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were incubated with either catalytic or equimolar amounts of
trypsin in [H218O]Tris·HCl buffer. LC-MS analysis clearly showed
that monocyclic SFTI-1 (the product of peptide splicing)
formed upon incubation of 5 with both catalytic and equimo-
lar proteinase. However, monocyclic SFTI-1 was detected only
when 1 was incubated with an equimolar amount of enzyme.
The hydrolysis and transpeptidation mechanism requires the
hydrolysis of the acyl–enzyme intermediate and subsequent
formation of a new acyl–enzyme intermediate. Only during the
hydrolysis is the atom 18O incorporated into the newly formed
carboxyl group of the N-terminal peptide. However, in all these
cases no product with +2 Da was detected, thus indicating no
incorporation of 18O into monocyclic SFTI-1. Thus, we conclude
that peptide splicing of SFTI-1 analogues by serine proteinase
proceeds by the direct transpeptidation mechanism, very simi-
larly to that catalyzed by the proteasome, as postulated by Vi-
gneron et al.[2] and confirmed by Mishto et al.[7] The question
remains as to why, in the case of 1 and 7, peptide splicing was
observed only with an equimolar amount of the enzyme. As
postulated by Vigneron et al.[2] a new peptide bond is formed
at the expense of proteolysis of a peptide bond within the
substrate. Assuming that the slowest step of peptide splicing
is the acyl–enzyme formation, the concentration of this inter-
mediate, when a catalytic amount is used, is below the limit of
detection by our method. With equimolar peptide/enzyme, the
concentration of the acyl–enzyme intermediate is significantly
higher, and in consequence splicing is observed. This can be
explained by fast nucleophilic attack of a-amino groups of the
second peptide chain on acyl–enzyme intermediate.
Experimental Section
Peptide synthesis: All peptides were synthesized manually by
a solid-phase approach with Fmoc chemistry. The synthetic proto-
cols were similar to those recently described.[15] In brief, 2-chlorotri-
tyl chloride resin (GL Biochem, Shanghai, China) was used as
a solid support. Peptide chains were elongated in consecutive
cycles of deprotection and coupling. Deprotection was performed
by piperidine (20% in DMF) with Triton X-100 (1%). Chain elonga-
tion was achieved with equimolar mixtures of the protected amino
acid derivatives, N,N’-diisopropylcarbodiimide (DIC), 1-hydroxyben-
zotriazole (HOBt), and reagents (3 equiv. relative to resin reactive
groups). A one-step procedure with TFA/phenol/triisopropylsilane/
H2O (88:5:2:5, v/v/v/v) was used to remove peptide from the resin.
Peptides were purified on Discovery BIO Wide Pore C8 HPLC
column (10 mm; 10250 mm; Sigma–Aldrich). The purity of the
final preparation was evaluated with a ProStar HPLC system
(Varian) and a Discovery BIO Wide Pore C8 column (5 mm; 4.6
250 mm; Sigma–Aldrich).
Intermolecular disulfide bridge formation: Disulfide bridges were
formed essentially as described previously.[20] In brief, the C-termi-
nal fragment of the inhibitor (0.01 mmol) in aqueous acetic acid
(0.1m, 4 mL) and 2,2’-dithiodipyridine (0.02 mmol) in isopropanol
(4 mL) were mixed and stirred for 1 h at ambient temperature. The
peptide derivative active in the thiol function was purified by RP-
HPLC and lyophilized. Dithiopyridyl-peptide and the N-terminal
fragment (0.02 mmol) were dissolved in sodium acetate (0.1m,
pH 5, 6 mL) for 1 h. The resulting two-peptide-chain analogues
were purified by RP-HPLC and lyophilized.
Proteinase susceptibility assay: SFTI-1 analogue (240 mm) was in-
cubated at ambient temperature with equimolar bovine b-trypsin
in Tris·HCl buffer (100 mm, pH 8.3) containing CaCl2 (20 mm) and
Triton X-100 (0.005%). Aliquots were extracted periodically (after
1.5, 3, and 24 h) and analyzed by HPLC and MS (MALDI-TOF) as de-
scribed below. Selected SFTI-1 analogues (1 and 5) were incubated
in [H218O]Tris·HCl (pH 8.3). 18O-enriched water (97%) was purchased
from Campro Scientific (Berlin, Germany).
Native, bicyclic SFTI-1 assumes a conformation in solution,
which remains practically unchanged in the complex with the
cognate proteinase.[19] We wondered if such a predefined con-
formation is a prerequisite for efficient interaction with the
enzyme, that is, whether the two-peptide-chain SFTI-1 ana-
logues evaluated in this study are also characterized by a rigid,
predefined conformation. The NMR data clearly indicate that
1 does not assume a defined conformation in solution. Lack of
the essential NOE signals (other than sequential ones) along
with high temperature coefficients and vicinal coupling con-
stants support the absence of a defined secondary structure.
Therefore, unlike native SFTI-1, the folding of 1 into a confor-
mation specific for SFTI-1 is induced by its interaction with the
target enzyme. This indicates that, at least in the case of SFTI-
1 analogues, peptide splicing is a sequence- not structure-spe-
cific.
HPLC and MS analysis: Proteinase-catalyzed reaction products
were analyzed by a ProStar HPLC system and a Discovery BIO Wide
Pore C8 column (5 mm; 4.6250 mm; Sigma–Aldrich) with TFA
(0.1% in water; solvent A) and acetonitrile (80% in A; solvent B):
linear gradient 10–90% B over 40 min; flow rate, 1 mLminÀ1. The
eluate was monitored at 226 nm. MS analysis of the reaction prod-
ucts was carried out using with a Biflex III MALDI-TOF spectrometer
(Bruker) and a-CCA as a matrix, and a TripleTOF 5600+ system
with DuoSpray Ion Source and microLC (AB SCIEX, Framingham,
MA).
Co-crystallization: Bovine pancreatic trypsin was obtained from
BioShop (Burlington, Canada). Crystals were prepared by the sit-
ting-drop vapor-diffusion method: protein solution (1 mL) contain-
ing trypsin (2.57 mm), STFI-1 variant (2.57 mm), and CaCl2 (0.5 mm)
in HEPES (1.25 mm, pH 7.0) was mixed with an equal volume of re-
servoir solution containing sodium acetate trihydrate (0.1m,
pH 4.6), ammonium sulfate (0.3m), and poly(ethylene glycol) 8000
(30%) and allowed to equilibrate. Co-crystals of the complex ap-
peared after 48 h and continued to grow for several days.
In conclusion, this study provides solid evidence on protei-
nase-catalyzed peptide splicing in two-chain SFTI-1 analogues,
obtained by complementary methods, MS and X-ray crystallog-
raphy. Splicing occurred both in analogues containing a cleava-
ble peptide bond at the non-prime site, as well as in those
that do not. Using H218O we were able to show that peptide
splicing of SFTI-1 catalyzed by proteinases proceeds according
to the direct transpeptidation mechanism; water molecules are
not able to efficiently compete with the a-amino groups of
peptides for nucleophilic attack on the acyl–enzyme intermedi-
ate.
Crystal structure solution: The crystals were flash-frozen without
cryoprotection. The diffraction data were collected at EMBL beam-
line P13 at PETRA III (DESY, Hamburg, Germany). Data were indexed
and integrated by using XDS.[21] Further computations were per-
formed using programs in the CCP4 package.[22] The data were
ChemBioChem 2015, 16, 2036 – 2045
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