Capture of the potentially posi-tively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme by modell
Trang 1inhibitor relaxes the strict intrinsic selectivity
Tim J Winterburn1, Lowri H Phylip1, Daniel Bur2, David M Wyatt1, Colin Berry1and John Kay1
1 School of Biosciences, Cardiff University, UK
2 Actelion Pharmaceuticals Ltd, Allschwil, Switzerland
Gene-encoded inhibitors of aspartic proteinases are
rather rare in nature Thus, there is a need to
under-stand the mechanisms of action of the few that are
known, in order to exploit their therapeutic potential
[1] We have described previously one such inhibitor:
the IA3 protein from Saccharomyces cerevisiae [1–4]
This remarkable polypeptide not only is a highly
potent inhibitor of its target enzyme, saccharopepsin,
but also appears to be completely specific for this sole
target proteinase [1,2] Crystal structures solved for
complexes of IA3 with saccharopepsin revealed an
unprecedented mechanism of action [2,3] IA3 from
S cerevisiaeconsists of 68 residues but all of the inhib-itory activity towards saccharopepsin resides within the N-terminal half or segment of the polypeptide [2,3] The free inhibitor is essentially unstructured [5,6] but, upon contacting its target enzyme, residues 2–32 become ordered and adopt an alpha helical conforma-tion occupying the active site cleft of the proteinase [2,3] This absolute selectivity for saccharopepsin was shown to be conferred by a combination of the K18 and D22 residues in the S cerevisiae IA3 sequence
Keywords
aspartic proteinase inhibition; IA3; inhibitor
engineering; Pichia aspartic proteinase;
specificity relaxation
Correspondence
J Kay, School of Biosciences, Cardiff
University, Museum Avenue, Cardiff CF10
3US, UK
Fax: +44 029 20 87 41 16
Tel: +44 029 20 87 41 24
E-mail: kayj@cardiff.ac.uk
(Received 30 March 2007, revised 23 May
2007, accepted 25 May 2007)
doi:10.1111/j.1742-4658.2007.05901.x
Yeast IA3 aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, sac-charopepsin Even aspartic proteinases that are very closely similar to saccharopepsin (e.g the vacuolar enzyme from Pichia pastoris) are not sus-ceptible to significant inhibition The Pichia proteinase was selected as the target for initial attempts to engineer IA3 to re-design the specificity The
IA3polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence Alterations made by deletion or exchange
of the residues in the C-terminal segment of these polypeptides had only minor effects By contrast, extension of each of these wild-type and
chimaer-ic polypeptides at its N-terminus by an MK(H)7MQ sequence generated inhibitors that displayed subnanomolar potency towards the Pichia enzyme This gain-in-function was completely reversed upon removal of the exten-sion sequence by exopeptidase trimming Capture of the potentially posi-tively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme
by modelling, may increase the local IA3 concentration and create an anchor that enables the N-terminal segment residues to be harboured in clo-ser proximity to the enzyme active site, thus promoting their interaction In saccharopepsin, some of the counterpart residues are different and, consis-tent with this, the N-terminal extension of each IA3 polypeptide was with-out major effect on the potency of interaction with saccharopepsin In this way, it is possible to convert IA3 polypeptides that display little affinity for the Pichia enzyme into potent inhibitors of this proteinase and thus broaden the target selectivity of this remarkable small protein
Abbreviations
Nph, L -nitrophenylalanine; PpPr, vacuolar aspartic proteinase from Pichia pastoris; Z, L -norleucine.
Trang 2coupled with the requirement for an alanine residue to
be present at position 213 in saccharopepsin [1]
A wide range of other aspartic proteinases of plant,
parasite, vertebrate and fungal origin has been shown
previously to be resistant to inhibition by IA3 [2]
Included among these enzymes are a number with
sequences that are very closely related to that of
sac-charopepsin (e.g the vacuolar aspartic proteinase from
P pastoris; PpPr) [1] This shares a sequence identity
of 77% with saccharopepsin and essentially all of the
active site residues of saccharopepsin that contact the
helical IA3 inhibitor are identical in PpPr PpPr also
has the crucial Ala residue present at position 213 in
its sequence Despite this close relatedness, the two
enzymes differ drastically in their susceptibility to
inhi-bition by IA3 Accordingly, it was of considerable
interest to examine whether IA3 could be adapted to
loosen its stringent specificity and, in this way, begin
the process of engineering it to target aspartic
protein-ase(s) other than saccharopepsin Since PpPr is not
inhibited effectively by IA3 yet is so closely related to
saccharopepsin, it was an obvious choice as the initial
new target enzyme In the present study, we show that,
inter alia, inhibitors with subnanomolar potency
against PpPr, can be generated by simply attaching a histidine-rich extension at the N-terminus of the IA3 polypeptide This dramatic alteration in behaviour may be explained by the positively ionisable histidine residues initiating additional contacts outside the active site that promote occupation of the active site of the target proteinase by the inhibitory segment
For ease of interpretation, residues in the inhibitors are denoted by single letter abbreviations while those from the proteinase are indicated in the three-letter code
Results and Discussion
Wild-type IA3and PpPr
We have reported previously that, at the standard pH
of 4.7 that we have justified and used consistently in all of our earlier studies [1–4], wild-type IA3 from
S cerevisiae has an inhibitory potency against saccha-ropepsin that is so tight that the Ki value lies at or beyond the limits of accurate determination using the assay methodology available It has thus been esti-mated as < 0.1 nm [1–4] and, in comparative terms,
S cerevisiae IA3 is ineffective against PpPr (1; Fig 1)
1
2
3
4
5
6
7
8
9
10
M
M
M
M
(H) ZQ
(H) ZQ
N K D E
34 24 22
K (n M )
55 ± 11
15 ± 3
100 ± 20 NI
3 ± 0.5
4 ± 0.5
15 ± 5
280 ± 30
2 ± 0.2
10 ± 1
E
N K D H
N K D
S
2
Fig 1 Inhibition at pH 4.7 of PpPr by wild-type and chimaeric forms of IA 3 from S cerevisiae and S castellii Sequences of IA 3 from S cere-visiae and S castellii (detailed in Fig 2) are depicted schematically by dark-shaded and open boxes respectively, with residues at positions
1, 2, 18, 22 and 68/81 identified individually Inhibitors 1-3 and 5 were recombinant proteins containing an additional LE(H)6 sequence attached to the C-terminal residue (E68 for 1 & 5; H81 for 2 & 3) Inhibitors 4 and 6-10 were synthetic peptides of the indicated length In these forms of IA3, L -norleucine (Z) was substituted for methionine NI = no inhibition at 2 l M
Trang 3We have also reported previously that our constant
searching of the sequence databases for orthologues of
S cerevisiae IA3 enabled us to identify the
counter-part polypeptide from Saccharomyces castellii [1] To
determine whether the S castellii polypeptide might
be a more effective inhibitor of PpPr upon which
to base initial protein engineering studies, it was
produced in recombinant form in Escherichia coli
and purified to homogeneity as described in the
Experimental procedures The S castellii IA3 was,
however, only marginally more effective as an inhibitor
of PpPr than its S cerevisiae counterpart (cf 2 and 1;
Fig 1)
The effect of C-terminal segment residues on the
inhibitory activity of the N-terminal segment
The sequences of IA3 from S castellii and S cerevisiae
are aligned in Fig 2 These show only 45% identity in
the N-terminal ‘segment’ (residues 2–32) that has been
demonstrated previously to contain the inhibitory
activity towards saccharopepsin [1–4] Residues 33–35
are identical in both sequences and form a link
between the inhibitory N-terminal ‘segment’ and
resi-dues of the C-terminal ‘segment’ The C-terminal
seg-ment (residues 36–81; Fig 2) from S castellii IA3 is
considerably longer than its counterpart (residues
36–68) in the S cerevisiae polypeptide and differs
sub-stantially in sequence (Fig 2) To establish whether the
respective C-terminal segments might have an influence
(beneficial or detrimental) on any inhibitory activity
that might be intrinsic to the N-terminal segments,
chimaeric proteins were engineered in which residues
35–81 and 35–68 in the respective polypeptides were
replaced by their counterparts from the other
sequence The chimaera that consisted of residues 1–34
from S cerevisiae IA3fused to residues 35–81 from the
S castellii sequence remained as poor an inhibitor of
PpPr as the wild-type S cerevisiae IA3 (cf 3 and 1;
Fig 1) A shorter variant of the S cerevisiae
poly-peptide which terminated at residue 34 and so was
completely devoid of any residues whatsoever to
correspond to positions 35–68⁄ 81, did not inhibit PpPr
either (4; Fig 1) Thus, the N-terminal segment of
S cerevisiae IA3 does not have any significant effect
on PpPr, irrespective of the absence, presence or
nature of the residues contributing the C-terminal segment
Against saccharopepsin, S cerevisiae-based inhibi-tors 3 and 4 both had Ki values of < 0.1 nm at
pH 4.7, just as reported previously for the full-length, wild-type S cerevisiae polypeptide (inhibitor 1) [2–4] Entirely in keeping with these earlier findings, the nat-ure and indeed presence or absence of residues beyond position 34 in this sequence would appear to have no influence on inhibition of saccharopepsin
The reciprocal chimaera, which consisted of residues 1–34 from S castellii IA3 fused to residues 35–68 from the S cerevisiae polypeptide, was slightly more effect-ive as an inhibitor of PpPr than the wild-type
S castellii IA3 (cf 5 and 2; Fig 1), with the measured
Kifalling into the single digit nanomolar range Since these two polypeptides differ only in the nature of their C-terminal segments, it would appear that the C-terminal segment (residues 35–81) from S castellii
IA3 has a slight detrimental effect on the inhibitory activity against PpPr that is intrinsic to its own N-ter-minal segment This interpretation was examined by producing a shorter variant of the S castellii sequence that lacked any C-terminal segment and so consisted only of residues 2–34 This had a comparable inhibi-tory potency to that of the chimaera (cf 6 and 5; Fig 1) The detrimental effect of S castellii residues 35–81 may result from adverse interaction(s) occurring either within the full-length S castellii polypeptide (residues 1–81) itself or between the C-terminal seg-ment of the polypeptide and PpPr at a remote site far removed from the active site cleft where the N-terminal segment might be expected to bind Furthermore, because the chimaeric inhibitor 5 had a comparable potency to that of inhibitor 6 which was devoid of any C-terminal segment, the C-terminal segment (residues 35–68) from S cerevisiae IA3 would appear, once again, to be inert, this time being without influence on the inhibitory activity against PpPr that is intrinsic to the N-terminal segment of the S castellii polypeptide Against saccharopepsin, the S castellii N-terminal segment (inhibitor 6) is a potent inhibitor (Ki¼ 0.4 ± 0.1 nm at pH 4.7) and the interactions of this type of inhibitor variant within the active site cleft of the enzyme have been documented previously [4] The counterpart N-terminal segment (residues 2–34) from
Species Sequence
S cerevisiae
S castellii
Fig 2 Alignment of the sequences of IA3from S cerevisiae and S castellii Identical residues are boxed in black.
Trang 4S cerevisiae IA3 (inhibitor 4) is even more effective
than inhibitor 6 against saccharopepsin (Ki< 0.1 nm)
[1–4] This behaviour stands in stark contrast to that
observed against PpPr where inhibitor 6 was >
500-fold more effective than inhibitor 4 (Fig 1)
Conse-quently, the effect of exchanging residues within the
inhibitory sequence of 6 was examined Replacement
of the S castellii residues 24–34 by the corresponding
residues from S cerevisiae IA3had only a small
(three-to four-fold) adverse effect on inhibi(three-tory potency
against PpPr (cf 7 and 6; Fig 1) However, when the
key residues K18 and D22 that have been shown to be
so important in restricting the activity of S cerevisiae
IA3to saccharopepsin as its sole target proteinase were
introduced into the S castellii sequence in place of the
intrinsic M18⁄ K22 pair, the inhibitory activity against
PpPr was essentially destroyed (cf 8 and 6; Fig 1)
Thus, it would appear that the residues at positions 18
and 22 again play a decisive role, allowing effective
inhibition of PpPr by the S castellii polypeptide
Changes in other locations, including the ‘remote’
attachment of residues 35–81 from its own C-terminal
segment, cause only minor perturbation of the
inhibi-tory potency intrinsic to the N-terminal segment
The effect of extending the N-terminal segment
Since the above-described adaptations in the
C-ter-minal segment were without major influence, the
effect of extending the inhibitory segment (residues
2–34) at its N-terminal end was investigated next
Careful consideration was given to the design of the
N-terminal extension sequence that was to be
intro-duced Insufficient amounts of PpPr were produced
for crystallization attempts to be a realistic possibility;
thus, the design process was informed by a 3D model
for PpPr that was generated based on the crystal
structures that have been reported previously for
saccharopepsin complexed with different variants of
S cerevisiae IA3 [2,3] The two proteinases share 77%
sequence identity, and they are likely to have closely
similar 3D structures Inspection of the PpPr model
identified a patch of negatively ionisable amino acids
on the surface of the enzyme, adjacent to the end of
the active site cleft where the N-terminal residues of
an inhibitory IA3 helix would be expected to bind
(Fig 3A) Extension of the inhibitory sequence of IA3
at its N-terminus by four amino acids (residues X8–
X11, Fig 3B) was estimated to generate a polypeptide
that would make few beneficial contacts, whereas a
seven amino acid extension (consisting of residues
X5–X11, Fig 3B) would be long enough to make
some of the predicted contacts with the side-chains of
residues such as Asp161, Asp164 and Glu17 on the surface of PpPr; and an extension of nine amino acids (residues X3–X11) would exploit the potential binding site offered by this patch to the full (Fig 3B) Consequently, IA3 variants with four (HHZQ) and seven (HHHHHZQ) residue extension sequences, respectively, were designed initially to introduce the appropriate number of potentially positively charged (at the experimental pH of 4.7) histidine residues (at positions X8–X9 or positions X5–X9, respectively) followed by a norleucine residue (indicated by Z, at position X10) and a glutamine (residue X11) in place
of the naturally occurring N-terminal (methionine)
A
B
Fig 3 Representation of PpPr and the extension residues of IA3 (A) Negatively ionisable surface residues (red) adjacent to the edge
of the active site of PpPr; the active site is occupied by a putative helical IA3inhibitor with the residue at its N-terminus serving as a potential attachment point for an extension; (B) potential interac-tions of the indicated negatively ionisable surface residues (red)
of PpPr with several positively ionisable amino acids of the MK(H) 7 MQ extension sequence (residues X1–X11, respectively) The C-alpha representation of the helical inhibitory segment occu-pying the active site of the proteinase is depicted in yellow.
Trang 5residue of IA3 which has been shown previously to
be unimportant for inhibitory activity [1,2] The logic
for introduction of the Q residue is explained below;
inspection of the PpPr model revealed an additional
pocket that might accommodate the side-chain of
straight-chained residues such as norleucine (in
syn-thetic peptides) or methionine (in recombinant
pro-teins) at position X10 (Fig 3B)
These two extension sequences were introduced at
the N-terminus of inhibitor 7 This inhibitor was
selec-ted initially because it consists only of residues 2–34
and was thus free of any potential ‘complications’ that
might have been contributed to binding by the
pres-ence of residues 35–68⁄ 81 Since it was also only a
weak inhibitor of PpPr, any improvement in inhibitory
potency should be readily quantifiable The
polypep-tide containing the short, four-residue (HHZQ)
exten-sion had a comparable potency to that of its parent
(i.e non-extended inhibitor 7) (cf 9 and 7; Fig 1)
However, the longer (H)5ZQ-extended variant showed
a seven-fold improvement in potency against PpPr
(cf 10 and 7; Fig 1)
Since this seven-residue extension was already
suffi-cient to engender an improvement of inhibitory
potency against PpPr, the extension sequence was
lengthened further to include all seven histidine
resi-dues indicated by the model The additional two
histi-dine residues (at positions X3 and X4; Fig 3B) were
introduced downstream from a methionine and a
lysine residue (at positions X1 and X2, respectively),
the logic for which will be substantiated below These
four MKHH residues were thus introduced upstream
from the (H)5-containing extension described above to
generate the sequence MK(H)7MQ (Fig 3B)
Coinci-dentally, this extension contains sufficient histidine
res-idues to enable it to be used as an affinity tag for
purification purposes In all of our previous studies
with IA3 [1–4], recombinant protein versions such as
inhibitors 1–3 and 5 (Fig 1) were purified to
homogen-eity from E coli lysates by nickel-chelate
chromatogra-phy, facilitated by a LE(H)6 tag that was positioned
at the C-terminus of each polypeptide This tag was
shown to have no effect on the potency of inhibition
of saccharopepsin [2,3] This C-terminal His-tag could
thus be deleted and introduced instead within the
extension sequence at the N-terminus of each desired
polypeptide
The 11 amino acid-containing sequence MK(H)7MQ
was thus introduced as the N-terminal extension
attached to residue 2 of S castellii IA3, as described in
the Methods section The resultant, recombinant
pro-tein (and the others to be described) were purified
from E coli cell lysates just as readily as their
C-ter-minally tagged predecessors using exactly the protocol described previously for the latter [1–4] The N-termin-ally extended S castellii protein showed a potency against PpPr that was improved by 150-fold compared
to its counterpart with the tag at the C-terminus of the polypeptide (cf 11; Fig 4; 2, Fig 1) With such a dra-matic benefit from extension of the inhibitory segment
at its N-terminus, it was clearly of importance to establish whether this enhanced potency was influenced
to any extent by the presence⁄ absence and nature of the amino acid residues contributing the C-terminal segment to this polypeptide Consequently, the residues (35–81; Fig 2) comprising the C-terminal segment were systematically deleted, in blocks of 12⁄ 13 residues
at a time Truncation of the N-terminally tagged
S castellii polypeptide (inhibitor 11) at residue Q68 generated inhibitor 12 which corresponded in overall length to S cerevisiae IA3 Although this resulted in a seven-fold weakening in potency against PpPr (cf 12 with 11; Fig 4), a subnanomolar Ki value was still recorded for inhibitor 12 Further truncation at resi-dues Y57 and K45, respectively (inhibitors 13 and 14; Fig 4) did not cause any further significant loss of inhibitory potency against PpPr Thus, in contrast to the detrimental effect that was described above when residues 35–81 were attached in the full-length, C-terminally tagged inhibitor 2, the presence of residues 69–81 at the C-terminus of the N-terminally tagged S castellii polypeptide appears to confer a benefit to the inhibition of PpPr (cf inhibitors 11 and 12; Fig 4) This was substantiated by the data obtained for the chimaeric inhibitor 15 (Fig 4) which was identical in length to inhibitor 12 but contained residues 35–68 from S cerevisiae IA3as the C-terminal segment in place of the counterpart S castellii residues
of inhibitor 12 Both inhibitors had comparable Ki values against PpPr (15 and 12; Fig 4) Consequently, the nature of residues 35–68 at the C-terminus of these N-terminally tagged polypeptides would appear to be unimportant for inhibition Quantitatively, the magni-tude of the beneficial effect conferred by residues 69–81 from the S castellii sequence is small compared
to that achieved by location of the full-length extension
at the N-terminal end of the polypeptide The benefit of these alterations at each extremity of the polypeptide chain, must of necessity arise from contacts that are made outwith the active site of the enzyme
The effect of removal of the N-terminal extension The full N-terminal extension sequence, MK(H)7MQ, was designed to consist of an even number of residues upstream from the Q residue This enables their
Trang 6removal by the action of a diamino exopeptidase,
cath-epsin C, which cleaves off dipeptides sequentially from
the N-terminus of a polypeptide [7] The first
methion-ine (X1) in the extension sequence is necessary for
translation initiation and the lysine is located at
position X2 so that in the event of removal of the
N-terminal methionine residue by an E coli
amino-peptidase, the new N-terminal lysine residue would
prevent any digestion by cathepsin C [7]; such a
desM(X1)-extended IA3 would still contain its His-tag
and so could be removed by nickel-chelate
chromato-graphy Unlike an N-terminal lysine residue, glutamine
(at position X11; Fig 3) does not in itself constitute a
stop point for cleavage by cathepsin C However, if
dipeptide removal by cathepsin C is performed in the
presence of an excess of glutamine cyclotransferase,
once an N-terminal glutamine residue is newly exposed,
it is rapidly converted into pyroglutamic acid Further
digestion by cathepsin C is thus prevented, leaving the
cyclised Q as the N-terminal residue (replacing the
nat-urally occurring Met1) of each IA3 polypeptide
Appli-cation of this trimming treatment to the longest and
shortest variants with the wild-type S castellii sequence
(inhibitors 11 and 14) and to the chimaeric
inhib-itor 15, generated polypeptides 11T, 14T and 15T,
respectively, each with a pyrrolidone carboxylic acid
residue (cyclised Q) at its N-terminal end (Fig 4) Each trimmed polypeptide was purified as described in the Experimental Procedures section by passage through a nickel-chelate column to remove any residual parent
IA3 with its intact histidine tag together with the two enzymes used in the trimming procedure which are also both C-terminally His-tagged The purity, identity and concentration of each trimmed polypeptide was deter-mined by MALDI-TOF mass spectrometry and amino acid analysis As a representative example, the spectra for one of these inhibitor pairs (14⁄ 14T) are depicted in Fig 5 The mass peak observed in the spectrum for the parent inhibitor 14 (Fig 5A) corresponds to the theor-etical value for the N-terminally extended S castellii
IA3variant terminating at K45 as its C-terminus After treatment with the cathepsin C⁄ glutamine cyclotrans-ferase enzyme combination, this peak was completely absent in the 14T sample Instead, a peak with a smal-ler mass (4983 Da; Fig 5B) was observed which corres-ponds to that expected (4983 Da) for the trimmed IA3 polypeptide devoid of the histidine-rich extension but with an N-terminal pyroglutamate residue The compo-sition of 14T determined by amino acid analysis was (residues⁄ mol) Asp 4.9 (5); Thr 1.0 (1); Ser 4.7 (5); Glu 6.7 (8); Gly 1.8 (2); Ala 5.6 (6); Val 1.3 (1); Met 2.3 (5); Leu 1.0 (1); Phe 1.2 (1); Lys 8.5 (8), with the theoretical
11
11T
12
13
14
14T
15
15T
16
16T
*Q
MK(H) MQ
0.1 ± 0.1
30 ± 4
0.7 ± 0.1
K
Saccharopepsin PpPr
MK(H) MQ
MK(H) MQ
0.3 ± 0.2
4 ± 0.3
S
Y MK(H) MQ
MK(H) MQ
MK(H) MQ
S
*Q S
1.1 ± 0.1 2 ± 0.3
0.9 ± 0.1 0.9 ± 0.4
30 ± 10 10 ± 2
0.4 ± 0.1 0.7 ± 0.2
15 ± 1 1.5 ± 0.2 0.6 ± 0.2 <0.1
100 ± 10 <0.1
E S
E S
E N
2
*Q
*Q
Fig 4 Inhibition at pH 4.7 of PpPr and S cerevisiae (saccharopepsin) by variant forms of IA 3 from S castellii and S cerevisiae Sequences
of IA3from S castellii and S cerevisiae are depicted by open and dark-shaded boxes respectively, with residue 2 and the C-terminal residue
of each length variant identified individually The MK(H) 7 MQ extension was positioned upstream from residue 2 at the N-terminus of inhibi-tors 11-16 Inhibiinhibi-tors 11T, 14T, 15T & 16T were generated by removal of this extension by cathepsin C trimming to leave a cyclised Q resi-due (= *Q) at the N-terminus of each polypeptide.
Trang 7values given in parentheses Histidine was absent,
indi-cating the purity of the trimmed polypeptide that
resulted from the chromatographic procedures (see
Experimental procedures) and substantiating the
complete absence of His-tagged parent polypeptide or
any partially-processed intermediate Directly
compar-able results were obtained for all of the other inhibitor
pairs described in Fig 4 (data not shown for brevity)
For the chimaeric 15⁄ 15T pair and the shortest
14⁄ 14T pair, removal of the N-terminal extension in
this way resulted in an approximately 35-fold loss in
potency against PpPr (Fig 4) In the case of the
full-length S castellii protein, however, an even larger loss
in potency (approximately 300-fold) against PpPr resulted from trimming off the N-terminal extension (cf 11T and 11; Fig 4) Indeed, the trimmed polypep-tide showed a potency against PpPr that was compar-able to that measured for the original S castellii protein tagged at its C-terminus (cf 11T; Fig 4; 2, Fig 1) Inhibitors 11T and 2 differ only in having (1)
a cyclised Q in place of the Met1 residue at the N-ter-minus and (2) the C-terminal LE(H)6 tag It would thus appear that appending the His-tag at the C-termi-nus of the authentic S castellii polypeptide is without significant effect By contrast, introduction of the histi-dine-rich extension at the N-terminal end of the
S castellii polypeptide transforms it into an inhibitor with subnanomolar potency against PpPr
Since IA3from S cerevisiae had been shown above to
be an even poorer inhibitor of PpPr, the effect of extend-ing this polypeptide at its N-terminus was also exam-ined Introduction of the MK(H)7MQ extension at the N-terminal end of wild-type S cerevisiae IA3resulted in
an improvement of approximately 100-fold in inhibitory potency against PpPr relative to the C-terminally tagged polypeptide (cf 16; Fig 4; 1, Fig 1) This modification thus transformed the ineffective polypeptide 1 into a highly potent inhibitor with a subnanomolar Ki value against PpPr (16; Fig 4) Once again, however, this gain
in potency was completely lost upon removal of the N-terminal extension by treatment with cathepsin C The resultant, trimmed S cerevisiae IA3 reverted to being as mediocre an inhibitor of PpPr as the original construct with its C-terminal tag (cf 16T; Fig 4; 1, Fig 1)
Binding effects
An explanation for these effects may be advanced based on remote interactions that occur outwith the active site cleft of the target proteinase Free IA3 is predominantly unstructured [5,6] Neither S cerevisiae nor S castellii IA3 show any significant intrinsic affin-ity for PpPr (inhibitors 1 and 16T and 2 and 11T) and
so the E + I« EI equilibrium lies well to the left When the extension with its multiple, positively ionisa-ble histidine residues (at pH 4.7) is attached at the N-terminus of these polypeptides, the potential capture
by the residues of the largely negatively charged surface adjacent to the edge of the active site cleft (Fig 3A) may increase the local inhibitor concentra-tion and help to locate the residues of each N-terminal
IA3segment in closer juxtaposition to the active site of the enzyme This anchoring function of the extension residues may allow inhibitory sequences, which, by
A
B
Fig 5 MALDI-TOF mass spectrometry analysis of S castellii IA3
terminating at K45 before (A) and after (B) removal of the
N-ter-minal extension by cathepsin C ⁄ glutamine cyclotransferase (A) The
peak at 6327 Da corresponds to that expected theoretically
(6334 Da); the 3168 Da peak is most likely the doubly charged ion.
(B) The observed mass peak coincides with that predicted
(4983 Da) for the trimmed polypeptide The 2496 Da peak is
prob-ably the doubly charged ion and no peak is present at 6327 Da
cor-responding to the parent, untrimmed peptide.
Trang 8themselves are suboptimal, to reside long enough in
the vicinity of the enzyme matrix to consolidate the
helical arrangement that ensures a successful, tight
interaction with the enzyme Whereas two histidine
residues (X8 and X9; Fig 3B) were insufficient for this
purpose, five residues (X5–X9) resulted in an increase
in potency of almost an order of magnitude against
PpPr, with the X5 and X6 histidine residues potentially
establishing contacts with Asp164 and Glu17,
respect-ively, of the enzyme (Fig 3B) Addition of a further
two histidine residues (X3 and X4) and a lysine at X2
consolidated this effect even more, resulting in a
further, more substantial gain in potency
Since neither Glu17 nor Asp164 is conserved in the
sequence of saccharopepsin, the validity of this
inter-pretation was examined by determination of inhibition
constants for the interaction of the N-terminally
exten-ded inhibitors with saccharopepsin The potencies of
inhibitors 9 (containing two histidines) and 10 (five
his-tidines) against this enzyme were closely similar and
comparable to that of the parent inhibitor 7 (Ki¼
0.4 ± 0.1, 0.3 ± 0.1 and 0.8 ± 0.1 nm, respectively)
Further lengthening to include all seven histidine
resi-dues of the MK(H)7MQ sequence resulted in extended
inhibitors that were only two- to ten-fold more potent
against saccharopepsin than their respective, trimmed
counterparts (cf 15T and 15, 14T and 14 and 11T and
11; Fig 4) Indeed, the trimmed S castellii polypeptide
(inhibitor 11T; Fig 4) had a potency against
saccharo-pepsin that was identical to that reported previously
[1] for the C-terminally histidine-tagged counterpart
(inhibitor 2) of this sequence (Ki¼ 4 ± 0.1 nm)
Saccharomyces castellii IA3 is thus a weaker inhibitor
of saccharopepsin than S cerevisiae IA3 (K < 0.1 nm)
[1–4] From this evidence, it would appear that Glu17
and Asp164 may be responsible, at least in part, for
facilitating the substantially increased binding of
N-ter-minally extended inhibitors to PpPr because these two
are among the few amino acids in this region with a
number of negative-ionisable residues, that are not
conserved in saccharopepsin Site-directed mutagenesis
to introduce each of these residues, separately and
together, in place of their wild-type counterparts in
saccharopepsin would enable further substantiation of
this interpretation Consistent with this conclusion,
however, the N-terminally extended and trimmed
vari-ants of S cerevisiae IA3 were both potent inhibitors of
saccharopepsin, to the extent that each Ki value was
too tight for accurate quantitation (inhibitors 16⁄ 16T;
Fig 4) For this pair of proteins, the interactions made
by residues 2–34 of S cerevisiae IA3 upon
encounter-ing the active site of saccharopepsin, are already
opti-mized and so are sufficient by themselves to facilitate
tight, specific binding of this helical N-terminal seg-ment of IA3 The E + I« EI balance thus lies far to the right and the addition of further residues at the N-terminus or beyond residue 34 of the inhibitory segment is superfluous However, in the case of PpPr, the serendipitous positioning of negatively ionisable residues in a patch adjacent to but remote from the active site provides a capture site for positively ionisa-ble residues in the N-terminal extension By this device, it is thus possible to transform IA3 polypep-tides with little intrinsic affinity for PpPr into inhibi-tors with subnanomolar potency against this enzyme
as a target proteinase For aspartic proteinases that do not possess this fortuitous surface feature and which are more distantly-related to saccharopepsin, including those of clinical⁄ agricultural relevance, it would appear likely that changes will need to be made within the inhibitory sequence of the N-terminal segment itself in order to re-target the inhibitory activity of IA3
Experimental procedures
Saccharopepsin and the vacuolar aspartic proteinase from
P pastoris (PpPr) were produced in recombinant form and purified from each culture medium, as described previously [1] The N-terminal sequence determined for the purified PpPr was Ala-Ser-His-Asp-Ala-Pro-Leu-Thr-Asn-Tyr-Leu-Asn, which corresponds to that of the mature form of the proteinase predicted by the DNA sequence
Wild-type IA3polypeptides from S cerevisiae and S cas-tellii were produced in E coli with an additional LE(H)6 sequence attached at the C-terminus and purified to homo-geneity by nickel-chelate chromatography, as described pre-viously [1–4] Chimaeric and N-terminally extended IA3 variants were produced by engineering cassette versions of the DNA encoding S cerevisiae IA3in the pET-22b expres-sion plasmid (Novagen, Milton Keynes, UK) An unwanted SacI site near the 3¢-end of the IA3 coding sequence was removed and an NheI site was introduced as a silent muta-tion in the codons for Ala34–Ser35 (GCT AGTfi GCT AGC) by separate site-directed mutageneses using the Quikchange Kit (Stratagene, Amsterdam, the Netherlands),
as described previously [1] Digestion of the resultant plas-mid (J35-pET22b) with NdeI–NheI enabled removal of the bases encoding S cerevisiae residues 1–34 whereas digestion with NheI–XhoI permitted excision of the nucleotides enco-ding residues 35–68 The respective excised fragments were replaced with DNA encoding the corresponding residues 1–34 (inhibitor 5) or 35–81 (inhibitor 3) from S castellii
IA3 Each relevant segment was amplified by PCR using
S castellii IA3 DNA as template and oligonucleotide pairs containing the appropriate restriction enzyme sequence (Table 1) The authenticity of each construct was confirmed
by sequencing In this way, pET22b plasmids were
Trang 9gener-ated encoding chimaeric polypeptides which consisted,
respectively, of residues 1–34 from S castellii followed by
residues 35–68 from S cerevisiae IA3 (inhibitor 5, Fig 1)
or residues 1–34 from S cerevisiae followed by residues
35–81 from S castellii (inhibitor 3, Fig 1)
To generate IA3polypeptides each extended at its
N-ter-minus and devoid of the LE(H)6 tag at the C-terminus, a
further cassette vector was engineered by making use of the
XbaI site that is located in the cloning region of pET-22b
upstream from both the NdeI and ribosome binding sites
pET-22b carrying S cerevisiae IA3 DNA as the insert was
digested with XbaI–NdeI and gel purified to remove the
excised 43 bp fragment It was replaced by a pair of
syn-thetic oligonucleotides (newpet TOP⁄ BOT; Table 1) which
reconstituted the sequence between the XbaI and NdeI sites
and introduced the additional bases required to encode
most [MK(H)6HM] of the desired N terminal extension in
the correct frame, restoring the NdeI site (CAT ATG)
which coincidentally encodes the HM residues (at positions
X9–X10) of the extension sequence Clone screening was
faci-litated by the introduction of a new HindIII site (AAG
CTT) between the ribosome-binding and NdeI sites and the
new expression vector engineered in this way was called
newpet-22b To introduce the final Q residue (at position
X11) of the desired extension and to remove the C-terminal
LE(H)6 tag from the required constructs, oligonucleotide
primers were designed to anneal to the 5¢- and 3¢-ends of
the target DNA encoding S cerevisiae, S castellii or
chimaeric IA3of each desired length Each forward primer
consisted of an NdeI consensus sequence followed by a Gln
codon before continuing inframe at the codon for residue 2
of the relevant IA3sequence Each reverse primer encoded
stop codons in all three frames after the final desired IA3
codon to ensure the appropriate target polypeptide length PCRs were performed with the high-fidelity PfuUltraTM polymerase (Stratagene) Following gel purification, each amplicon was treated with NdeI and XhoI, prior to ligation into the newpet-22b vector that had been similarly digested Sequencing confirmed the authenticity of each construct In this way, pET-22b plasmids encoding inhibitors 11–16, each with an N-terminal MK(H)6HMQ extension (Fig 4) were generated The oligonucleotides used for each PCR employed in this series are listed in Table 1
Treatment to remove the N-terminal extension from each extended IA3 polypeptide was carried out using the TAG-ZymeTMsystem, first described by Pedersen et al [7], accord-ing to the manufacturer’s instructions (Qiagen, Crawley, UK) Briefly, this involved pretreatment of the DAPaseTM (cathepsin C; 100 mU) with an equal volume of 20 mm cyste-amine-HCl for 5 min at room temperature, prior to mixing with 6 U (120 lL) of Q cyclaseTMand samples of 1–1.5 mg
of each purified, N-terminally tagged recombinant IA3 This mixture was incubated at pH 7.0 in the presence of 5 mm EDTA to chelate any free Ni2+ ions After 2 h at 37C, DAPaseTM and Q cyclaseTM, which are both C-terminally His-tagged, were removed, together with any residual IA3 protein with intact tag by absorption onto a nickel-nitrilotri-acaetic acid agarose column, equilibrated in 20 mm sodium phosphate buffer, pH 7.0⁄ 150 mm NaCl Flow through fractions (usually 8· 0.9 mL) were pooled, concentrated by centrifugation in a Vivaspin-2 spin concentrator fitted with
a 3000 Da molecular mass cut-off membrane (Vivascience, Sartorius, Epsom, UK) and the released dipeptide fragments were removed by gel filtration on a Sephadex G-25 column, equilibrated in 25 mm sodium phosphate buffer, pH 6.5 containing 50 mm NaCl Fractions containing trimmed IA3
Table 1 Construction of mutant forms of IA 3 from S cerevisiae and S castellii The indicated pairs of forward (F) and reverse (R) oligonucle-otide primers were used to introduce the desired changes in S castellii or S cerevisiae IA 3 , thus generating each of the identified variants.
(R) CCGCTCGAGATGATCCATCAATTCATCTTTATCTTG
(R) CTAGCTAGCCATGTTTTTCATTCCTTCACTAGC
(R) CCGCTCGAGCGGCTATCTATCTAATGATCCATCAATTCATCTTTATC
(R) CCGCTCGAGCGGCTATCTATCTATTGTTCTTGCTTCCCAGCACC
(R) CCGCTCGAGCGGCTATCTATCTAATACGAATCTTGAGCTTTCTTTTC
(R) CCGCTCGAGCGGCTATCTATCTATTTTGTCTTCATTTTTTCCTTACTTTC
(R) CCGCTCGAGCGGCTATCTATCTACTCCTTCTTATGCCCCGCC
(R) CCGCTCGAGCGGCTATCTATCTACTCCTTCTTATGCCCCGCC
Trang 10were identified by SDS⁄ PAGE, pooled and concentrated, if
necessary, in a Vivaspin concentrator as above In this way,
inhibitors 11T, 14T, 15T and 16T were generated from their
respective parents 11, 14, 15 and 16
Synthetic peptide forms of IA3 (inhibitors 4, 6–10) were
obtained from Alta Biosciences (Birmingham, UK) and
contained l-norleucine residues in place of methionine,
where appropriate, as described previously [1–4] Inhibition
assays were performed at pH 4.7 as described previously
[1–3] The chromogenic peptide substrate used was
Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu (where the scissile peptide
bond is indicated by the asterisk and Nph represents
l-nitrophenylalanine) and was purchased from Alta
Bio-sciences N-terminal sequencing was performed by
automa-ted Edman degradation (Alta Biosciences) Samples for
amino acid analysis were hydrolysed in vacuo for 24 h at
110C in 6 m HCl No attempt was made to correct the
values obtained for methionine to include the products of
oxidation, methionine sulfoxide and methionine sulfone
MALDI-TOF mass spectrometry was performed at the
University of Dundee ‘Fingerprints’ Proteomics Facility,
UK MALDI mass spectra were generated using a Voyager
DE-STR MALDI-TOF MS system (Applied Biosystems,
Foster City, CA, USA) with delayed extraction in positive
ion reflectron mode Samples (diluted to a final
concentra-tion of 2 pmolÆlL)1) were applied to a MALDI sample
plate and supplemented with 1.0 lL of a 5 mgÆmL)1
solu-tion of a-cyano-4-hydroxy-trans-cinnamic acid matrix
(Sig-ma, Poole, UK) plus 10 mm ammonium di-hydrogen
phosphate in 50% (v⁄ v) acetonitrile in 0.1% (v ⁄ v)
trifluoro-acetic acid, mixed and allowed to air dry prior to analysis
The mass spectrometer was internally calibrated using a
matrix ion at 568.13 Da and mass measurement accuracy
was typically ± 0.01% The resultant data were analysed
using the massXpert computer program [8] Modelling
calculations were carried out on an SGI Octane
work-station (Silicon Graphics, Geneva, Switzerland) with
dual R12000 processors, using the moloc program (Gerber
Molecular Design, Amden, Switzerland), as reported
pre-viously [1,4]
Acknowledgements
Supported by awards (to J.K.) from the UK
Biotech-nology and Biological Sciences Research Council
(grant numbers 72⁄ C13544 and 72 ⁄ 0014846) We are
very grateful to our colleagues Jakob Winther and
Anette Bruun (formerly of the Carlsberg Laboratory,
Copenhagen, Denmark) for help with production of recombinant PpPr; to John Fox, Alta Biosciences, Bir-mingham, for provision and analysis of synthetic pep-tide variants of IA3; and to Doug Lamont and Kenny Beattie, University of Dundee, for carrying out mul-tiple mass spectrometry analyses of the IA3 variants The endless patience, tolerance and skill of Marian Williams in the production and revision of the manu-script is hugely appreciated
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