Native thrombocidin-1 and unfolded thrombocidin-1 exert antimicrobial activity via distinct structural elements.
ABSTRACT Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.
- SourceAvailable from: Hans J Vogel[Show abstract] [Hide abstract]
ABSTRACT: Chemokines are best known as signaling proteins in the immune system. Recently however, a large number of human chemokines have been shown to exert direct antimicrobial activity. This moonlighting activity appears to be related to the net high positive charge of these immune signaling proteins. Chemokines can be divided into distinct structural elements and some of these have been studied as isolated peptide fragments that can have their own antimicrobial activity. Such peptides often encompass the α-helical region found at the C-terminal end of the parent chemokines, which, similar to other antimicrobial peptides, adopt a well-defined membrane-bound amphipathic structure. Because of their relatively small size, intact chemokines can be studied effectively by NMR spectroscopy to examine their structures in solution. In addition, NMR relaxation experiments of intact chemokines can provide detailed information about the intrinsic dynamic behavior; such analyses have helped for example to understand the activity of TC-1, an antimicrobial variant of CXCL7/NAP-2. With chemokine dimerization and oligomerization influencing their functional properties, the use of NMR diffusion experiments can provide information about monomer-dimer equilibria in solution. Furthermore, NMR chemical shift perturbation experiments can be used to map out the interface between self-associating subunits. Moreover, the unusual case of XCL1/lymphotactin presents a chemokine that can interconvert between two distinct folds in solution, both of which have been elucidated. Finally, recent advances have allowed for the determination of the structures of chemokines in complex with glycosaminoglycans, a process that could interfere with their antimicrobial activity. Taken together, these studies highlight several different structural facets that contribute to the way in which chemokines exert their direct microbicidal actions.Frontiers in Immunology 01/2012; 3:384.
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ABSTRACT: Platelets have been shown to cover a broad range of functions. Besides their role in hemostasis, they have immunological functions and thus participate in the interaction between pathogens and host defense. Platelets have a broad repertoire of receptor molecules that enable them to sense invading pathogens and infection-induced inflammation. Consequently, platelets exert antimicrobial effector mechanisms, but also initiate an intense crosstalk with other arms of the innate and adaptive immunity, including neutrophils, monocytes/macrophages, dendritic cells, B cells and T cells. There is a fragile balance between beneficial antimicrobial effects and detrimental reactions that contribute to the pathogenesis, and many pathogens have developed mechanisms to influence these two outcomes. This review aims to highlight aspects of the interaction strategies between platelets and pathogenic bacteria, viruses, fungi and parasites, in addition to the subsequent networking between platelets and other immune cells, and the relevance of these processes for the pathogenesis of infections.Future Microbiology 11/2013; 8:1431-51. · 4.02 Impact Factor
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ABSTRACT: Up to date, perception of platelets has changed from key players in coagulation to multitaskers within the immune network, connecting its most diverse elements and crucially shaping their interplay with invading pathogens such as fungi. In addition, antimicrobial effector molecules and mechanisms in platelets enable a direct inhibitory effect on fungi, thus completing their immune capacity. To precisely assess the impact of platelets on the course of invasive fungal infections is complicated by some critical parameters. First, there is a fragile balance between protective antimicrobial effects and detrimental reactions that aggravate the fungal pathogenesis. Second, some platelet effects are exerted indirectly by other immune mediators and are thus difficult to quantify. Third, drugs such as antimycotics, antibiotics, or cytostatics, are commonly administered to the patients and might modulate the interplay between platelets and fungi. Our article highlights selected aspects of the complex interactions between platelets and fungi and the relevance of these processes for the pathogenesis of fungal infections.Thrombosis and haemostasis. 07/2014; 112(3).
Native Thrombocidin-1 and Unfolded Thrombocidin-1 Exert
Antimicrobial Activity via Distinct Structural Elements□
Paulus H. S. Kwakman,aJeroen Krijgsveld,bLeonie de Boer,aLeonard T. Nguyen,cLaura Boszhard,a
Jocelyne Vreede,dHenk L. Dekker,eDave Speijer,fJan W. Drijfhout,gAnje A. te Velde,hWim Crielaard,i
Hans J. Vogel,cChristina M. J. E. Vandenbroucke-Grauls,a,jand Sebastian A. J. Zaata1
cBiochemistryResearchGroup,DepartmentofBiologicalSciences,UniversityofCalgary,T2N1N4Calgary, Alberta, Canada
Background: The properties required for antimicrobial activity of chemokines are unclear.
Results: Native thrombocidin-1 requires a three-dimensional positive patch for activity, but unfolded thrombocidin-1 is active
through the N-terminal linear peptide regions.
Conclusion: Native thrombocidin-1 and unfolded thrombocidin-1 exert activity via distinct structural elements.
Significance: Folded and unfolded antimicrobial chemokines can exert activity through different structural elements.
Chemokines (chemotactic cytokines) can have direct antimi-
crobial activity, which is apparently related to the presence of a
distinct positively charged patch on the surface. However,
despite the loss of their positive patch, thus questioning the
importance of this patch for activity. Thrombocidin-1 (TC-1) is
a microbicidal protein isolated from human blood platelets.
TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-
antimicrobial activity. We assessed the structure-activity rela-
tionship for antimicrobial activity of TC-1. Reduction of the
charge of the TC-1-positive patch by replacing lysine 17 with
alanine reduced the activity against bacteria and almost abol-
ished activity against the yeast Candida albicans. Conversely,
or size resulted in a 2–3-fold increased activity against Staphy-
lococcus aureus, Escherichia coli, and Bacillus subtilis but did
TC-1 resulted in loss of the folded conformation, but this dis-
ruption of the positive patch did not affect antimicrobial activ-
ity. Using overlapping 15-mer synthetic peptides, we demon-
strate peptides corresponding to the N-terminal part of TC-1
to have similar antimicrobial activity as intact TC-1.
Although we demonstrate that the positive patch is essential
for activity of folded TC-1, unfolded TC-1 retained antimi-
crobial activity despite the absence of a positive patch. This
activity is probably exerted by a linear peptide stretch in the
N-terminal part of the molecule. We conclude that intact
TC-1 and unfolded TC-1 exert antimicrobial activity via dis-
tinct structural elements.
innate and adaptive immunity. The general chemokine struc-
rallel ?-strands, and a C-terminal ?-helix. Chemokines have a
fide bonds (1). Correct folding is essential for specific interac-
tions of chemokines with their receptors (1).
In addition to their chemotactic activity, many chemokines
also have direct antimicrobial activity (2–4). Antimicrobial
chemokines have a distinct three-dimensional amphipathic
structure consisting of a hydrophobic region and a positive
crobial peptides. An amphipathic structure is supposedly
required for microbicidal activity, with hydrophobic domains
being essential for membrane interactions and cationic
domains providing selective interaction with the negatively
charged outer surfaces of microorganisms (6–8).
Strikingly, several antimicrobial chemokines as well as disul-
fide-containing antimicrobial peptides retain antimicrobial
activity when linearized (9–12). Moreover, reduction and
unfolding are even required to reveal the full antimicrobial
activity of human ?-defensin 1 (13). Upon linearization, these
proteins can lose their characteristic ?-sheet secondary struc-
tures (14, 15) that will substantially affect their three-dimen-
ity and suggest that other structural elements, at least in the
linearized proteins, are involved in this antimicrobial activity.
supplemental Tables S1 and S2.
1To whom correspondence should be addressed: Academic Medical Center,
Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Tel.: 31-20-
5664863; Fax: 31-20-5669609; E-mail: firstname.lastname@example.org.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 50, pp. 43506–43514, December 16, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
43506 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER50•DECEMBER16,2011
by guest, on May 7, 2013
Supplemental Material can be found at:
Thrombocidins are microbicidal proteins of human blood
platelets (2) derived from platelet chemokines that contribute
to innate immunity (17). Thrombocidin-1 (TC-1),2the most
potent thrombocidin, only differs from the chemokine NAP-2/
CXCL7 by a C-terminal truncation of an alanine and aspartate
residue. This truncation is required to reveal the microbicidal
activity of TC-1 (2).
antimicrobial activity of TC-1. To determine the role of the
positive patch, we constructed TC-1 substitution variants with
an augmented or a decreased positive charge of the patch. To
study activity of the unfolded protein, we disrupted the three-
and we confirmed change of structure by NMR.
As unfolding did not reduce antimicrobial activity of TC-1,
we studied the activity of short linear peptides covering the
entire TC-1 sequence. We identified peptides derived from the
TC-1 N-terminal region with antimicrobial activity similar to
the activity of full-length TC-1. Thus, our data demonstrate
a part of the N-terminal region seems responsible for the anti-
occurs in vivo (13), it will be relevant to compare structure-
function relationships of other antimicrobial proteins in their
native and their unfolded conformation.
Antimicrobial activity was assessed against the laboratory
strains Bacillus subtilis ATCC6633, Staphylococcus aureus
42D, Escherichia coli ML-35p (18), and clinical isolates of Can-
dida albicans and Cryptococcus neoformans (2).
Overlapping 15-mer peptides covering the entire TC-1
(19). Peptides were named after their first amino acid and the
position of this amino acid in TC-1.
The x-ray resolved structure of NAP-1 (Protein Data Bank
(21). As all substitutions are located at the protein surface, and
all TC-1 variants only had marginally reduced Ramachandran
scores (supplemental Table S1), the designed substitutions
would not significantly alter the fold of the protein. RasWin
Molecular Graphics version 184.108.40.206 was used for ribbon repre-
sentation of TC-1 (22). Swiss model was used for prediction of
the three-dimensional structure of TC-1 substitution variants
(25) were used for electrostatic potential and hydrophobicity
DNA coding for TC-1 was obtained in a two-step PCR
protocol. Primers were designed based on the cDNA
sequence of platelet basic protein (PBP) (26). First a PBP
amplicon was amplified from the human bone marrow
cDNA library (Clontech) using Pfu polymerase (Stratagene)
and oligonucleotides PBP forward and PBP reverse primers,
respectively (supplemental Table S1); primers were from
Applied Biosystems. This PBP amplicon served as a template
to generate the product coding for TC-1, with primers con-
taining NdeI and BamHI restriction sites to allow ligation of
the digested PCR product to NdeI-BamHI-digested pET9a
expression vector (Novagen). The ligation products were
used to transform E. coli BL21DE3(LysS) cells (Merck) by
heat shock. Individual colonies on selective LB agar plates
were checked for the presence of an insert by colony PCR
using a direct primer recognizing the T7 promoter sequence
of the pET9a vector (TAATACGACTCACTATAGGG) and
the reverse primer for TC-1 (supplemental Table S2). Both
strands of three positive clones were sequenced. Bacteria
containing the correct construct were stored in glycerol
broth at ?80 °C until further use.
Sequences coding for TC-1 variants were generated by PCR
using primers containing the desired substitutions. For TC-1
S12K, G13K, and I14K (Fig. 1), primers PKPET S12K, PKPET
G13K, and PKPET I14K, respectively, were used as forward
primers in combination with reverse primer PKPET-1 (supple-
mental Table S2). For TC-1 D42K and D49K substitutions,
PKPET-1 was used as a forward primer, and PKPET D42K and
PKPET D49K as the respective reverse primers. PCR products
were cloned into pCR2.1, and the ligation product was used to
selective LB agar were checked for the presence of a correct
TC-1 insert. After digestion with NdeI and BamHI, the TC-1
gene-containing fragments of these clones were purified from
agarose gels using QiaExII (Qiagen), ligated to NdeI and
BamHI-digested pET9a, and the resulting clones were checked
for correct inserts as described above.
TSB, trypticase soy broth.
FIGURE 1. Primary amino acid sequence of NAP-2, TC-1, and TC-1 substitution variants. Substituted residues in TC-1 variants are indicated in black.
by guest, on May 7, 2013
E. coli BL21DE3(lysS) cells containing pET9a-derived con-
structs were cultured in 0.5–2 liters of LB medium with chlor-
amphenicol ? kanamycin (50 ?g/ml each). When cultures had
reached an absorbance of 0.3 at 620 nm, isopropyl ?-D-thiogal-
actoside (Roche Applied Science) was added to a final concen-
tration of 0.5 mM to induce expression of the cloned genes.
volume) and kept at ?20 °C overnight. Bacteria were lysed by
ultrasonication (5 min, 0 °C); cell debris was removed by cen-
trifugation (1 h, 150,000 ? g), and the supernatant was diluted
5-fold in 50 mM HEPES, pH 8.0. Recombinant proteins were
purified in a two-step procedure adapted from a method
described previously for native thrombocidins (2). As a first
step sonicates were applied to a 5-ml SP fast flow column (GE
Healthcare) equilibrated in 50 mM HEPES, pH 8.0, at a flow of
using continuous acid urea-PAGE. Protein concentrations
were determined with a BCA protein assay (Pierce) and molec-
by MALDI-TOF and Q-TOF analyses.
Activity of reduced TC-1 was assessed by electrophoretic
analysis and gel overlay (see below). Protein (?1 mg/ml) was
pretreated in 100 mM Tris-HCl, pH 8.5, containing 6 M guani-
othreitol (DTT, Sigma) was added (1 mg/ml final concentra-
tion), and incubation was allowed to proceed for 4 h under
tion of iodoacetamide (Sigma; 4 mg/ml final concentration) to
prevent reoxidation. Alkylated protein was purified by HPLC
using a Hypersil PEP C18 RP column (150 ? 4.6 mm; Alltech),
lyophilized, and dissolved in 0.01% acetic acid. Efficiency of
alkylation was confirmed by mass spectrometry. Protein con-
centration was determined using a BCA protein assay (Pierce).
in the supplemental material.
1H NMR and far-UV circular dichroism
acquire far-UV circular dichroism spectra for TC-1 samples
prepared for these and the NMR experiments. Either native
H2O/D2O) or with SDS-d25micelles (10 mM) at a protein con-
centration of 0.014 mM and at pH 6.3. Reduced TC-1 was pre-
pared by boiling TC-1 for 15 min followed by incubation with
10 mM DTT-d10for 1 h at room temperature with gentle shak-
ing. For each sample, 10 CD scans were accumulated and aver-
aged, scanning from 260 to 190 nm at a 200 nm/min scanning
100[?]obs/(lcn), where [?]obsis the observed ellipticity in mil-
lidegrees; l is the path length of the cuvette in centimeters; c is
the molar concentration, and n is the number of residues in
One-dimensional1H NMR spectra were acquired for the
solution or with SDS micelles on a Bruker Avance 500 MHz
equipped with a Cryo-ProbeTM. 64 scans were acquired at 298
K with a spectral width of 8013 Hz. Water suppression was
performed using an excitation sculpting pulse sequence (27).
Microbicidal Assay—Microbicidal activity was assayed
essentially as described by Harwig et al. (28). Overnight cul-
in fresh TSB and cultured for 3 h at 37 °C (bacteria) or 30 °C
absorbance at 620 nm was measured, and the microorganisms
were diluted to 2 ? 106CFU/ml in incubation buffer, based on
To determine the concentration of protein or peptide
required to kill at least 99.9% of the inoculum (LC99.9), 25-?l
aliquots of 2-fold serially diluted peptide in incubation buffer
were prepared in wells of a polypropylene microtiter plate
(Costar, Corning), and to each of the wells 25 ?l of a microbial
suspension containing 2 ? 106CFU/ml was added. To assess
the influence of physiological salt concentrations, 0.9% (w/v)
NaCl was added to incubations. All tests were performed in
at 37 °C, duplicate 10-?l aliquots were plated on blood agar
plates. The plates were inspected for growth after 24 h.
Radial Diffusion Assay—Microbial suspensions were pre-
pared as described for the liquid bactericidal assay. An inocu-
lum of 107CFU was mixed with 20 ml of nutrient-poor agarose
(0.03% (w/v) TSB in 10 mM sodium phosphate buffer, pH 7.0,
mm in diameter were punched in the agarose to which 2.5-?l
allowed to diffuse into the agarose for 3 h at 37 °C (bacteria) or
30 °C (yeasts). Subsequently, the agarose was overlaid with 20
ml of double-strength nutrient agar (6% TSB, 1% Bacto-agar
(Difco), 45 °C), and plates were incubated overnight at 37 °C
(bacteria) or 30 °C (yeasts). To assess the influence of physio-
logical salt concentrations, 0.9% (w/v) NaCl was added to both
the nutrient-poor agarose and double strength nutrient agar.
ured to calculate the area of growth inhibition.
for 8 min in 10 mM phosphate buffer, pH 7.0, and placed on a
plate with B. subtilis-inoculated nutrient-poor agarose (see
under “Radial Diffusion Assay”), and the plate was incubated
for 3 h. After removal of the gel, the agarose was overlaid with
double-strength nutrient agar and treated as described for the
radial diffusion assay.
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Analysis of Positive Patch of TC-1—Analysis of TC-1 by elec-
composed of the residues Arg-54, Lys-56, Lys-57, Lys-61, and
Lys-62 in one face of the C-terminal ?-helix; residue Lys-17 in
the turn between the N-terminal segment and the first
?-strand, and residues Lys-41, Arg-44, and Lys-45 in the turn
this positive patch, residue Lys-17 appears to fulfill a central
role by bridging the positive charges of the loop between the
second and third ?-strand and those of the C-terminal ?-helix
ity of TC-1—Because Lys-17 occupies a central position in the
TC-1 positive patch, substitution of this residue with an
uncharged amino acid would disrupt this patch. Electrostatic
potential plots show that a substitution of lysine 17 by alanine
(K17A) would indeed have a profound effect on the positive
(Fig. 2B) to assess the importance of the positive patch for anti-
microbial activity of TC-1.
Recombinant TC-1 (rTC-1) had antimicrobial activity
had reduced activity against B. subtilis, S. aureus, and E. coli
important for antimicrobial activity of TC-1, particularly
against C. albicans.
Design of TC-1 Substitution Variants with Augmented Posi-
tive Patches—As a complementary approach to assess the
importance of the positive patch, we constructed TC-1 substi-
tution variants with augmented positive patches. The nega-
tively charged residue Asp-42 occupies a central position
within the positive patch (Fig. 2B), thus reducing the net posi-
tive charge of the patch. We constructed a TC-1 D42K substi-
tution variant that has a positive patch with substantially aug-
mented charge density as shown by an electrostatic potential
plot of a Swiss Model-generated three-dimensional structure
TC-1 (B) and TC-1 K17A (C) were calculated using CCP4mg. Positively and negatively charged surfaces are shown in blue and red, respectively. The left panels
correspond to the orientation of TC-1 as shown in A. The middle and right panels show the molecules rotated along their vertical axis by 90° and 180°,
assay with the indicated microorganisms, equimolar amounts (2.5 ?l of 250
?M preparations) of TC-1 (gray bars) and TC-1 K17A (white bars) were tested.
Activity was measured as the area of microbial growth inhibition.
DECEMBER16,2011•VOLUME286•NUMBER50 JOURNALOFBIOLOGICALCHEMISTRY 43509
by guest, on May 7, 2013
We also aimed to enlarge the size of the positive patch. The
and D49K substitution of TC-1 indeed strongly enlarges the
surface area of the positive patch (Fig. 4). TC-1 S12K, G13K,
and I14K substitutions result in increased cationicity of the
outer surface in the region between the Asp-42 and Asp-49
proximal to the native TC-1 positive patch (Fig. 4).
Antimicrobial Activity of TC-1 Variants with Augmented
was assessed in a radial diffusion assay, and the area of growth
inhibition was compared with that of unmodified TC-1. The
TC-1 D42K variant with a larger charge density of the positive
patch had a 2.6-fold larger zone of growth inhibition of S.
aureus and E. coli compared with TC-1, and 2-fold increased
inhibition zone of B. subtilis (Fig. 5). However, the inhibition
zone of C. albicans was reduced to 80% that of TC-1. TC-1
D49K, which has a larger positive patch size, produced a 3.4-
and a 2-fold larger zone of inhibition of B. subtilis and E. coli
(Fig. 5). The inhibition zone of C. albicans was reduced to 80%
that of TC-1.
Of the TC-1 variants with lysine substitutions in the N-ter-
minal segment, only TC-1 I14K had substantially increased
exhibited a slight increase in inhibition zones of B. subtilis.
TC-1. With E. coli, inhibition zones of S12K and G13K were 60
and 27% of those of TC-1, respectively. S12K and G13K com-
pletely lacked activity against C. albicans (Fig. 5).
Antibacterial Activity of Reduced TC-1—Because several
antimicrobial peptides and chemokines retain antimicrobial
activity as linearized molecules, we subsequently investigated
whether TC-1 would also retain antimicrobial activity upon
linearization. Native TC-1 was reduced by DTT treatment or
reduced and alkylated. Samples were run on acid urea-PAGE,
and the gels were either silver-stained or used for antibacterial
reduced/alkylated TC-1 migrated slower in acid urea-PAGE
compared with the native protein (Fig. 6, top panel), indicating
alkylation, performed to prevent refolding, did not affect pro-
reduced proteins and increases the molecular weight only
Structural data from NMR spectroscopy and circular dichr-
folded structure. The backbone amide region of the one-di-
mensional NMR spectrum of reduced TC-1 showed a profile
very different from that of folded TC-1 with a narrower distri-
bution of peaks (Fig. 7A), indicating loss of folded TC-1 struc-
three-dimensional positive patch. The far-UV CD spectrum of
folded TC-1 shows a profile consistent with its expected struc-
ture of a three-stranded ?-sheet and a C-terminal ?-helix (Fig.
7B). The corresponding spectrum for reduced TC-1 showed a
FIGURE 4. Electrostatic surface plots of thrombocidin variants with enhanced positive patches. Electrostatic surfaces were calculated using CCP4mg, in
2A. The right panels show the molecules rotated along their vertical axis by 90°.
FIGURE 5. Antimicrobial activity of TC-1 variants with augmented posi-
indicated variants were tested, and activity was measured as the area of
microbial growth inhibition. Activity of the TC-1 variants against B. subtilis, S.
size relative to that of TC-1. The areas of growth inhibition of these microor-
ganisms by TC-1 were 16.5, 30.0, 11.0, and 25.1 mm2, respectively.
by guest, on May 7, 2013
much less pronounced profile, indicating major unfolding and
supporting the NMR results. Given that many antimicrobial
peptides change conformation upon binding to their target
membranes (8, 15), the structures of native and reduced TC-1
were also measured in the presence of SDS micelles. The CD
and proton NMR spectra for TC-1 indicate that its native
?-sheet structure converts into more ?-helical elements in the
of ?-helical structures (Fig. 7B). A similar membrane-bound
conformation was induced for reduced TC-1 (Fig. 7B).
Interestingly, reduced TC-1 fully retained its antibacterial
activity in the overlay assay (Fig. 6, bottom panel). This was
confirmed using a quantitative liquid bactericidal assay, in
which both folded and reduced TC-1 had an LC99.9(the lethal
concentrations killing 99.9% of an inoculum) of 4 ?M for B.
ity of TC-1 against S. aureus and only slightly reduced activity
against E. coli and against yeasts. The concentration of
reduced/alkylated TC-1 required to kill E. coli and C. neofor-
mans was 2-fold higher than of folded TC-1 (Table 1), and the
area of growth inhibition of C. albicans in a radial diffusion
assay was reduced from 24 mm2for native to 20 mm2for
unfolded TC-1. When tested at physiological salt concentra-
tions, both native and unfolded TC-1 completely lacked anti-
microbial activity (Table 1).
TC-1-derived Peptides with Antimicrobial Activity—Our
studies with the TC-1 positive patch mutants clearly showed
the importance of the three-dimensional patch for the activity
of folded TC-1. It was therefore remarkable that unfolding of
markedly affect the antimicrobial activity. Several peptides
microbial activity (30–33). To investigate the possibility that
linear regions within the protein could be responsible for the
antimicrobial activity of linearized TC-1, we screened overlap-
ping 15-mer peptides covering the entire TC-1 sequence. Pep-
tides A1 and D51 indeed had bactericidal activity, causing an
None of the other peptides tested affected bacterial survival.
The active peptides A1 and D51 corresponded to stretches in
the N- and C-terminal part of TC-1 respectively. For both
regions, 15-mers shifting one residue at a time were synthe-
substantially more potent than peptides from the C terminus
(Table 1). Peptide L3 was almost as active as rTC-1 (Table 1).
Direct antimicrobial activity of chemokines is clearly associ-
ated with a distinct positive patch at the surface of the native
folded molecule in aqueous solutions (4). These chemokines
also have hydrophobic domains, and thus have an amphipathic
peptides (34). Such an amphipathic structure is deemed essen-
tial for antimicrobial activity; a positively charged region is
required for selective interaction with negatively charged
microbial outer surfaces, whereas a hydrophobic region allows
interaction with the apolar part of microbial membranes (35,
36). The mechanism of microbicidal action of these proteins is
of microbial membranes (37) or interference with intracellular
targets after translocation across the membrane are the most
common modes of action (7, 38).
Several antimicrobial peptides and antimicrobial chemo-
kines retain activity as linearized molecules despite the loss of
their characteristic ?-sheet structures (14, 15). This questions
the requirement of the three-dimensional patch of antimicro-
of native TC-1 were left untreated (U), were reduced in DTT (R), or were
reduced and alkylated (RA). Samples were analyzed in acid urea gels, which
FIGURE 7. Influence of linearization of TC-1 on three-dimensional struc-
ture. A, one-dimensional1H NMR spectra showing the backbone amide
in aqueous solution and in the presence of SDS micelles. B, corresponding
far-UV circular dichroism (CD) spectra of the TC-1 samples.
by guest, on May 7, 2013
bial chemokines and shows that other structural elements can
also be sufficient for antimicrobial activity of these proteins.
Our goal was to investigate the structural elements involved in
antimicrobial activity of TC-1.
TC-1 is the most potent antimicrobial protein of human
platelets (2). It differs from the chemokine NAP-2/CXCL7 by
only a two-amino acid C-terminal truncation. This truncation
is required for strong antimicrobial activity (2), but it does not
atively charged C terminus folds back over the positively
charged surface and may thus interfere with antimicrobial
activity (40). The C-terminal truncation generating TC-1
reduces the negative charge of the C terminus and thus abol-
ishes interference of the C terminus with the positive patch
turn between the second and third ?-strand, in one face of the
C-terminal ?-helix, and a single residue (Lys-17) in the turn
prior to the first ?-strand. Residue Lys-17 occupies a central
a large positive patch. Disruption of the positive patch in TC-1
by substitution of the central cationic residue Lys-17 with an
alanine substantially reduced the antimicrobial potency, and
vice versa the augmentation of the positive patch enhanced the
antimicrobial activity of TC-1. These results clearly demon-
strate the importance of this positive patch for the activity of
The TC-1 K17A substitution had differential effects on the
activity against various microorganisms. This substitution
nearly abolished antifungal activity but only slightly reduced
antibacterial activity. Conversely, augmentation of the positive
patch at several positions enhanced antibacterial activity but
not antifungal activity. A similar phenomenon has been
described for variants of human ?-defensins where small mod-
ifications in amphipathic structures result in substantially
altered antimicrobial specificity (41, 42). It has been proposed
that pathogen specificity has driven evolutionary diversifica-
tion of defensin genes (43). Identification of functional sites
involved in pathogen specificity is important to improve our
understanding of the mechanism of action of antimicrobial
with enhanced activity.
In the presence of SDS micelles, TC-1 loses most of its char-
acteristic ?-sheet structure and adopts a conformation with
higher ?-helical content. Similar conformational changes have
been reported for hBD-3 and GCP-2/CXCL6 in membrane-
mimicking conditions (14, 15, 44). It has been suggested that
these structural modifications are involved in the mode of
the distinct hydrophilic positive surface patch likely is required
for association with the negatively charged outer surface of
microorganisms. Upon close contact with microbial mem-
branes, conformational changes likely result in exposure of
more apolar residues. The resulting increased hydrophobicity
could facilitate subsequent steps in the mode of action of these
molecules, i.e. to insert into or transverse the microbial
structure, indicating that the structure of native TC-1 is rigidly
held in place by two disulfide bonds. The positive patch of
native TC-1 is formed by residues of several secondary struc-
tural elements, including residues of the ?-sheet. This implies
that the patch is disrupted in reduced TC-1. Despite the lack of
a positive patch, reduced and unfolded TC-1 had antimicrobial
activity comparable with the native protein. This indicates that
unfolded TC-1 exerts antimicrobial activity via a distinct mode
Interestingly, linear 15-mer peptides corresponding to the
N-terminal part of TC-1 had antimicrobial activity similar to
that of intact TC-1. Peptides corresponding to the C-terminal
region only had activity at high concentrations. This latter
result is in accordance with the previous observation that a
peptide corresponding to the entire TC-1 C-terminal ?-helical
activity of reduced and unfolded TC-1.
Similar to TC-1, several chemokines and antimicrobial pep-
tides, including hBD-3, also retain antimicrobial activity after
linearization (9–12, 32). For instance, variants of hBD3 lacking
disulfides are still antimicrobial, despite the loss of most of the
Microbicidal activity of TC-1 and TC-derived peptides
Peptides were named after the first residue and its position in TC-1. RA indicates reduced/alkylated.
B. subtilisE. coliS. aureusC. neoformans
AELRCMC. . . . LAGDES
AELRCMC. . . . LAGDES
AELRCMC. . . . LAGDES
AELRCMC. . . . LAGDES
aTesting was done with 0.9% NaCl.
43512 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER50•DECEMBER16,2011
by guest, on May 7, 2013
structural elements of the native protein (14, 15). Like TC-1,
tein might thus be involved in the activity of linear hBD-3. In a
both cysteines of the CC sequence had similar antimicrobial
activity of the linear CCL28 variant by suggesting spontaneous
folding into the native conformation (9). As CCL28-derived
peptides have antimicrobial activity equivalent to the intact
folded protein (9), the activity of linear CCL28 might also be
due to peptide regions in the unfolded protein, similar to the
case of linearized TC-1.
Human ?-defensin 1 (hBD-1) even requires reduction and
unfolding to unmask its antimicrobial activity (13). Reduced
colocalize with thioredoxin, the enzyme system likely involved
in hBD-1 reduction (13). The necessity for unfolding to reveal
hBD-1 activity indicates that the native protein lacks the char-
acteristics required for antimicrobial activity, whereas the lin-
ear molecule does exert such activity. Possibly the reduced
protein can spontaneously adopt a conformation with antimi-
crobial activity or, alternatively, linear peptide stretches within
its sequence are responsible for activity of linearized hBD-1.
Because linearization of chemokines and antimicrobial pro-
teins occurs in vivo, linear peptides might also be generated by
proteolytic degradation of such linearized proteins. A C-termi-
proteolysis by cathepsin D (47). Thus, generation of such pep-
tides requires proteolysis. Interestingly, proteolysis is a com-
mon and important mechanism in modulation of biological
activity of chemokines (48–50). Generation of NAP-2/CXCL7
and connective tissue activating peptide-III (CTAP-III) from
their parent molecule, platelet basic protein, requires N-termi-
C terminus is required to convert NAP-2/CXCL7 and CTAP-
III into the microbicidal proteins TC-1 and TC-2, respectively
(2). Proteolysis of chemokines is also an important mechanism
to dampen inflammatory responses (48). In the case of antimi-
crobial chemokines, proteolysis would abolish the antimicro-
bial activity of the intact proteins. However, the generation of
antimicrobial peptides from processed chemokines would
allow the reduction of chemokine activity while maintaining
antimicrobial effects, constituting yet another mechanism of
chemokine-mediated fine-tuning of host defenses.
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