Dimerization of NaD1 and antifungal activity 1 Dimerization of the plant defensin NaD1 enhances ...

25 Pages·2012·0.83 MB·English

by  Marc Kvansakul

JBC Papers in Press. Published on April 17, 2012 as Manuscript M111.331009
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M111.331009
Dimerization of NaD1 and antifungal activity

Dimerization of the plant defensin NaD1 enhances its antifungal activity

Fung T. Lay1,2,#, Grant D. Mills1,2,#, Ivan K.H. Poon1, Nathan P. Cowieson3, Nigel Kirby3, Amy A.
Baxter1,2, Nicole L. van der Weerden1,2, Con Dogovski1, Matthew A. Perugini1, Marilyn A.
Anderson1,2, Marc Kvansakul1,4 and Mark D. Hulett1,2,4

1From the Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University,
Melbourne, VIC, 3086, Australia

2Hexima Limited, Melbourne, VIC, 3000, Australia

3Australian Synchrotron, 800 Blackburn Road, Clayton, VIC 3168, Australia

*Running title: Dimerization of NaD1 and antifungal activity
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4To whom correspondence should be addressed: Mark D. Hulett, Tel.: [phone]; Fax: +61-3- nlo
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9479-1266; E-mail: [email protected] or Marc Kvansakul, Tel.: [phone]; Fax: +61-3- e
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9479-1266; E-mail: [email protected]. Department of Biochemistry, La Trobe Institute for fro
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Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia h
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Keywords: defensins, plant defense, innate immunity, X-ray crystallography, antimicrobial w
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peptides, small angle X-ray scattering, cell permeabilization, peptide interactions, NaD1, fungi .jb
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Background: NaD1 is a potent antifungal plant to high-resolution (1.4 and 1.58 Å, b/
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defensin from Nicotiana alata flowers. respectively), both of which contain NaD1 in g
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Results: NaD1 forms dimers as determined by a dimeric configuration. Using protein es
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X-ray crystallographic, biophysical and crosslinking experiments as well as small n
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biochemical approaches. angle X-ray scattering analysis and analytical ec
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Conclusion: Dimerization of NaD1 enhances ultracentrifugation, we show that NaD1 b
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its fungal cell killing. forms dimers in solution. The structural r 2
5
Significance: Understanding the molecular basis studies identified lysine at position 4 (K4) as , 2
0
1
of NaD1 antifungal activity helps define critical in formation of the NaD1 dimer. This 8
defensin function and has potential application was confirmed by site-directed mutagenesis of
for improving plant resistance against K4 that resulted in substantially reduced
agronomically important fungal pathogens. dimer formation. Significantly, the reduced
ability of the K4 mutant to dimerize
SUMMARY correlated with diminished antifungal
The plant defensin, NaD1, from the activity. These data demonstrate the
flowers of Nicotiana alata, is a member of a importance of dimerization in NaD1 function
family of cationic peptides that displays and has implications for the use of defensins
growth inhibitory activity against several in agribiotechnology applications such as
filamentous fungi including Fusarium enhancing plant crop protection against
oxysporum. The antifungal activity of NaD1 fungal pathogens.
has been attributed to its ability to _______________________________________
permeabilize membranes, however the Plants are continually exposed to a myriad of
molecular basis of this function remains potentially damaging microorganisms,
poorly defined. In this study, we have solved phytophagous insects and environmental
the structure of NaD1 from two crystal forms stresses. As a counter-measure, plants have
1
Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
Dimerization of NaD1 and antifungal activity
developed sophisticated innate defense crystallography and presented as a dimer in the
mechanisms for protection. These range from asymmetric unit (20).
preformed physical barriers such as the cell wall Importantly, plant defensins display a variety
to the constitutive or inducible expression of of biological functions despite their conserved
chemical compounds including secondary three-dimensional structures. Best characterised
metabolites and defensive proteins. is their ability to inhibit fungal pathogens (9,21-
Of the defense proteins that are produced by 25). A number of defensins have also been
plants, the defensins represent one of the largest described with antibacterial activity (26-28) or
families. They are small (~5 kDa), basic and can act as inhibitors of protein synthesis (29-32),
cysteine-rich and are abundantly expressed in "-amylases (33,34), proteases (35,36) or ion
various plant tissues. As a result of recent channels (37,38). It is interesting to note that the
advances in whole-genome sequencing and latter functions are more consistent with a role in
proteomics, coupled with improved defense against insect pests. Furthermore, some
bioinformatic analyses, there has been a huge defensins have been implicated in plant
expansion in the number of described defensins, tolerance to environmental stresses such as high
many of which are encoded by multigene levels of zinc (39), salt (40,41) or cold (42).
families (1-5). Hence, the varied biological functions of plant
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Plant defensins are distinguished from other defensins make them attractive candidates for ow
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protein families based on the number and exploitation in agribiotechnological applications lo
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arrangement of cysteines, the configuration of to confer enhanced resistances to pathogens, e
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the disulfide bridges (CI–CVIII, CII–CV, CIII–CVI insect herbivory or environmental stresses in from
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sheet tethered via three disulfide bonds to an "- into two classes. Class I defensins encode a w
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helix in a configuration known as the cysteine- precursor protein with an ER signal peptide and .jb
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stabilized "! (CS"!) motif. The fourth disulfide a mature defensin domain whereas class II .o
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bond links the N- and C-terminal regions, defensins encode a precursor with an additional b/
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producing a pseudocyclic protein that renders C-terminal propeptide (CTPP) (7,9). At least for g
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the protein highly stable to chemical and thermal three class II defensins, namely NaD1, PhD1 st o
denaturation (6,7). Interestingly, the Petunia and PhD2 (where the protein has been n D
hybrida floral defensins PhD1 and PhD2 have a characterized), the CTPP is removed from the ec
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fifth disulfide bond (8-10). Apart from the mature, biologically active protein (8-10). b
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cysteines, only a limited number of other NaD1 is a defensin from the flowers of the r 2
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residues are conserved among members of this ornamental tobacco, Nicotiana alata (9). It has , 2
0
1
family. They include a serine (position 8), two been suggested that its primary function is to 8
glycines (position 13 and 34), an aromatic protect the reproductive tissues against damage
residue (position 11) and a glutamic acid from potential fungal pathogens (9,24,25). This
(position 29) (numbering relative to the radish is corroborated by in situ hybridization studies
defensin Rs-AFP2) (7). An amino acid sequence on N. alata flowers demonstrating accumulation
alignment of select representative examples of of NaD1 transcripts in tissues surrounding the
plant defensins is given in Fig. 1. reproductive organs. Immunoblot analysis with
Despite the low primary sequence identity NaD1-specific antibodies also revealed high
between different plant defensins, their three- levels of the protein in floral tissues (9).
dimensional structures are remarkably similar. Furthermore, NaD1 exhibits potent in vitro
To date, the structures of thirteen defensins have antifungal activity against a number of
been elucidated (8,10-20). All of these defensin pathogenic filamentous fungi including
structures, with the exception of one, were Fusarium oxysporum f. sp. dianthi (Race 2), F.
solved by solution 1H-NMR spectroscopy as oxysporum f. sp. vasinfectum and Botrytis
monomeric proteins (7). More recently, the cinerea (9,24,25). Recent studies have
structure of the SPE10 defensin from demonstrated that the antifungal activity of
Pachyrrhizus erosus seeds was solved by X-ray NaD1 involves specific interactions with the
2
Dimerization of NaD1 and antifungal activity
fungal cell wall, followed by permeabilization of density was obtained for all residues of the entire
the plasma membrane and entry of NaD1 into molecule. Two chains of NaD1 were built for
the cytoplasm. It has been proposed that NaD1- crystal form A. NaD1 crystal form B was solved
mediated membrane permeabilization, together by molecular replacement using monomeric
with the recognition of intracellular targets, NaD1 from crystal form A as a search model
leads to fungal cell death (24,25). However, the with PHASER (48). The final models for both
molecular basis of this process has not been crystal forms were built with Coot (49) and
defined. refined with Phenix to a resolution of 1.4 Å
Previously, the structure of monomeric NaD1 (form A) and 1.58 Å (form B). All data
was determined by solution 1H-NMR collection and refinement statistics are
spectroscopy (10). Here we report the high- summarized in Table 1. Refinement yielded
resolution crystal structures of NaD1 from two R and R values of 11.9% and 13.7% for
work free
crystal forms, both of which contain NaD1 in a crystal form A, and 20.1% and 23.2% for crystal
dimeric configuration. Using crosslinking form B, respectively. The coordinates have been
experiments as well as small angle X-ray deposited in the Protein Data Bank (accession
scattering analysis and analytical codes 4aaz and 4ab0, respectively). Figures were
ultracentrifugation, we demonstrate that NaD1 prepared using PyMol (DeLano Scientific).
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forms dimers in solution. Dimerization of NaD1 Expression of NaD1 and NaD1 Mutants in ow
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is critical for antifungal activity as a mutant that Pichia pastoris—NaD1 and NaD1 mutants were lo
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does not dimerize has a greatly reduced ability to recombinantly expressed in the methylotrophic e
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kill the filamentous fungus Fusarium yeast Pichia pastoris essentially as described by fro
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oxysporum. Cabral et al. (50). Briefly, DNA encoding the h
mature region of NaD1 was cloned into the ttp://w
EXPERIMENTAL PROCEDURES pPIC9 expression vector (Invitrogen) directly in- w
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Protein Purification—NaD1 was purified frame with the "-mating factor secretion signal .jb
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from whole Nicotiana alata flowers up to the using the restriction enzymes XhoI and NotI. An .o
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petal coloration stage of flower development as alanine was added to the N-terminus of the b/
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described in van der Weerden et al. (25). NaD1 sequence to ensure efficient cleavage of g
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Crystallization and Data Collection—NaD1 the signal at the Kex2 cleavage site. The st o
crystals were obtained by the sitting drop vapour glutamic acid at position 2 and the lysine at n D
diffusion method in 20% PEG 1500 and 10% position 4 of the mature NaD1 sequence were ec
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succinate-phosphate-glycine buffer pH 9, and mutated to alanine using the Phusion site- b
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native data was collected from two different directed mutagenesis kit (Finnzymes) to r 2
5
crystal forms, A and B, as previously described generate mutants rNaD1(E2A) and , 2
0
1
(43). rNaD1(K4A), respectively. After transformation 8
Heavy Atom Derivatization and Derivative into E. coli TOP10 cells, the pPIC9-NaD1,
Data Collection—Initial attempts to solve the pPIC9-NaD1(E2A) and pPIC9-NaD1(K4A)
structure of NaD1 by molecular replacement plasmids were isolated and linearized using SalI
using either the NMR structure of NaD1 (PDB to allow integration at the his4 locus of the P.
1MR4) or other plant defensin structures failed. pastoris genome. Linearized DNA was
Consequently, NaD1 crystals from crystal form transformed into electrocompetent yeast as
A were soaked in mother liquor supplemented described by Chang et al. (51) and His+
with 0.2 M 5-amino-2,4,6-triiodoisophthalic acid transformants were selected for by plating onto
(I3C) (44) and 10% ethylene glycol for 5 min. MD agar (13.4 g/L yeast nitrogen base [YNB],
Derivative diffraction data were collected at 100 400 µg/L biotin, 10 g/L dextrose and 15 g/L
K using a wavelength of 1.5 Å at the Australian agar). A single His+ colony was used to
Synchrotron (beamline 3ID1) and processed inoculate 200 mL of BMG (100 mM potassium
using XDS (45). phosphate, pH 6.0, 13.4 g/L YNB, 400 µg/L
Model Building and Refinement—I3C sites biotin, 1% [v/v] glycerol) and incubated with
were found with ShelX (46) and refined using constant shaking at 30°C until the OD reached
600
Phenix (47). Clear and continuous electron ~3.0. The cell mass was collected by
3
Dimerization of NaD1 and antifungal activity
centrifugation (1,500 g, 10 min) and from the high-resolution models using the
resuspended into 1 L of BMM (100 mM program Moleman2 (Uppsala Software Factory).
potassium phosphate, pH 6.0, 13.4 g/L YNB, Analytical Ultracentrifugation—
400 µg/L biotin, 0.5% [v/v] methanol) to induce Sedimentation velocity experiments were
expression. Expression was continued for 72 h conducted in a Beckman model XL-A analytical
with constant shaking at 30°C after which time ultracentrifuge at a temperature of 20°C.
the cell mass was removed by centrifugation Samples of rNaD1 and rNaD1(K4A) were
(10,000 g, 10 min) and the supernatant collected. studied at a concentration of 0.5 mg/mL (94 "M)
rNaD1, rNaD1(E2A) and rNaD1(K4A) were solubilized in PBS, pH 7.4. 380 "L of sample
isolated from the supernatant using ion- and 400 "L of reference solution were loaded
exchange chromatography using SP sepharose as into a conventional double sector quartz cell,
described for isolation of NaD1 from plant which were subsequently mounted in a Beckman
tissue. 4-hole An-60 Ti rotor, and centrifuged at a rotor
Fungal Growth Inhibition Assays—The speed of 40,000 rpm overnight. Data were
ability of NaD1, rNaD1, rNaD1(E2A) and collected at a single wavelength (237 nm) in
rNaD1(K4A) to inhibit the growth of Fusarium continuous mode at 15 min intervals using a
oxysporum f. sp. vasinfectum was assessed as step-size of 0.003 cm without averaging. Solvent
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described previously (25). density (1.0053 g/mL at 20°C) and viscosity ow
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Protein Crosslinking—NaD1 at 125–500 (1.0184 cp) and an estimate of the partial lo
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µg/mL was crosslinked through primary amino specific volume of rNaD1 (0.727 mL/g) and ed
groups by the addition of 6.25 mM rNaD1(K4A) (0.725 mL/g) were computed fro
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bis[sulfosuccinimidyl] suberate (BS3; Thermo using the program SEDNTERP (52). h
Scientific) in a buffer containing 20 mM sodium Sedimentation velocity data at multiple time ttp://w
phosphate and 150 mM NaCl, pH 7.1, at room points were fitted to a continuous sedimentation w
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temperature for 30 min. The samples were coefficient [c(s)] distribution and a continuous .jb
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reduced with dithiothreitol and denatured, and mass [c(M)] distribution model (53-55) using the .o
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subjected to SDS-PAGE prior to Coomassie program SEDFIT, which is available at b/
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Brilliant Blue staining. NaD1, rNaD1, www.analyticalultracentrifugation.com. g
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rNaD1(E2A) and rNaD1(K4A) were also st o
crosslinked with 6.25 mM BS3 or with 10 mM RESULTS n D
Bis-N-succinimidyl-[pentaethylene glycol] ester Crystal Structure of NaD1—The structure of ec
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(BS[PEG]5; Thermo Scientific) at final protein NaD1 (crystal form A) was solved by single be
concentrations of 250 µg/mL or 375 µg/mL, heavy atom isomorphous replacement with r 2
5
respectively, as described above. anomalous signal and refined to a resolution of , 2
0
1
Small Angle X-Ray Scattering—SAXS data 1.4 Å. A second crystal form (form B) was 8
was collected at the SAXS/WAXS beamline at solved by molecular replacement using
the Australian Synchrotron. NaD1 at 0.125, monomeric NaD1 from the previous
0.25, 0.5, 1 and 2 mg/mL in distilled water at pH experimentally determined structure of form A.
7.0 was measured in a Q range between 0.035 As expected, NaD1 adopts the cysteine-
and 0.6 Å-1 at 12 KeV and with a 1.6 meter stabilized "! (CS"!) motif formed by a triple-
camera length. Normalization was achieved via stranded antiparallel #-sheet and a single $-helix
an integrating beam stop. Data was measured on that is tethered to the sheet via three disulfide
a Pilatus 1M camera (Dectris) and absolute bonds (Fig. 2A). The fourth disulfide bond
scaled using distilled water. To control for reinforces the N- and C-terminal regions of the
radiation damage, the samples were measured in molecule. Superimpositions of monomeric
a 1.5 mm quartz capillary and flowed past the NaD1 from crystal form B with chain 1 or all 20
beam while 10 ! 1 second exposures were chains within the ensemble of lowest energy
measured on samples and blanks. The exposures NMR structures for NaD1 (PDB code 1MR4)
were compared for agreement before being are shown in Figures 2B and C, respectively. As
averaged. The radius of gyration was calculated evident from Figure 2C, there are no appreciable
differences between the crystal and NMR
4
Dimerization of NaD1 and antifungal activity
structures for NaD1, with the crystal structure of this dimer presents a weaker acidic and
fitting well within the NMR ensemble. hydrophobic surface (Fig. 3D).
Moreover, superimposition of the monomers Protein Crosslinking Studies—To investigate
from crystal form A or B with the NMR the possibility that NaD1 forms a dimer in
structure of NaD1 yields RMSD values of 1.0 – solution, we utilized chemical crosslinking
1.3 Å over all 47 C" backbone atoms. studies to directly assess the configuration of the
Two Alternative Dimeric Forms—NaD1 is dimer in solution using
present as a dimer in the asymmetric unit of both Bis(sulfosuccinimidyl)suberate (BS3). BS3
crystal forms (Fig. 3). In crystal form A, the "1- contains an amine-reactive N-
!2 loop of one monomer faces the "-helix of the hydroxysulfosuccinimide (NHS) ester at each
other monomer (Fig. 3A). However, there are no end of an 8-carbon (11.4 Å) spacer arm. Once
protein-protein contacts at the dimer interface, the NHS esters react with primary amines
with all interactions being mediated by ordered (present in the side chain of lysine residues and
water molecules. Consequently, it is possible the N-terminus of each polypeptide) at pH 7–9,
that this dimeric form only exists in the crystal it forms stable amide bonds, along with release
and is not sufficiently stable to be observed in of the N-hydroxysulfosuccinimide leaving
solution. In contrast, the dimeric configuration group. D
of crystal form B is formed by the association of As illustrated in Fig. 4A, NaD1 migrates as a ow
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the !1-strands from the two participating single protein band at ~5 kDa (monomer) on loa
reducing SDS-PAGE in the absence of BS3. de
monomers (Fig. 3B). This creates an extended !- d
sheet leading to the formation of a more compact When the BS3 crosslinker is added, an additional from
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stabilized by three hydrogen bonds, two from NaD1 dimer (~10 kDa) (Fig. 4A). The protein w
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K4–K4 and one from S35–C47. Additionally, a band corresponding to the dimer becomes more .jb
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salt bridge is found between C47–R40 (Fig. 3C). pronounced with increasing amounts of NaD1 .org
Electrostatic Surfaces of the NaD1 Dimers— being used in the crosslinking reaction. At the b/
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The biological activity of NaD1 has been linked higher concentrations (e.g. 500 µg/mL NaD1 + gu
to an activity at the fungal cell wall (24,25). To BS3), multimers of 15 and 20 kDa are also est o
investigate the possibility of NaD1 surface apparent, consistent with the expected sizes of a n D
interactions with components of the fungal cell trimer and tetramer of NaD1, respectively. ece
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wall, we examined the electrostatic surface Based on our structural studies that suggested be
potential of the two different dimer a pivotal role for K4 in NaD1 dimerization, we r 25
configurations (Fig. 3C–F). generated a K4A mutant of NaD1 in P. pastoris , 20
1
to examine whether dimerization would be 8
The NaD1 dimer in crystal form A has two
affected. As controls, rNaD1 and rNaD1(E2A)
distinct negatively charged pockets at the ends
(glutamic acid at position 2 is not involved at the
of the dimer, with two small areas of local
dimer interface) were also produced.
positive charge (Fig. 3C,E). In contrast, the
These proteins were subjected to BS3
charge distribution on the surface of the NaD1
crosslinking. As shown in Fig. 4B, rNaD1 or
dimer from crystal form B is markedly different.
rNaD1(E2A) were able to form dimers. In
A continuous surface of positive charge is
contrast, dimerization was abolished in the
present on one side of the dimer that begins on
rNaD1(K4A) mutant.
the first monomer and continues to the end of
When using BS3, three potential crosslinks
the second monomer (Fig. 3F). The two K4
can be formed in dimer form A (K17–K22;
residues make key contributions to the middle of
K28–K45; K17–K28) and one in dimer form B
the positively charged face of the dimer (Fig.
(K4–K4). Since mutation of K4 could prevent
3G). Flanking either side of the positive charge
crosslinking with BS3 in dimer form B and thus
are two well-separated negatively charged
lead to an apparent loss of crosslinking that is
pockets (Fig. 3F). Furthermore, the opposite side
not correlated with loss of dimerization we also
performed chemical crosslinking using
5
Dimerization of NaD1 and antifungal activity
BS[PEG] , which has a spacer arm of 21.7 Å Two elements of the scattering data indicate
5
(Fig 4C). BS[PEG] enables additional cross- that NaD1 is largely a dimer in solution. Firstly
5
linkages in dimer form B between K28–K45 and the molecular mass, calculated from I(0) on the
K45–K45. When using BS[PEG] , rNaD1 again absolute scattering scale across the concentration
5
revealed crosslinked dimers on SDS-PAGE, range, was around 8.9 kDa. This corresponded to
whereas rNaD1(K4A) showed substantially an oligomerization state around 1.7, suggesting
reduced amounts of dimer. Overall our chemical that more of the protein is in a dimeric state than
crosslinking results suggest that rNaD1(K4A) a monomeric state. In addition, the radius of
has a significantly lower propensity to form gyration was calculated from the high-resolution
dimers compared to the wild-type rNaD1. structures of monomer and dimer. The Rg of the
Circular dichroism analysis of NaD1, rNaD1, monomeric structure is calculated to be 10 Å
rNaD1(E2A) and rNaD1(K4A) indicated that while the dimer is 15 Å. The measured Rg from
there was no major spectral differences between the data is 13.2 Å or somewhere between
the proteins suggesting that they were similarly monomer and dimer.
folded (data not shown). Therefore the reduced Sedimentation Velocity Analysis of rNaD1 and
dimerization of rNaD1(K4A) is not a result of rNaD1(K4A) —Sedimentation velocity studies
incorrect folding and provides evidence to of rNaD1 and rNaD1(K4A) were employed in
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support the importance of K4 in NaD1 the analytical ultracentrifuge to further validate ow
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dimerization. the observations from our chemical crosslinking lo
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Antifungal activity of NaD1, rNaD1 and experiments and SAXS analysis. The absorbance e
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rNaD1 mutants—To determine whether versus radial position profiles at different time fro
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dimerization of NaD1 contributes to its points were initially fitted to continuous h
antifungal activity, the ability of rNaD1(K4A) to sedimentation coefficient [c(s)] distribution ttp://w
inhibit the growth of F. oxysporum f. sp. model (Fig. 7). The c(s) distribution analyses w
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vasinfectum was assessed and compared to yielded excellent fits to the data as demonstrated .jb
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rNaD1 and rNaD1(E2A) (Fig. 5). The inhibitory by the low rmsd values of 0.00464 for rNaD1 .o
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activity of rNaD1 and rNaD1(E2A) was similar and 0.00410 for rNaD1(K4A), as well as the low b/
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to native NaD1 (IC50 value of <1.15 "M). In Runs test Z values of 1.50 for rNaD1 and 0.86 gu
contrast, there was a significant decrease in the for rNaD1(K4A). The resulting c(s) distributions es
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ability of rNaD1(K4A) to inhibit fungal growth, show that both rNaD1 and rNaD1(K4A) exist as n
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with an approximate five-fold increase in its IC50 a mixture of monomers (s ~ 0.57-0.77 S) and ece
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value (5 µM) relative to rNaD1 and dimers (s ~ 1.05-1.26 S), however rNaD1(K4A) b
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rNaD1(E2A). shows significantly less propensity to dimerize r 2
5
SAXS Analysis—Since the solution NMR relative to rNaD1, which predominantly exists as , 2
0
1
structure of NaD1 indicated that the molecule a dimer. 8
was present as a monomer during the spectra
acquisition (at pH 4) (10), we sought to establish DISCUSSION
if NaD1 could be present as a dimer in solution The formation of dimers and/or higher order
under more physiologically relevant conditions. oligomers in antimicrobial peptides has been
For this purpose, we utilized small angle X-ray proposed as a contributing factor to their ability
scattering (SAXS) analysis. The NaD1 protein to disrupt biological membranes (56-61). In this
was measured at five concentrations, 0.125, study, we employed a number of biophysical
0.25, 0.5, 1 and 2 mg/mL. The scattering curves approaches to examine the three-dimensional
show a similar shape at each concentration (Fig. and quaternary structures of NaD1, both in solid
6A). The lowest angle regions of the scattering state (i.e. crystals) and in solution. In all
curves conform to a straight line on a Guinier examined environments, NaD1 formed dimers
plot (Fig. 6B) and the calculated radius of and smaller amounts of higher order oligomers.
gyration does not vary significantly across the This is consistent with previous observations
concentration range, indicating an absence of made for various other plant defensins. For
significant concentration effects (Table 2). example, Terras et al. (1992) demonstrated using
SDS-PAGE analysis that unreduced radish
6
Dimerization of NaD1 and antifungal activity
defensins Rs-AFP1 and Rs-AFP2 had apparent decorated with negatively charged glycoproteins
molecular masses of 15 (trimer) and/or 20 kDa (64,65) and dimeric NaD1 may be important for
(tetramer) (22). This was in contrast to the initial binding to these cell wall components as a
reduced and S-pyridylethylated derivatives of precursor step for oligomerization.
both proteins which migrated as single bands An important functional link between NaD1
with a relative molecular mass of ~5 kDa. This dimerization and antifungal activity was strongly
was similarly demonstrated for other plant suggested by site-directed mutagenesis of the
defensins, namely Dm-AMP1, Dm-AMP2, Hs- lysine at position 4 (K4). As described, the
AFP1, Ah-AMP1 and Ct-AMP1 (21). Melo et solved structure of NaD1 crystal form B
al. (2002) also demonstrated that the cowpea indicated that K4 plays a crucial role in the
defensin, Cp-thionin, could exist as dimers along monomer-monomer interaction to form the
with smaller quantities of other multimers by dimeric conformation. Indeed, the substitution of
MALDI-TOF mass spectrometry (35). K4 with alanine resulted in a markedly reduced
More recently, Song and colleagues ability of NaD1 to form dimers, as shown by
(20,62,63) provided experimental evidence chemical crosslinking and sedimentation
including a crystal structure to support a dimeric velocity analyses. The importance of the NaD1
configuration for the plant defensin SPE10. dimeric configuration for its antifungal activity
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Similar to our study, the authors observed a was demonstrated as the K4A mutant displayed ow
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dimer of SPE10 in the asymmetric unit (20). greatly reduced capacity to kill the filamentous lo
a
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However, unlike the !-sheet to !-sheet fungus F. oxysporum with an approximate five- e
d
configuration of NaD1 crystal form B (Fig. 8A), fold increase in its IC50 value compared to native from
the two SPE10 monomers are arranged in a side and recombinant NaD1. Examination of the h
by side manner with the "-helix of one amino acid sequences of all class II defensins ttp://w
monomer contacting the !-sheet of the second described to date (7) indicates that K4 is totally w
w
monomer (Fig. 8B), resulting in an extended and conserved. As such, it is tempting to speculate .jb
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slightly twisted molecular surface. Dimerization that all class II defensins may have the ability to .o
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buries 606 Å2 of solvent accessible surface area dimerize and that dimerization may be important b/
y
and results in the formation of intermolecular for their respective functions (e.g. by enhancing g
u
hydrogen bonds involving R36–D21, R40–D22, their antifungal activity). est o
E4–K25, N5–N26, N17–D37 and G18–D37 It is interesting to compare the findings n D
(20). described herein for NaD1 dimerization with ec
e
m
To date, the relevance of dimer formation in defensins from other species such as those from b
e
plant defensins has not been examined. mammals. The mammalian defensins are defined r 2
5
However, it has been suggested that for some under three distinct structural subfamilies, "-, !- , 2
0
1
cationic antimicrobial proteins (CAPs), such as or # (theta), based on their differences in size, 8
the mammalian defensins, dimeric the placement and connectivity of their (six)
configurations represent the functional cysteines, the nature of their precursors and their
biological units (56-61). Furthermore, the sites of expression (66-71). It is worth noting
dimeric protein unit may undergo orchestrated here that plant defensins do not share primary or
oligomerization to form larger assemblies that three-dimensional structures with these
are pertinent to the ability of the given CAP to mammalian namesakes except that they all have
kill. This hypothesis is certainly central to an implicated role in innate host immunity.
models in which CAPs operate via the creation Indeed, it was in the context of their defense role
of oligomeric pores. that the name “defensin” was originally coined
Considering that oligomerization of defensins (72,73).
may play a key role for their biological function, Human "-defensins or human neutrophil
and that their initial site of activity is either a cell peptides (HNPs) exhibit broad antimicrobial
wall or plasma membrane, our observation of a activity against Gram-negative and Gram-
dimer coupled with an extended positively positive bacteria, fungi and enveloped viruses
charged surface area on one side of the NaD1 (74) and have also been linked with adaptive
dimer is significant. Fungal cell walls are immunity as immunostimulating agents (67,68).
7
Dimerization of NaD1 and antifungal activity
The three-dimensional structures of several "- could be needed to drive octamer formation (59).
and !-defensins have been solved (57-59,61,75- As NaD1 is known to interact with components
82). Interestingly, the structures of several (as yet undetermined) on the fungal cell wall and
human defensins, including HNP-1, HNP-3 and membrane, together with the observation that
HBD-2, indicated the presence of dimers and/or cations (e.g. Ca2+) can abrogate antifungal
multimers in the asymmetric units. For HNP-1, activity (25), it would be plausible to suggest
Wei et al. (61) recently suggested a functional that electrostatic attraction mediated by anionic
link between multimerization and antibacterial surface components (such as phospholipids,
activity as mutants with reduced capacity to self- sphingolipids or glycoproteins), may be
associate exhibited poor bactericidal activity. important.
HNP-3 (PDB code 1DFN) consists of a It is known that for the seed defensins from
triple-stranded antiparallel !-sheet that forms a radish (Rs-AFP2) and dahlia (Dm-AMP1),
six-stranded sheet as part of a symmetrical dimer interactions with specific sphingolipids
(57). The dimer is stabilized by four direct (glucosylceramide and mannose-(inositol-
phosphate) -ceramide, respectively) on fungal
hydrogen bonds between the !2-strands of the 2
plasma membranes is required for their
monomers, three hydrogen bonds mediated
antifungal activity (83,84). They are able to
through water molecules and several D
hydrophobic interactions (Fig. 8C). This is permeabilize the fungal membrane and induce ow
somewhat reminiscent of the !-sheet packing Ca2+ influx and K+ efflux. Given that fungi grow nloa
d
from the tip and require the maintenance of an e
observed for NaD1 crystal form B. Moreover, d
HNP-3 was also able to form dimers and higher intracellular Ca2+ concentration gradient to drive from
osolilguotimone rcso nbdyit ioenqs uwilhibicrihu mm atccheendtr itfhuagta utisoend foinr pcoolllaeraizgeude s g(1ro9w96th) su(g8g5e,8s6te)d, tThahte vtihsese ng rowanthd http://w
the crystallisation (except that PEG8000 was inhibition may be due to dissipation of this w
w
omitted) (57). gradient (87). Interestingly, other plant defensins .jb
c
HBD-2 consists of a triple-stranded from alfalfa and corn have been reported to .org
antiparallel !-sheet and an "-helix but in an block ion channels, although interaction with b/
fungal Ca2+ channels has not yet been y g
"!!! arrangement (!"!! for NaD1) (59), which u
demonstrated (37,38). It is noteworthy that es
was observed in two crystal forms (PDB codes t o
scorpion toxins, which are bone fide ion channel n
1FD3, 1FD4). The orthorhombic form contained D
blockers, also display the CS"! motif that is ec
four monomers in the asymmetric unit, formed e
m
conserved in the plant defensins (88-90). b
by two topologically identical dimers (Fig. 8D). e
Numerous models have been proposed in the r 2
Its dimer interface is also mediated by two 5
literature to account for the potential molecular , 2
hydrogen bonds and several hydrophobic (van 0
1
mechanisms of action for various cationic 8
der Waals) interactions from the !1-strands of
antimicrobial peptides on target membranes
the monomers, resulting in a six-stranded !-
(reviewed by (91)). Based on the crystal
sheet for the dimer as in NaD1 and HNP-3. The
structure of HNP-3, Hill et al. (57) proposed
monoclinic form contained two octameric
three possible models for HNP-3–membrane
assemblies within the asymmetric unit, which
interaction: (i) a wedge model, (ii) dimer pore
were proposed to be the stable, native quaternary
model and a (iii) general pore model. In all
structure of HBD-2 for activity (59), although no
proposed models, the dimer is the protomeric
additional evidence for the presence of such an
unit, building from a single dimer, to two
oligomer in solution was presented. Intriguingly,
stacked dimers onto a large annular pore
subsequent dynamic light scattering experiments
involving multiple dimers. Intriguingly, these
revealed that concentrated solutions of HBD-2
models (or variations thereof) are conceivable
mainly formed dimers with other aggregated
for HNP-3 and other dimer-forming CAPs such
forms (59). This led Hoover and colleagues
as NaD1 and may indeed represent transition
(2000) to suggest that additional stabilizing
states. As such, factors including protein
interactions such as those provided by the
concentration, the local environment of the
negative charged target (bacterial) membrane
target membrane (e.g. lipid composition), the
8
Dimerization of NaD1 and antifungal activity
volume of the molecule, its structure, and its 11. Almeida, M. S., Cabral, K., Kurtenbach,
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The dimerization of NaD1 results in the 13. Bruix, M., Gonzalez, C., Santoro, J.,
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permeabilization and entry of NaD1 into the cell and Rico, M. (1993) Biochemistry 32,
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15. de Paula, V. S., Razzera, G., Barreto-
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