Supporting Information A bestatin-based chemical biology strategy reveals distinct roles for malarial M1- and M17-family aminopeptidases Michael B. Harbut 1 , Geetha Velmourougane 1 , Seema Dalal 2 , Gilana Reiss 1 , James Whisstock 3 , Ozlem Onder 4 , Dustin Brisson 4 , Sheena McGowan 3 , Michael Klemba 2 , and Doron C. Greenbaum 1 * 1 Department of Pharmacology, University of Pennsylvania, 433 S. University Avenue, 304G Lynch Laboratories, Philadelphia, PA 19104-6018, USA. 2 Department of Biochemistry, Virginia Polytechnic Institute and State University, 306 Engel Hall, Blacksburg, VA 24061 3 Department of Biochemistry and Molecular Biology and Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, VIC 3800, Australia. 4 Department of Biology, University of Pennsylvania, 326 Leidy Laboratory, Philadelphia, PA 19104.
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Supporting Information
A bestatin-based chemical biology strategy reveals distinct roles for
malarial M1- and M17-family aminopeptidases
Michael B. Harbut 1, Geetha Velmourougane1, Seema Dalal2, Gilana Reiss1,
James Whisstock3, Ozlem Onder4, Dustin Brisson4, Sheena McGowan3, Michael
Klemba2, and Doron C. Greenbaum1*
1Department of Pharmacology, University of Pennsylvania, 433 S. University
Avenue, 304G Lynch Laboratories, Philadelphia, PA 19104-6018, USA.
2Department of Biochemistry, Virginia Polytechnic Institute and State University,
306 Engel Hall, Blacksburg, VA 24061
3Department of Biochemistry and Molecular Biology and Australian Research
Council Centre of Excellence in Structural and Functional Microbial Genomics,
Monash University, Clayton, VIC 3800, Australia.
4Department of Biology, University of Pennsylvania, 326 Leidy Laboratory,
Philadelphia, PA 19104.
Supplementary Figures S1 : Full blots from Figure 1E and attempted labeling of PfAPP by MH01. (a) Complete blots of the IP/Westerns from Figure 1E show that PfA-M1-YFP is processed and Pf-LAP-YFP migrates as a single band. (b) A parasite line expressing a YFP-tagged PfAPP protein was incubated with MH01 followed by immunoprecipitation for biotin (MH01) and western blot analysis for YFP, which confirmed is not targeted by MH01. The reciprocal experiment involving the immunoprecipitation of PfAPP-YFP (after incubation of parasites with MH01) using a YFP-specific antibody revealed no biotinylation of the protein (second panel). S2 : Morphological and biochemical profiling of bestatin and PheLeu. (a) In vitro kinetic evaluation of the PheLeu ABP. (b) Activity-based profiling using a fluorescent version of MH01 reveals no change in prob specificity relative to the biotinylated MH01.
a
30
40
115
80
50
kDa
65
b
0.25 0.50 1.0 1.0
bestatin+
PfA-M1PfA-M1
PfA-M1
Pf-LAP
probe (µM)
88 nM 42 nMPheLeu
* (Pf-LAP)K i (PfA-M1)K iABP Structure KK i */
0.48H2N
OH
O
NH
O
S3 : Stereo diagrams of inhibitors bound to active site of PfA-M1 and Pf-LAP. (a) 1.8 Å PfA-M1-BTA and (b) 2.0 Å Pf-LAP-PNAP. Inhibitors BTA and PNAP are colored in green, carbon atoms of PfA-M1 and Pf-LAP residues are colored grey. Zinc is shown as black spheres. Hydrogen and metallo-bonds are indicated (dashed black lines). Electron density shown is a composite omit map contoured to 1.0 σ and calculated using CNS. For clarity only electron density of each ligand, zinc ions and Pf-LAP carbonate ion (b) is shown.
S4 : Cartoon diagrams of the biologically functional Pf-LAP hexamer colored by chain: A (green); B (cyan); C (wheat); D (yellow); E (salmon); F (grey). (a) The cartoon diagram of PNAP bound to active sites of biological unit of Pf-LAP. PNAP is colored in magenta. Zinc ions are shown as black spheres. (b) cartoon diagram of Pf-LAP-PNAP where chains D-F are excluded to show PNAP binding to active sites of chains A-C that line the interior cavity of the hexamer.
S5 : Bestatin and PheLeu probe treatments do not cause DV swelling. (a) Parasites expressing YFP-tagged plasmepsin II, which localizes to the DV, were treated with bestatin or PheLeu probe (each 10 µM) and imaged by fluorescence microscopy. (b) Quntitation of DV size of the treated PMII-YFP parasites indicates a lack of DV swelling upon treatement with either compound. (c) A concentration-response curve showing the effect of bestatin-treatment on P. falciparum culture in I-Media versus media containing all amino acids.
S6 : DV-localised endoproteases are not inhibited by BTA. Activity assays for falciparin 2/2’, DPAP1 and PfAPP show that no inhibition occurs at 30 µM BTA. S7 : Inhibition of PfA-M1 causes DV swelling but does not prevent proteoytic cleavage of full length Hb. Parasites treated with BTA (1 mM) and PNAP (0.25 mM) are capable of initiating Hb degradation, as shown by an absence of full length Hb subunits (17 kDa) in both untreated and BTA-treated parasites, in contrast to parasites treated with E64-d, which disrupts the initial endoproteolytic cleavage of Hb.
S8 : Small dipeptide species accumulate in BTA-treated parasites. (a) The LC trace identifies a peak that increases in the lysates of BTA-treated parasites. (b)The MS profile identifies the species with a molecular weight of 282 Da, which may corresponds to a Thr-Tyr dipeptide from Hb. Another MS trace identifies a putative Hb-derived His-Lys dipeptide, with a molecular weight of 283. (d) A table of the putative dipeptides species identified in BTA-treated parasites.
Supplementary Table 1 | Data Collection and refinement statistics
aValues in parentheses refer to the highest resolution shell.
bAgreement between intensities of repeated measurements of the same reflections and can be defined as: ∑(Ih,i – <Ih>)/∑ Ih,i, where Ih,i are individual values and < Ih > is the mean value of the intensity of reflection h.
Data collection rPfA-M1_BTA Pf-LAP-PNAP Space Group P212121 P212121 Cell dimensions (Å) a=75.5, b=108.8,
B factors (Å2) Mean main chain 16.6 16.3 Mean side chain 21.8 22.4 Mean ligand 42.4 53.0 Mean water molecule 32.2 28.2 r.m.s.d. bonded Bs Main chain Side chain
1.61 3.94
1.80 4.47
Supplementary Methods Determination of Ki and Ki
* values
Bestatin has been shown to be a slow-binding inhibitor of leucine
aminopeptidases, including Pf-LAP, with the slow step involving a conformational
change of the initially-formed low affinity enzyme-inhibitor complex (EI) to form a
tight complex (EI*; Scheme X)1-3. Ki is the dissociation constant for the initial
enzyme-inhibitor complex whereas Ki* is the overall inhibition constant and is
defined as [E][I]/([EI]+[EI*]) 4. Ki* values were determined for the di-Zn form of Pf-
LAP3 in 50 mM Tris-HCl pH 8.0 containing 50 mM ZnCl2, 250 mM Leu-AMC,
0.1% Triton X-100, 180 ng/mL Pf-LAP and inhibitor at 25 °C. Changes in
fluorescence upon mixing of substrate and inhibitor with enzyme were monitored
in 96 well plates using a Victor3 microplate fluorometer. Progress curves were
followed for 160 minutes and fit to the equation for slow-binding inhibition [P] = vst
+ (vo – vs)(1 – e(-kobs
t)/kobs, where vo is the initial rate, vs is the steady-state rate
and kobs is an apparent first-order rate constant for the formation of the high
affinity enzyme-inhibitor complex, EI*4 At the inhibitor concentrations necessary
to produce well-defined progress curves (at least two half-times), vs was typically
< 5% of the uninhibited velocity making steady-state approaches to determining
Ki* (i.e. Dixon plot) unfeasible. Instead Ki
* was determined from plots of kobs vs.
[I]. In the case of bestatin, the data defined a hyperbolic curve. k6 was
determined from the relationship k6 = vs/vo*kobs and was added to the data set
(kobs = k6 when [I] = 0) to better define the hyperbolic curve. Data were fit by non-
linear regression to the equation kobs = k6 + k5[I]/(Kiapp + [I]), where Ki
app = Ki(1 +
[S]/Km). Under our assay conditions the Km for Leu-AMC was 1.1 mM. Ki* was
calculated from the relationship Ki* = k6Ki/(k5 + k6) where Ki and (k5 + k6) were
determined from curve fits and k6 was determined as described above. For
bestatin probe, Phe-Ala probe and BTA probe, plots of kobs vs. [I] were linear, a
situation that can arise if Ki is much greater than the inhibitor concentrations used
in the assays3,5. In these cases kobs vs. [I] plots were fit by linear regression
yielding a slope of k6/Ki*app; determination of k6 as described above enabled
calculation of Ki*app and thus Ki*. With BTA probe, the combination of low affinity
for Pf-LAP and insolubility at high micromolar concentrations restricted the range
of kobs values that could be determined compared to those for bestatin and Phe-
Ala probes. However, sufficient data were available to allow an estimate for the
Ki* value to be made.
Scheme 1:
k3 k5 E + I EI EI*
k4 k6
X-ray Crystallography
Diffraction images were processed using MOSFLM6 and SCALA7 from the CCP4
suite8. 5% of each dataset was flagged for calculation of RFree9 with neither a
sigma nor a low-resolution cut-off applied to the data. Subsequent
crystallographic and structural analysis was performed using the CCP4i
interface10 to the CCP4 suite8, unless stated otherwise. The inhibitor complex
was initially solved and refined against the unbound PfA-M1 and Pf-LAP
structure (protein atoms only) as described previously11 and clearly showed
unbiased features in the active site for both structures. Superposition of BTA into
the Pf-LAP active site was performed using the X-ray crystal structure of Pf-LAP-
bestatin (3KR4.pdb) where the bestatin scaffold was used to superpose BTA.
Superposition of PNAP into the PfA-M1 active site was performed using the X-
ray crystal structure of PfA-M1-bestatin (3EBH.pdb) where the bestatin scaffold
was used to superpose PNAP. Pymol12 was used to produce structural
ESIMS of BodipyLysacetic azide C24H33BF2N8O3 Calculated Mass 530.27, found
553.1 (M+Na+).
IV) Synthesis of BodipyBTA click probe (41):
NH
HN
HN
OOH2NO
OOO
HNHN
OOOH
H2N
OO
NN
N
O
HN
HN
O NH2O
NB NF FH3C
CH3
The synthesis of the BodipyBTA click probe was done employing the same
protocol for the click reaction as for the BiotinBTA click probe using BTA alkyne
and BodipyLysacetic azide 40. The product 41 was obtained in 45% yield as a
colourless solid. ESIMS: 1520.5 [M+H]+; HRMS: found [M+H]+ 1520.7716.
C78H101BF2N15O14+ requires 1520.7714.
V) Synthesis of BodipyPheNaph click probe (42):
NH
HN
HN
OOH2NO
OOO
HNHN
OOOH
H2N
O
NN
N
O
HN
HN
O NH2O
NB NF FH3C
CH3
The synthesis of the BodipyPheNaph click probe was synthesized employing the
same procedure for the click reaction, as for the BiotinBTA click probe using
PheNaph alkyne 23 and BodipyLysacetic azide 40. The product 41 was obtained
in 42% yield as a colourless solid. ESIMS: 1540.7 [M+H]+; HRMS: found [M+H]+
1540.7800. C81H101BF2N15O13+ requires 1540.7759.
Supplementary references 1. Wilkes, S.H. & Prescott, J.M. The slow, tight binding of bestatin and
amastatin to aminopeptidases. J Biol Chem 260, 13154-62 (1985). 2. Stack, C.M. et al. Characterization of the Plasmodium falciparum M17
leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development. J Biol Chem 282, 2069-80 (2007).
3. Maric, S. et al. The M17 leucine aminopeptidase of the malaria parasite Plasmodium falciparum: importance of active site metal ions in the binding of substrates and inhibitors. Biochemistry 48, 5435-9 (2009).
4. Morrison, J.F. & Walsh, C.T. The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 61, 201-301 (1988).
5. Rich, D.H., Moon, B.J. & Harbeson, S. Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes. J Med Chem 27, 417-22 (1984).
6. Leslie, A.G.W. Joint CCP4+ESF-EAMCB Newsletter on Protein Crytallography.
7. Evans, P. Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62, 72-82 (2006).
8. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50, 760-3 (1994).
9. Brunger, A.T. Assessment of phase accuracy by cross validation: the free R value. Methods and applications. Acta Crystallogr D Biol Crystallogr 49, 24-36 (1993).
10. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol Crystallogr 59, 1131-7 (2003).
11. McGowan, S. et al. Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase. Proc Natl Acad Sci U S A 106, 2537-42 (2009).
12. DeLano, W.L. The PyOL Molecular Graphics System. (DeLano Scientific, San Carlos, CA USA, 2002).
13. Androulakis, S. et al. Federated repositories of X-ray diffraction images. Acta Crystallogr D Biol Crystallogr D64, 810-4 (2008).
14. Onder, O., Turkarslan, S., Sun, D. & Daldal, F. Overproduction or absence of the periplasmic protease DegP severely compromises bacterial growth in the absence of the dithiol: disulfide oxidoreductase DsbA. Mol Cell Proteomics 7, 875-90 (2008).