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headgroup is shifted by a methylene unit, which demands an
exceptional binding mode. Therefore, we set out to elucidate
the PSFꢀs mode of action, commencing with the synthesis of
the CFZ and ONX 0914 PSF counterparts 1 and 2 (Figure 1).
Next, we determined crystal structures of yeast CP (yCP):1
with resolutions up to 2.1 ꢁ by performing crystal soaking
experiments with incubation times of 1–6 h prior to data
collection (see Table ST1).
dehydroxylation of Thr1 is rather an integral part of the
inhibition mechanism than an artificially base-induced event.
Consistently, the FoÀFc electron density map clearly depicts
the intramolecular displacement product (S,S)-aziridine-T1’
(Figure 3b), thus excluding the elimination product (E)-
dehydrobutyrine (Dhb) (Figure S1b, Supporting Informa-
tion). Moreover, cyclization initiated by Thr1N is confirmed
Based on the identical headgroups of the PSF and the
common serine protease inhibitor phenylmethanesulfonyl
fluoride (PMSF), we anticipated a similar inactivation
mechanism through the formation of a covalent adduct on
Thr1 upon attack of Thr1Og on the sulfur atom. Unexpect-
edly, after incubation of the crystal for 2 h, the yCP:1
structure revealed empty substrate binding channels (2.1 ꢁ
resolution, Rfree = 19.7%, PDB ID 4R17). Instead, the FoÀFc
map displayed negative electron density at the Thr1 side chain
of subunit b5, disclosing a chemical modification of the
catalytic center (see Figure S1a), whereas b1 and b2 remained
identical to the apo state. To identify a short-lived reaction
intermediate of 1 at b5 we conducted time-resolved intact
protein mass spectrometry using LC-ESI-LTQ-FT-MS anal-
ysis on various CP types incubated with 1 and 2 (Figure 2).
Figure 2. Deconvoluted intact-protein mass spectra of the b5c and b5i
subunits following treatment of cCP with 1 (left panel) and iCP with 2
(right panel) (25 mm) after incubation for 12 h. The species labeled
“ÀH2O” represent either the aziridine or the crosslinked state
(expected mass difference: 18.0 Da). The insets feature enlargements
of the major species. The species labeled “+2ÀHF” represents the
covalently modified intermediate of the b5 subunits prior to dehydrox-
ylation (expected mass difference: 572.7 Da). See Figure S5 for spectra
of b5 of untreated cCP and iCP.
Figure 3. Comparative X-ray analysis of the b5 active site after time-
dependent soaking experiments of yCP crystals with 1. The 2FoÀFc
electron density maps (blue mesh, contoured at 1s) show distances in
ꢂ as black dashed lines. The active site triad Thr1 (T1), Asp17 (D17),
and Lys33 (K33) has been excluded prior to phasing. Stereoviews of
(a)–(d) are depicted in Figure S6. a) Subunit b5 of the apo structure
with unmodified T1 (yellow).[18] b) Aziridine-T1’ (yellow) formation on
b5 after 2 h soaking time with 1. The trajectory of the nucleophilic
attack of K33 (green) is shown as a pink dashed arrow (PDB ID 4R17).
c) K33-T1’-crosslink (yellow) formation on subunit b5 after 6 h soaking
time (PDB ID 4R18). d) Superposition of the apo structure with
unmodified T1 (light gray), the aziridine-T1’ intermediate (I, yellow),
and the K33-T1’-crosslink (II, yellow). The structural rearrangement of
T1 upon its conversion into intermediate I and crosslink II is illustrated
with a pink dashed arrow.
Spectra with incubation times up to 2 h confirmed the
formation of a covalent adduct on the b5 subunit of all
applied CP types with an observed mass increase correspond-
ing to the ligand upon fluorine release (see Figure 2 and
Figure S2). Furthermore, we observed a formal loss of a water
molecule (À18 Da) at the b5 subunit which was validated by
multiple experiments and different mass spectra deconvolu-
tion algorithms (see Figures S3 and S4 and Tables ST2 and
ST3).
These findings suggest either an addition–elimination
reaction as described for PMSF, which for example converts
the active Ser195 of thrombin into dehydroalanine,[17] or an
addition–displacement mechanism comprising sulfonylation
of Thr1Og, followed by an intramolecular substitution by
Thr1N to yield an aziridine. While the PMSF-induced
elimination reaction requires strong alkaline conditions, our
experiments were carried out at pH 6.8–7.5, indicating that
by the inverted stereoconfiguration of the methyl group in
(S,S)-aziridine-Thr1’, implying an SN2-like displacement. To
analyze the stability of the aziridine ring we conducted further
soaking experiments with extended incubation times up to 6 h
at pH 6.8. The 2FoÀFc electron density map of the yCP:1
structure revealed a SN2-type ring-opening of the aziridine-
Thr1’ by attack of the amino group of Lys33 (Lys33Ne),
yielding an intramolecular crosslink in b5 (Figure 3c; 2.4 ꢁ
resolution, Rfree = 19.5%, PDB ID 4R18). This Lys33-Thr1’
bond proves the presence of a polarity-inversed Thr1
intermediate and is certainly surprising, since the function
of Lys33Ne is to maintain the pKa of Thr1Og, and hence
2
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Angew. Chem. Int. Ed. 2014, 53, 1 – 6
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