Supplementary Material:
Solution structure of the phytotoxic protein PcF: the first characterized member of the
Phytophthora PcF toxin family
Giuseppe Nicastro, Giuseppe Orsomando, Elena Ferrari, Lucia Manconi, Filomena Desario, Adolfo
Amici, Alessia Naso, Armando Carpaneto, Thelma A. Pertinhez, Alberto Spisni, and Silverio
Ruggieri
METHODS
Bioassay
Tomato seedling bioassays were set as described (1,2) using protein samples vacuum-dried and
re-dissolved in 200 μL distilled water.
Disulphide Bonding Determination
Pure PcF (65 nmol) was dissolved in 40 μL of 0.1 M sodium citrate, pH 3.0, 6 M guanidine/HCl
(buffer A). Partial reduction was performed by adding 260 μL of 0.1 M TCEP (Sigma) in buffer A
(20 min, room temperature). First derivatization was performed by adding 300 μL of 0.5 M NEM
(Fluka) in buffer A (30 min, room temperature). NEM-labeled species were separated by C18HPLC (SupelcosilTM LC-18-T, 4.6 x 250 mm, 5 μm, 300 Å, Supelco), performed at 4 °C, 1.3
mL/min, by a two-step acetonitrile gradient (0-23% for 5 mL, then 23-39% for 77 mL) in 0.1%
trifluoroacetic acid in water. Individual peaks were treated as described [3] to achieve complete
reduction and Pam-alkylation of residual cysteines. Further desalting by HPLC (Superdex Peptide
PE 7.5/300, Amersham Pharmacia Biotech) was performed at 0.5 mL/min in 0.1% trifluoroacetic
acid, 30% acetonitrile in water, and the collected dual labeled PcF species were quantified by 280nm
= 4,470 M-1 cm-1. Each polypeptide (≥ 0.5 nmoles) was immobilized onto a PVDF membrane, then
loaded onto an automated Edman sequencer (Procise 491, Applied Biosystems) for one-shot
sequencing. Released amino acids were identified as phenylthiohydantoin derivatives using NEMand Pam-cysteine standards (3,4).
15N
Relaxation Measurements and Analysis
T1, T2, and 1H-15N-NOE values were acquired using pulse sequences adapted from standard
schemes (5) and incorporating appropriate suppression of cross-correlation (6). T1 and T2
experiments were acquired with recycle delays of 5 s. Relaxation delays of 10, 50, 100, 200, 300,
400, 500, 600, 700, 800, 900, 1000, and 1200 ms were employed for T1 measurements, and 10, 20,
30, 35, 40, 50, 55, 60, 75, 90, 100, 120, 130, and 150 ms for T2. Intensities were extracted using
Nonlinear Spectral Lineshape Modelling, and fitted to single exponential using routines within
NMRPipe (7). Heteronuclear NOE spectra were acquired with 5 s of on-resonance saturation for the
NOE experiment, and 5 s recycle delay (no saturation) for the reference spectra. 1H-15N-NOE
values for each given residue were calculated as the intensity ratio (I/I0) of the 1H-15N correlation
peak in the presence (I) and absence (I0) of proton saturation during the relaxation delay. The
15
N
heteronuclear relaxation parameters were first analyzed by a model-free approach using the
TENSOR2 program (8), that is based on the Woessner description of the molecular diffusion (9-12)
and on the Lipari-Szabo model-free analysis of local flexibility (13,14). The internal mobility is
summarized by the parameters S2 (amplitude of the internal motion) and e (effective correlation
time constant of the motion). The rotational diffusion tensor of the molecule, represented by the
ratio R2/R1 (T1/T2), was measured only in the secondary structured regions, because of the
negligible contribution of the internal motions to R1 and R2 (9). From this value we derived the
contribution of the global motion to R1, R2, and heteronuclear NOE, and the remaining
contributions were fitted to the local motional parameters (S2 and e). When necessary, we
introduced the chemical shift exchange term (Rex) to fit R2 (11), or the extended motion factors (S2f
and S2s, for the fast and slow motions, respectively) (15). As a result, five models of internal motion
with increasing complexity were iteratively applied to obtain a measured relaxation rate within a
95% confidence limit (16). The five models tested are in order: (S2); (S2, e); (S2, Rex); (S2, e, Rex);
(S2f, S2s, e). Relaxation parameters were further analyzed by a reduced spectral density mapping
approach, that allows the characterization of the frequency of motion, of the overall tumbling rates,
and of the conformational exchange contributions, without any assumption about the shape of the
molecule or the nature of its motions (17-20). Spectral density functions J(0), J(N) and J(H) were
calculated, and analyzed as described (19).
RESULTS
Assignment of disulphide bonds and biological activity
The
three
disulphide
bridges
were
subjected
to
partial
reduction
with
tris-(2-
carboxyethyl)phosphine (TCEP), followed by alkylation with N-ethylmaleimide (NEM) and HPLC
separation. Under these conditions, seven species were obtained, P1 corresponding to the nonreduced protein, and P2-P7 to the progressively reduced and NEM-alkylated proteins (Fig. S1
bottom). The latter species were sequenced after full reduction and labeling of the residual cysteines
with 2-propionamide (Pam). As a result, the selective Pam-alkylation pattern of P4, P5 and P6
species (Fig.S1 middle) unambiguously identified the disulphide connectivities Cys6-Cys40, Cys11Cys44, and Cys26-Cys39 (Fig. S1 top). The alkylation pattern of the remaining species was, in some
cases, less clear, but never conflicting with the other results.
The disulphide pattern of PcF (Fig. S1 top) differs from any other in the SwissProt database,
being the 1st cysteine bridged to the 5th, the 2nd to the 6th, and the 3rd to the 4th.
Noteworthy, P2-P7 species showed low or null toxicity upon tomato seedlings bioassay (Table
S1), thus suggesting that integrity of each disulphide bridge is crucial for bioactivity.
Backbone Dynamics
The
15
N relaxation data analyzed by TENSOR2 yielded 45 R1, 42 R2 and 49 1H-15N-NOE
values. Figure S2 (panels A-F) reports the plots of 1H-15N-NOE, R1, and R2 values, the order
parameter S2, the effective correlation time for internal motions e, and the conformational
exchange contributions Rex. A complete set of the three 15N relaxation values could not be obtained
for some residues due to a significant overlap in the 1H-15N HSQC spectra (Asp2, Tyr5, Gln7, Glu17,
Gly30, Gly33, Thr48). As shown in Figure S2G, 22 residues fit to the simplest model of internal
mobility, which requires only S2 (model 1, see METHODS); residues Leu4, Cys6, Leu32, Ala50, and
Ala52 require an effective internal correlation time on fast timescale, e (model 2); residues Ile9,
Gly10, Thr13-Tyr15, Lys31, Asp34-Asp35, Gln43 and Gly45 require a second term with Rex > 5 s-1,
suggesting conformational exchanges in the μs-ms timescale (models 3 and 4); residues Ala8, Ser46,
Thr47, and Ser51 require the extended model-free formalism (model 5). The heteronuclear relaxation
data appear fairly constant throughout the PcF structured regions. On the other hand, low or even
negative 1H-15N-NOE values, together with small R2, are observed at both N-terminus (Glu1-Leu4)
and C-terminus (Ser46-Ala52), indicating their pronounced structural flexibility (Fig. S2C). R2 values
above average are observed for residues Ile9, Gly10 and Thr13-Tyr15, clustered before helix α1, and
for residues Asp34-Asp35, located at the loop-α2 helix link. Such high R2 values, together with the
need of the Rex term, indicate the residues experience slow (μs-ms timescale) conformational
exchange. The helices 1 and 2 have an average S2 of 0.92, which suggests a restricted backbone
motion (Fig. S2D). The residues located in the inter-helical loop, Gln29-Asp34, on the other hand,
show a reduced S2 averaging of about 0.80, suggesting a slightly higher flexibility (Fig. S2D). As
for the region Leu4-Glu17, the S2 values ranging from 0.6 to 0.9, (Fig. S2D), suggest this stretch may
undergo coherent fluctuations.
From the relaxation parameters measured at 14.1 T, the spectral density functions J() for each
of the 42 1H-15N bond vectors were mapped at the three frequency values 0,
15
N and 1H, yielding
the corresponding J(0), J(N) and J(H) values (Fig.S3). J(0) reveals that most of the residues in the
structured regions have values corresponding to J(0) = (2/5) c (dotted line in Fig. S3A), thus
indicating that the frequency of their motion corresponds to the overall tumbling frequency of the
molecule. The J(0) values drop at both N- and C- terminus, confirming their enhanced internal
mobility. An increase in J(0) is observed for the segments Ile9-Tyr15 (except Cys11) and Asp34Asp35, suggesting they undergo a significant conformational exchange process. Similarly, in the
J(N) profile (Fig. S3B), the largest values are found in the structured regions, while smaller values
characterize both protein termini, confirming their motion is in the ns time scale. Intermediate
variable values, though, characterize the stretch Leu4-Glu17 and the loop Gln29-Asp34, in agreement
with the expectation these regions experience a higher degree of flexibility than the helical
stretches. The J(H) values (Fig. S3C) are consistent with this structural model.
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resonance relaxation in macromolecules. 1. Theory and range of validity. J. Am. Chem. Soc.
104: 4546-4559.
(14) Lipari G, Szabo A (1982b) Model-free approach to the interpretation of nuclear magnetic
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Table S1 Biological activity of the partially reduced and NEM-alkylated PcF species
Arrangement of
NEM-linked
Withering activity on
PcF species
cysteines
residues
tomato seedlings
(SS – SH)
(n. per mol)
(score)
P1
3-0
0
5
P2
2-2
2
1
P3
uncertain
2 or 4
2
P4
1-4
4
1
P5
1-4
4
0
P6
1-4
4
1
P7
0-6
6
0
Untreated protein
3-0
0
5
P1 to P7, PcF species corresponding to peaks in Figure S1; SS, oxidized cystines; SH, reduced cysteines; Score,
arbitrary scale from 0 (symptomless) to 5 (complete necrosis).
(YVEF)EDPLYCQAIGCPTLYSEANLAVSKECRDQGKLGDDFHRCCEEQCGSTTPASA
1
-4
6
11
26
3940
44
COOH
COOH
COOH
COOH
44
NEM 44
Pam
44
NEM
40
Pam 40
NEM
40
NEM
39
NEM 39
NEM
39
Pam
Pam 26
Pam
Pam/
NEM
Pam/
NEM
Pam/
NEM
26
NEM 26
NEM
26
Pam
NEM 11
Pam
11
NEM 11
Pam
11
NEM
Pam
Pam/
NEM
6
Pam 6
NEM
6
NEM
44
NEM 44
40
Pam 40
39
Pam 39
26
11
6
COOH
6
NH2
NH2
NH2
NH2
NH2
P1
250
Abs 210 nm
200
150
P3
P2
100
P4P5 P6
50
P7
0
0
30
35
40
45
50
55
60
65
Retention time (min)
Figure S1 Assignment of disulphide bonds. (Bottom) C18-HPLC profile of the PcF protein after
partial reduction with TCEP and NEM-derivatization. The arrow indicates the native protein.
(Middle) Edman sequencing results of the protein species after complete reduction and Pamderivatization, showing, for clarity, only the modified cysteines. Peak P1 (fully Pam-alkylated) and
peak P7 (fully NEM-alkylated), useless for S-S reconstruction, are omitted. (Top) Reconstructed
disulphide bonding pattern of PcF: the connectivities Cys6-Cys40, Cys11-Cys44, and Cys26-Cys39 are
deduced from peaks P4, P5, and P6, each showing one of the three disulphide bridges with both
cysteines Pam-alkylated.
NOE (I/IO)
1
A
0
1H-15N
-1
-2
R1(1/s)
B
2
1
R2(1/s)
36
30
24
18
12
C
6
S2
0.8
D
0.6
0.4
0.2
E
te(ns)
3
2
Rex (s -1)
1
F
30
25
20
15
10
5
5
G
Model
4
3
2
1
0
10
20
30
40
50
Residue
Figure S2 Model-free formalism analysis. (A-C) Experimental
1
H-15N NOE heteronuclear
measurements and R1, R2 relaxation rates. (D-F) Dynamics parameters assuming an axially
symmetric rotational diffusion tensor: order parameter S2; internal correlation times e; chemical
exchange contribution Rex. (G) Selected models for each residue.
J(0) (10-8) (s/rad)
J(H) (10-11) (s/rad) J(N) (10-10) (s/rad)
A
1.0
0.5
B
4
3
2
1
C
3
2
1
0
10
20
30
40
50
Residue
Figure S3 Reduced spectral density mapping analysis. (A-C) Calculated values of J(0), J(N), and
J(H) are plotted versus the protein residues. In (A) the horizontal continuous dotted line indicates a
J(0) value of (2/5)c.