-PRN,
TRE-PRN and AAA-PRN (Ki values are 0.9, 0.8 and 0.4 nm,
The first reactive site does not inhibit trypsin
The wild-type first reactive site (TLE) of PI-2 is not inhibitory
towards trypsin. When the second reactive site is substituted by
alanines and the wild-type sequence is maintained in the first
reactive site (variant TLE-AAA), inhibition of trypsin is not
detected. We tried to engineer the ability to inhibit trypsin into
the first reactive site. First, variant TRE-AAA, which has its P1
leucine residue replaced by arginine, was created for this
purpose. This variant has a Ki for trypsin of 18 nm. Compared
with variant TLE-AAA, which has no detectable affinity for
trypsin, this is a significant improvement. However, compared
with the domain 2 inhibitors of trypsin, such as AAA-PRN
(Ki ˆ 0.38 nm), TRE-AAA is a poor inhibitor. Nevertheless,
the moderate antitryptic activity of the TRE-AAA variant
demonstrates that mutation of the P1 residue can change the
specificity of this reactive site.
To improve the inhibitory properties of the first loop towards
trypsin, we introduced more mutations. Contrary to our
expectations, variant PRN-AAA does not inhibit trypsin to a
detectable level, although the three central residues are identical to those in the wild-type second reactive site. This
appeared to be due not only to a different context for PRN in
the primary sequence. If in addition, residues P3 0 to P10 0 of
the first reactive site are replaced by those of the second
(PRN*-AAA), the inhibitor still does not inhibit trypsin.
Apparently, the conformation of the first domain does not
allow PRN to present a proper orientation of the reactive site to
trypsin, even if the entire sequence context of the second
domain is there.
Fig. 5. Titration of chymotrypsin with PI-2 variant AAA-AAA. In this
case, the microliters of inhibitor stock solution (horizontal axis) were added
to a final concentration of 500 nm chymotrypsin in an end volume of
200 mL. Enzyme activities (vertical axis) were monitored as breakdown of
SAAPLpNA during 10 min, as described in Materials and methods
(squares). Observed rates were corrected for the spontaneous hydrolysis
of substrate. The values for Ki and [I]stock, fitted to the observed activities by
nonlinear regression are indicated in the upper right corner of the plot. The
curve resulting by filling in these values in the equation described in
Materials and methods is drawn as a continuous line.
q FEBS 2000
PI-2 mutational analysis (Eur. J. Biochem. 267) 1981
respectively). The best binding variant of PI-2 (AAA-RRS;
Ki ˆ 0.15 nm) has a 13-fold better affinity than the AAA-ARA
mutant. Apparently the binding of the second reactive site to
trypsin is determined predominantly by the P1 arginine, and is
enhanced by P2 and P1 0 residues.
The second reactive site also inhibits chymotrypsin
The second reactive site is a strong inhibitor of chymotrypsin.
When the first reactive site is knocked out, as in AAA-PRN, the
second reactive site maintains a similar affinity (Ki ˆ 0.93 nm)
for chymotrypsin as the wild-type PI-2 (0.56 nm).
The arginine at the P1 position is sufficient to maintain a
reasonable affinity for chymotrypsin, because mutant AAAARA still has an affinity of 4.2 nm for chymotrypsin compared
with 70 nm for AAA-AAA. Most variants of the second
reactive site which use the P1 arginine have a better affinity for
chymotrypsin than AAA-ARA, except for AAA-RRS. The best
binding variant of chymotrypsin (AAA-VRS; Ki ˆ 0.24 nm)
has 17-fold better affinity than AAA-ARA (Ki ˆ 4.2 nm).
Apparent inhibitor concentrations and stoichiometry
The method of Green & Work [29] determines both the Ki value
and the concentration of inhibitor in the stock solution. Apparent inhibitor concentrations from both trypsin and chymotrypsin inhibition curves, as well as concentrations determined
by a protein assay, are given in Table 3. Values determined by
the Green & Work method correspond roughly to those determined by the protein concentration assay, if it is assumed that a
single inhibitor molecule can bind two enzyme molecules at the
Table 3. Reactive site and inhibitor concentrations in mm in the stock
solutions. ±, Not detected (the inhibition observed was not sufficient to
calculate a concentration of the inhibitor); ND, not determined.
Variant
[Reactive sites] [Reactive sites] [Inhibitor] Reactive sites
on trypsina
on chymotrypsinb proteinc
per moleculed
AAA-AAA
TLE-PRN
TRE-PRN
AAA-PRN
TLE-AAA
TRE-AAA
PRN-AAA
PRN*-AAA
AAA-ARA
AAA-HRS
AAA-VRS
AAA-KRS
AAA-SRH
AAA-RRS
±
23.0
41.4
18.2
±
32.5
±
±
0.505
0.615
0.599
0.295
1.49
0.196
a
^
Š 0.4
Š^ 0.8
^
Š 0.3
Š^ 0.6
Š^
Š
^
Š
^
Š
^
^
Š
Š
^
0.021
0.026
0.026
0.012
0.072
0.008
10.2
34.6
23.1
22.2
10.5
83.8
10.6
12.3
0.560
0.506
0.626
0.292
1.88
0.252
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
^
Š
0.4
1.2
1.3
0.3
0.7
16
2.5
0.7
0.078
0.091
0.11
0.028
0.20
0.023
8Š.9
24Š.9
25Š.2
23Š.2
5Š.9
22Š.5
10Š.3
4Š.7
NDf
NDf
NDf
NDf
NDf
NDf
±
0.9
1.7
0.8
±
1.7
±
±
1.0
1.2
1.0
1.0
1.0
1.0
Š:
Š:
Š:
Š:
:Š
Š:
:Š
:Š
Š:
Š:
Š:
Š:
Š:
Š:
1.1
1.5
0.9
0.9
1.8
3.7
1.0
2.6
1.1e
1.0e
1.0e
1.0e
1.3e
1.3e
Concentration of inhibitor as determined by titration of trypsin.
Concentration of inhibitor as determined by titration of chymotrypsin.
c
Concentration of inhibitor as calculated from protein determination.
d
Molar concentrations of trypsin and chymotrypsin inhibitor sites
calculated from titration on trypsin and chymotrypsin, relative to the molar
concentration of inhibitor, relative to the molar concentration of inhibitor
determined by the protein concentration assay. e The molar concentration of
inhibitor was not determined by the protein concentration assay, therefore,
the lowest molar concetration determined from the titration experiments
was set to 1.0.
b
same time. In one case (TRE-AAA) the data seem not to match.
Empie & Laskowski [30] described that accurate inhibitor
concentration values can be obtained by using high enzyme and
inhibitor concentrations in the Green & Work method, but
appropriate quantities of TRE-AAA were not available. Ratios
between observed concentrations of binding sites are indicated
in the last column of Table 3.
The mutation studies teach us that several inhibitor variants
have two binding sites for one of the enzymes. For instance, one
molecule of variant TLE-PRN has a single binding site for
trypsin (PRN), but two sites for chymotrypsin (both TLE and
PRN). The observed ratio is 0.9 : 1.5, which suggests that TLE
(binding chymotrypsin but not trypsin) and PRN (binding both
trypsin and chymotrypsin) can be occupied at the same time by
chymotrypsin. Conversely, 1 mol TRE-PRN binds 0.9 mol
chymotrypsin and 1.7 mol trypsin. This indicates that TRE
(binding trypsin but not chymotrypsin) and PRN (binding both
trypsin and chymotrypsin) can be occupied at the same time by
trypsin. When the first reactive site is AAA (as in variants
AAA-AAA and AAA-PRN), the number of reactive sites per
inhibitor molecule, for both trypsin and chymotrypsin, is close
to 1. This indicates that the AAA mutation in the first reactive
site abolishes binding of both enzymes completely. In contrast,
in a number of cases where the second domain is substituted by
alanines (as in variants TLE-AAA, TRE-AAA, PRN*-AAA),
there appear to be two binding sites for chymotrypsin left per
inhibitor molecule. This suggests that the second reactive-site
AAA can still bind chymotrypsin weakly. These observations
are in agreement with those made in the Ki determination and
indicate that PI-2 inhibitor molecules can bind two enzyme
molecules simultaneously.
DISCUSSION
A yeast expression system for PI-2
We investigated the inhibitory activity of the two domains of
potato inhibitor PI-2 to trypsin and chymotrypsin. PI-2 is
potentially useful, both as a tool for insect pest management
and as a cancer-preventive agent. However, for application,
some properties may benefit from engineering. For instance, for
insect pest management, its specificity could be altered to fit
PI-2-insensitive serine proteases that are deployed by pest
insects. For application in cancer prevention, better resistance
to cooking practices could be a desirable property, but also
improved affinity for the proposed chymotrypsin-like targets
[12]. For these purposes, understanding the determinants of the
specificity of PI-2 is crucial. Hence we set out to probe the role
of its individual domains.
As a first step in a mutagenesis study of PI-2, expression in
the yeast P. pastoris was established. In E. coli, PI-2 can be
produced in quantities up to 50 mg´L21 [26]. P. pastoris produces up to 250 times more PI-2 in a shaker flask. Such
amounts permit detailed analysis of the equilibrium constant.
The protein can be purified readily from the yeast medium
using a single ion-exchange chromatography step. The recombinantly produced protein is generally intact, as judged from its
mobility in SDS/PAGE. N-terminal sequence determination
reveals that up to four additional amino acids are found at
the N-terminus. These originate from the signal sequence of
the yeast a-factor, which is used to direct secretion of PI-2. The
glutamic acid-alanine repeat is normally cleaved by the
diaminopeptidase of P. pastoris [33], but apparently is not
removed in the case of PI-2. The poor removal in P. pastoris of
1982 J. Beekwilder et al. (Eur. J. Biochem. 267)
the glutamic acid-alanine sequence from the N-terminus has
been reported for other proteins [34].
Functional analysis of PI-2 produced in P. pastoris and E. coli
A clear picture emerges from the affinity studies of the alanine
substitution mutants of PI-2. The first domain of PI-2 can
inhibit only chymotrypsin, while the second domain can inhibit
both trypsin and chymotrypsin. We did not attempt to compare
this result with that of PI-2 purified from potato, because potato
PI-2 is a mixture of several protomers, all with slightly different
specificities, which are refractory to separation [5]. Published
values for some isolates of PI-2 suggest a Ki for chymotrypsin
of 20 nm [5] or 0.16 nm [35], but these values probably both
account for a mixture of PI-2 protomers with unknown
sequence.
Comparison of affinities of the yeast PI-2 with the more
qualitative data published previously [26] seems more appropriate, because these account for the same gene, isolated from
tubers of cv. Bintje [6]. With the E. coli-produced PI-2, the
effect of alanine substitutions was tested in both a qualitative
inhibition assay for trypsin and chymotrypsin, and in a phagedisplay selection system. When we compare the previous
results with the present analysis, there are matching results for
the second domain, i.e. it inhibits both trypsin and chymotrypsin. Regarding the first domain, a difference is observed
between the yeast material and the E. coli material. In the
qualitative inhibition assay with the E. coli-produced TLEAAA variant, no inhibitory activity to either of the tested
enzymes was observed [26]. Also, bacteriophage particles
carrying TLE-AAA bound only twofold more efficiently to
chymotrypsin than the inactive AAA-AAA variant, while the
AAA-PRN mutant bound 30 times better. In contrast, the TLEAAA mutant produced in P. pastoris accounts for a 2.1-nm
affinity for chymotrypsin.
A possible cause of the behavior of the E. coli-produced PI-2
is incorrect folding within the first domain. When purifying
PI-2 from E. coli, it was sometimes observed that the protein
would quickly lose activity after purification. Inhibitory activity
could be regained by treatment of the protein with a mixture
of oxidized and reduced glutathione, aimed at correct pairing
of disulfides ([36]; P. Bakker & M. A. Jongsma, unpublished
observations). This observation indicates that correct disulfide
bond formation is incomplete in the E. coli-produced material,
and could provide a reason for the lack of activity of domain 1.
As an alternative explanation, it can be considered that the
affinity for chymotrypsin of the yeast produced TLE-AAA
mutant may be due to the additional amino acids at the
N-terminus of the protein, which were found by protein
sequencing. Being in close vicinity (at the putative P5 position)
of the first domain, these amino acids might improve the
binding of the inhibitor to the protease. At present we are
studying ways to avoid the presence of the additional
N-terminal amino acids in Pichia-produced PI-2.
The properties of E. coli-produced PI-2 are of particular
relevance to the phage display approach for engineering PI-2
[21,26]. To introduce variability in order to select inhibitors
with novel specificities, proper folding of the randomized
domain is a prerequisite. The results of both production systems
correspond for the second domain. Therefore, it seems reasonable to assume that this domain is in its native conformation in
E. coli-produced PI-2. It is also relevant that the second domain
can inhibit both trypsin and chymotrypsin, and can tolerate
mutations around the P1 residue, which indicates that this
q FEBS 2000
domain is sufficiently flexible to be adapted to fit different
enzymes.
Domain 2 inhibits both trypsin and chymotrypsin
A clear difference between the two domains of PI-2 can be
observed. The first domain behaves as a typical inhibitor, which
inactivates either trypsin or chymotrypsin, depending on the P1
residue. The second domain of PI-2 appears to be a more
versatile inhibitor than the first. The native second domain
sequence, PRN, inhibits both trypsin and chymotrypsin. This
may be a property selected for in nature, because it can be
considered economically favorable to produce a single inhibitor
gene which can inhibit a range of enzymes. Remarkably, even
variants that were selected by phage display only for binding to
trypsin, still retain their activity against chymotrypsin, and this
activity seems to be a property connected to the presence of the
arginine at the P1 position. A limited impact of alanine
substitutions on the affinity for chymotrypsin at positions in
close proximity of the P1 residue has also been noted (but not
explained) in the case of the bovine pancreatic trypsin inhibitor
(BPTI) [37]. The present analysis cannot rule out the possibility
that other residues, outside the regions of PI-2 targeted here,
contribute significantly to the affinity for trypsin or
chymotrypsin.
The observed importance for both trypsin and chymotrypsin
inhibition of the P1 arginine is quite unusual. There is a wellknown specificity difference between trypsin, cleaving at the
C-terminal side of positively charged amino acids, and chymotrypsin, which cleaves preferentially at the C-terminal side of
apolar and aromatic residues [38]. The specificity is related to
the charge of the residue at the bottom of the S1 pocket of the
enzyme. This residue (189) is, in the case of chymotrypsin, a
neutral serine, and for trypsin a negatively charged aspartic
acid. In the case of the Kunitz-type inhibitor BPTI, the penalty
for loosing the S1±P1 contact is high: the affinity of BPTI for
chymotrypsin is over five orders of magnitude lower than that
for trypsin [39]. For PI-2 this is not the case, as the affinity of
the AAA-PRN variant for trypsin and chymotrypsin differs by
less than twofold.
In a recent paper, some light was shed on how positively
charged P1 residues are accommodated by chymotrypsin [40].
Two conformations of the side chain of P1 lysine have been
encountered. In the crystal structure of the BPTI±chymotrypsin
complex [41] the lysyl side chain bends and contacts Ser217 of
chymotrypsin. In the Lys18±OMTKY3±chymotrypsin complex
[40] the P1 lysine residue is embedded deeply in the S1 pocket.
The Ser217 of chymotrypsin is available only when P3 is a
proline residue (as in BPTI), whereas it is a cysteine in PI-2.
Therefore, it seems reasonable to assume that the P1 arginine
of PI-2 actually enters the S1 pocket. As appears from the
improved affinity of AAA-ARA relative to AAA-AAA, the P1
arginine contributes considerably to the affinity for chymotrypsin. This would suggest that the P1 arginine of PI-2 is not
protonated under our experimental conditions (pH 7.8), and
that it is energetically favorable to embed its side chain in the
apolar S1 pocket of chymotrypsin, like in the case of Lys18±
OMTKY3 [40].
Engineering trypsin inhibitory properties into the first
domain
The first domain of PI-2 was proposed to consist of the reactive
site loop, encoded in the first 27 amino acids, supported by
three cysteine bonds to the last 27 amino acids of PI-2 [22]. The
q FEBS 2000
observations made for the TLE-AAA and TRE-AAA variants
constitute experimental support for this proposal. The activity
of these variants indicates that the reactive site loop is held in
an inhibitory conformation, to which disulfides generally make
strong contribution [38]. Recently, experimental evidence has
been provided for disulfide bridging between the N-terminus
and C-terminus of a PI-2-like protein from tobacco flower
stigma [23].
When we tried to engineer the same inhibitory properties as
the second domain into the first domain, it became clear that
both domains cannot be interchanged. For instance, in contrast to
the second domain, the activity of the first domain does not
benefit clearly from the presence of an arginine at the P1
position. This is seen most clearly in the experiment in which
the P1 leucine of the first domain is changed into arginine. This
mutation reduces activity to chymotrypsin of the first domain,
and introduces only a modest activity to trypsin. By the leucineto-arginine mutation, the reactive site loop of the first domain
becomes identical to that of a PI-2 variant encoded by a
cDNA isolated from wounded tomato leaves [14] (Fig. 1). In
plant defense, this inhibitor might function to inhibit other
specificities than bovine trypsin or chymotrypsin.
The effect of point mutations at or around the P1 position on
the specificity of protease inhibitors from the Kazal family has
been researched extensively by Laskowski and colleagues
[30,40,42]. From their work it is apparent that the changes in
residues other than the primary recognition residue (P1), even
sequentially far from the reactive site, may exert large effects
on the specificity of inhibitors, provided these residues are in
contact with the enzyme. The crystal structure of PCI with
Streptomyces griseus proteinase B [22] reveals a number of
additional inhibitor residues that contact the protease, but are
not touched in the present mutagenesis study. These may
provide suitable targets for mutagenesis in the future.
In another approach, we introduced either the central
tripeptide or 13 amino acids of the reactive-site loop into the
reactive site of domain 1, as in mutants PRN-AAA and PRN*AAA. These mutations make the protein relatively unrecoverable, as we did not isolate strong bands of the correct molecular
mass. Analysis of the stoichiometry of binding indicates that
the PRN-AAA variant binds protease only to the second domain
(AAA), and that the PRN*-AAA has two weak binding sites for
chymotrypsin. This suggests that the properties of the first
domain are due not only to the protein sequence in the surface
loop, which can only provide a context for weak chymotrypsin
inhibition, as in PRN*-AAA, but also to its proper folding. Our
mutations probably affect the folding pathway of PI-2, and
thereby decrease the probability of proper disulphide formation
between the N-teminal and C-terminal residues. The second
domain is less susceptible to this because the protein backbone
is not interrupted as is the case for the first domain [22,23]. The
effect of mutations on function is therefore very different for
both domains and the second domain is most convenient for
grafting novel specificities.
ACKNOWLEDGEMENTS
This work was funded by the Dutch Technology Foundation and by the
Dutch Ministry of Agriculture by DWK program subsidy 282.
PI-2 mutational analysis (Eur. J. Biochem. 267) 1983
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
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