Review
The expanding scope of antimicrobial
peptide structures and their modes
of action
Leonard T. Nguyen, Evan F. Haney and Hans J. Vogel
Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada, T2N 1N4
Antimicrobial peptides (AMPs) are an integral part of the
innate immune system that protect a host from invading
pathogenic bacteria. To help overcome the problem of
antimicrobial resistance, cationic AMPs are currently be-
ing considered as potential alternatives for antibiotics.
Although extremely variable in length, amino acid com-
position and secondary structure, all peptides can adopt a
distinct membrane-bound amphipathic conformation.
Recent studies demonstrate that they achieve their anti-
microbial activity by disrupting various key cellular pro-
cesses. Some peptides can even use multiple
mechanisms. Moreover, several intact proteins or protein
fragments are now being shown to have inherent antimi-
crobial activity. A better understanding of the structure–
activity relationships of AMPs is required to facilitate the
rational design of novel antimicrobial agents.
Introduction
The emergence of multidrug-resistant ‘superbug’ bacteria
has created an urgent need for the development of novel
classes of antimicrobials. Unfortunately, the number of
new antibiotics in the pipeline of the big pharmaceutical
companies has been declining because they have shifted
their attention towards more lucrative areas of drug de-
velopment [1]. Since the initial discovery of antimicrobial
peptides (AMPs) in insects and animals in the 1980s, they
have been heralded as a promising alternative to today’s
antibiotics [2]. Ubiquitous in nature, AMPs form a key
component of an organism’s innate immune system. AMPs
typically have a broad spectrum of activity against patho-
genic bacteria and fungi, and often, their killing activities
extend to enveloped viruses, parasites and sometimes even
to cancerous cells. Many eukaryotic peptides act on bacte-
rial membranes or other generalized targets, in contrast to
most antibiotics, which usually target specific proteins.
This creates an advantage for AMPs because development
of microbial resistance by gene mutation is less likely [3].
AMPs obtained from higher eukaryote organisms are also
frequently referred to as ‘host defense peptides’, a term
that emphasizes their additional immunomodulatory ac-
tivities [2].
Traditionally, the search for novel AMPs involved the
identification of active peptides from natural sources. Such
studies are followed by the design of synthetic peptide
Corresponding author: Vogel, H.J. (vogel@ucalgary.ca).
464 0167-7799/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2011.05.001 Trends in Biotechnology, September 2011, Vol. 29, No. 9
analogs for structure–function studies. De novo peptide
design approaches have also been used, including high-
throughput combinatorial library screening, structure-
based modeling, predictive algorithms and the introduc-
tion of non-coded modifications to conventional peptide
chemistry [4,5]. Furthermore, AMPs are being discovered
through sequence searches for potential antimicrobial
fragments within large proteins. In fact, several proteins
have now been shown to exert direct antimicrobial activity
themselves, further widening the scope of the structural
frameworks for AMPs or antimicrobial proteins.
Generally, two physical features are common for AMPs: a
cationic charge and a significant proportion of hydrophobic
residues. The former property promotes selectivity for neg-
atively charged microbial cytoplasmic membranes over
zwitterionic mammalian membranes whereas the latter
facilitates interactions with the fatty acyl chains [6]. There
are also a few anionic AMPs, such as dermcidin, although
other biological activities seem to be more important for
these peptides [7]. Many linear AMPs are unstructured in
aqueous solution and require a membranous environment
to adopt a stable amphipathic conformation [8]. Although
the mode of action usually involves disrupting the integrity
of the bacterial cytoplasmic membrane in many ways
(Figure 1), other antimicrobial mechanisms have now been
characterized that target key cellular processes including
DNA and protein synthesis, protein folding, enzymatic ac-
tivity and cell wall synthesis [9–11]. A comprehensive list of
mechanisms utilized by AMPs is presented in Table 1.
Membrane interactions remain important even for intracel-
lular-targeting peptides because they must have a means of
translocation. The cationicity of the AMPs also promotes
interactions with negatively charged moieties on other bio-
molecules such as outer membrane lipids, nucleic acids and
phosphorylated proteins. During an infection, a host animal
may release multiple isoforms or structurally similar AMPs
that act by distinct mechanisms to achieve an overall syn-
ergistic effect [12]. Furthermore, for some of the better
studied AMPs, such as magainin and indolicidin, it has
been found that they can have multiple modes of action,
suggesting that they may work as ‘dirty’ antimicrobials that
rely on a multiple-hit strategy to increase their efficiency
and evade potential resistance mechanisms [3].
In this review, we aim to illustrate the wide diversity of
structural properties demonstrated by AMPs as well as the
growing number of ways in which they exert their activities.
(Figure_1)TD$IG][ Review Trends in Biotechnology September 2011, Vol. 29, No. 9

Toroidal pore

Disordered toroidal pore

Carpet model

+ Δd
+ +

Membrane
Membrane Conformational thinning/thickening
Barrel stave
adsorption change
+ + + +

0.2V

?
Electroporation
Charged lipid clustering
Bacterial membrane
ΔΨ

O
Non-lytic membrane
depolarization + + H
+ Non-bilayer intermediate

- -
Oxidized
Anion carrier lipid targeting
TRENDS in Biotechnology

Figure 1. Events occurring at the bacterial cytoplasmic membrane following initial antimicrobial peptide (AMP) adsorption. These events are not necessarily exclusive of
each other. In the classical models of membrane disruption, the peptides lying on the membrane reach a threshold concentration and insert themselves across the
membrane to form either peptide-lined pores in the barrel-stave model, solubilize the membrane into micellar structures in the carpet model, or form peptide-and-lipid-
lined pores in the toroidal pore model. In the revised disordered toroidal pore model, pore formation is more stochastic and involves fewer peptides. The thickness of the
bilayer can be affected by the presence of the peptides, or the membrane itself can be remodeled to form domains rich in anionic lipids surrounding the peptides. In more
specific cases, non-bilayer intermediates in the membrane can be induced; peptide adsorption to the membrane can be enhanced by targeting them to oxidized
phospholipids; a peptide may couple with small anions across the bilayer, resulting in their efflux; the membrane potential can be dissipated without other noticeable
damage; or conversely, in the molecular electroporation model, the accumulation of peptide on the outer leaflet increases the membrane potential above a threshold that
renders the membrane transiently permeable to various molecules including the peptides themselves.

Recent structure–function relationship studies will be pre-
sented within the framework of the three AMP structural
groups: a-helical peptides, b-sheet peptides and extended
peptides (Figure 2) [13].
a-Helical AMPs
The a-helical AMPs, such as magainin from the African
clawed frog, are amongst the most intensely studied AMPs
[14,15]. Numerous structure–function studies have indicat-
ed that membrane-bound a-helices with too large a hydro-
phobic surface become cytotoxic to mammalian cells [16].
Although these peptides are characterized by their struc-
ture, an extremely high propensity for the formation of the
a-helix, which may present a more continuous hydrophobic
surface, can become a contributing factor to cytotoxicity [17].
Indeed, peptides that remain antimicrobial but have low
cytotoxicity require a membranous environment to induce
proper folding and often have a proline- or glycine-induced
kink in the middle of the a-helix. The amphipathicity of the
peptides is usually segregated along the axis of the a-helix
such that the peptide can lie parallel to the membrane plane
during the initial lipid interactions, with the charged side
facing outward towards the phospholipid head groups and
the hydrophobic side embedded into the acyl tail core. The
properties of the a-helix tails sometimes influence the depth
of membrane insertion and, in turn, the antimicrobial activ-
ity [18].
The lengths of the a-helices are usually sufficient to span
the thickness of the membrane bilayers, with mismatches
between the hydrophobic portion of the helices and the lipid
acyl core strongly influencing the AMPs’ lateral and orien-
tational motions in the membranes [19,20]. The peptides are
thought to disrupt bacterial membranes through the forma-
tion of toroidal pores composed of loosely associated peptides
with interdigitating phospholipid head groups among them
[10]. Pore formation is often associated with a local change in

465
Review Trends in Biotechnology September 2011, Vol. 29, No. 9

Table 1. Different modes of action that have been described for AMPs
Major target Specific target/mode of action Example peptides a Refs
External proteins Autolysin activation Pep5, nisin [10]
Phospholipase A2 activation Magainin 2, indolicidin [10]
Outer surface lipids LPS permeabilization (Gram-negative) Cecropin P1 [69]
Lipid II (peptidoglycan precursor) Defensins, Nisin and other lantibiotics [34,35]
[70]
Outer membrane proteins (Gram-negative) Outer membrane protein I (OprI) SMAP-29, CAP-18 [71]
LPS-assembly protein D (LptD) inhibition Protegrin I peptidomimetic analogs [33]
Outer membrane protein F (OmpF) HP(2–20) analog [72]
Inner membrane Barrel-stave pore Alamethicin [10]
Detergent micellization Dermaseptin, cecropin [10]
Toroidal pore Magainin 2, melittin [10]
protegrin I [31]
Disordered toroidal pore Magainin analog, melittin [22]
Membrane thinning/thickening PGLa, LL-37 [21]
Charged lipid clustering Magainin analogs, Arg-rich peptides [26]
Non-bilayer intermediate formation Gramicidin S [73]
Oxidized phospholipid targeting Temporin L, indolicidin [27]
Anion carrier Indolicidin [49]
Non-lytic membrane depolarization Bovine lactoferricin, daptomycin [32]
[74]
Electroporation NK-lysin [42]
Integral membrane proteins Proton translocation-related proteins Clavanin A [28]
Nucleic acids DNA (general) Buforin 2, tachyplesin, indolicidin [10]
DNA (covalent interaction) Indolicidin [50]
Branched DNA WRWYCR [43]
RNA (general) Buforin 2 [10]
Intracellular proteins DnaK inhibition, GroEL chaperonin Pyrrhocoricin, drosocin, apidaecin [10]
20S proteasome, SH3-containing proteins PR-39 [46]
[47]
a
Peptides listed are not limited to their associated modes of action here and may employ multiple mechanisms simultaneously.

membrane thickness [21]. Recent molecular dynamics (MD)
simulations with a magainin analog and with melittin have
suggested that a re-evaluation of this model is in order
[22,23]. In the ‘disordered toroidal pore’, lipid molecules
are still curved inwards, but pore formation is more stochas-
tic and only one or two peptides are located near the center of
the water-permeable pore, with more peptides positioned at
the mouth of the pore on the external leaflet. As they
translocate to the interior of the cell, the peptides remain
mostly parallel to the bilayer plane, and only a small tilt is
observed. Interestingly, the peptides undergo partial
unfolding during translocation and their a-helicity is not
always maintained. In other MD simulations, similar tilted
orientations for a-helical peptides were observed and the
notion of imperfect amphipathicity was introduced [24].
This refers to the presence of some polar or charged residues
in the hydrophobic face of an AMP to pull lipid head groups
into the membrane interior, thereby facilitating pore forma-
tion. A perfect hydrophobic face on a peptide would favor a
fixed parallel orientation on a flat membrane surface. Im-
perfect amphipathicity is also a highlighted feature in the
proposed interfacial activity model [25], which is based on a
recent review of AMP literature. It closely resembles the
disordered toroidal pore model, with the key difference of the
interfacial activity model describing membrane disruption
without a need for peptide self-assembly.
The carpet model, where AMPs act more in a detergent-
like manner to break off the membrane lipids into micelle-
like structures, has also been described. This model
appears to be applicable mostly to the high concentrations
used in vitro, and is generally viewed as an extreme
extension of the toroidal pore model [10].
An interesting membrane perturbing effect induced by
some a-helical AMPs is that of lipid segregation, where
peptide-induced clustering of anionic lipids in bacterial
membranes causes slow leakage of intracellular contents
and/or membrane depolarization [26]. Alternatively, the
phase boundary effects between domains of differently
charged lipids may compromise the overall membrane
stability. Moreover, many bacterial membranes are mostly
composed of phosphatidylglycerol and phosphatidyletha-
nolamine lipids, where the latter does not like to form
stable bilayers after peptide-induced demixing. In the case
of Gram-positive bacteria such as Staphylococcus aureus,
which have cytoplasmic membranes that are mainly com-
posed of the negatively charged lipids phosphatidylglycerol
and cardiolipin, lipid segregation is not expected and this is
reflected in lower toxicity of some AMPs against these
species [26].
The targeting of oxidized lipids represents a unique
membrane-associated means for AMPs to enhance their
activity. The a-helical AMPs temporin B and L intercalate
more efficiently into membranes containing an oxidized
phosphatidylcholine lipid, probably via Schiff base forma-
tion between the peptide amino groups and lipid aldehyde
groups [27]. With the release of reactive oxygen species
during phagocytosis, it is possible that lipid oxidation could
increase bacterial membrane susceptibility to AMPs.

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(Figure_2)TD$IG][ Review Trends in Biotechnology September 2011, Vol. 29, No. 9

(a)

LL-37 Bovine
Magainin 2 lactoferrampin

(b)

Bovine lactoferricin Protegrin I

Human β-defensin-3

(c)

RRWQWR
Indolicidin
Tritrpticin
TRENDS in Biotechnology

Figure 2. An overview of the major structural classes of antimicrobial peptides (AMPs). (a) a-Helical peptides, (b) b-sheet peptides and (c) extended peptides. All of these
structures were solved by solution NMR spectroscopy in the presence of detergent micelles, except for the b-sheet peptides, which were studied in aqueous solution.
Positively charged side chains are colored in blue, negatively charged side chains in red and remaining side chains in grey. PDB IDs: magainin 2, 2MAG; LL-37, 2K6O; bovine
lactoferricin, 1LFC; protegrin 1, 1PG1; human b-defensin-3, 1KJ5; tritrpticin, 1D6X; indolicidin, 1G89.

Finally, the Phe-, His- and Gly-rich peptide clavanin A
has a unique dual pH-dependent mode of action [28]. At
neutral pH, it permeabilizes membranes like regular a-
helical AMPs. At slightly acidic pH, clavanin A is still
membrane disruptive. However, this does not occur via
lipid interactions, but rather through inhibitory interac-
tions with proteins involved in maintaining the pH gradi-
ent across the membrane.
In contrast to most a-helical peptides, buforin II, a
peptide derivative from histones of the Asian toad Bufo
bufo gargarizans, does not cause membrane disruption [9]
although its cell entry can be affected by membrane lipid
composition [29]. After translocation, buforin II accumu-
lates in the cytoplasm where it is known to exert its
antimicrobial activity by adapting an extended or polypro-
line II conformation to accommodate interactions with
nucleic acids [30].
b-Sheet AMPs
The b-sheet AMPs include several b-hairpin peptides in
addition to the defensin mini-proteins. Many of these
peptides disrupt bacterial membranes via the formation
of toroidal pores as observed for the porcine peptide prote-
grin I. This peptide forms oligomeric transmembrane b-
barrels in anionic membranes, but forms b-sheet aggre-
gates on the surface of cholesterol-containing membranes
[31].
Additional evidence suggests that non-lytic mecha-
nisms are also employed by certain b-sheet peptides. Al-
though tachyplesin from horseshoe crabs is generally
recognized as a membrane active peptide, it is also capable
of binding to the minor groove of DNA, which may interfere
with DNA–protein interactions [10]. The b-hairpin peptide
bovine lactoferricin can act synergistically with other an-
timicrobial agents by affecting the transmembrane poten-
tial and proton-motive force, resulting in inhibition of ATP-
dependent multi-drug efflux pumps [32]. Following trans-
location, bovine lactoferricin localizes to the cytoplasm
where it can also inhibit DNA, RNA and protein synthesis.
A series of peptidomimetic analogs of protegrin I featuring
stabilized b-hairpins are no longer membrane active, but
employ a new bactericidal mechanism involving a specific
protein target that affects outer membrane biosynthesis
[33]. Although these peptide analogs are remarkably po-
tent, their spectrum of activity is narrowed to Pseudomo-
nas aeruginosa strains, which express the susceptible
homolog of this b-barrel protein. Finally, although the
human defensins are membrane active, some of them
can also bind to the peptidoglycan precursor lipid II to
inhibit cell wall biosynthesis enzymes in Staphylococci
[34,35].
The b-hairpin or b-sheet conformations of these pep-
tides are often stabilized by disulfide bridges between
conserved Cys residues. However, these covalent bonds

467
Review
do not appear necessary for antimicrobial activity. Linear
derivatives of tachyplesin and lactoferricin retain antimi-
crobial activity while losing hemolytic activity [32,36].
Solid state NMR studies of tachyplesin and two linearized
analogs, in which the four Cys residues were substituted by
either Phe or Ala residues, show that all three peptides are
positioned at the membrane interface and adopt similar b-
sheet conformations on anionic membranes [37]. However,
this does not correlate with their activities: the Cys-to-Phe
peptide is equally active as tachyplesin whereas the Cys-
to-Ala peptide is inactive. Peptide dynamics, rather than
their structures, provide a reasonable explanation for
these functional differences. Whereas the active tachyple-
sin peptide shows enough mobility in the liquid-crystalline
phase of the membrane to disturb lipid packing, the inac-
tive analog is immobilized on the membrane and stays
highly aggregated.
At 45 residues, human b-defensin-3 (HBD-3) is rather
large as an AMP, containing an a-helical turn followed by a
three-stranded antiparallel b-sheet. This scaffold is held
together by three disulfide bonds in a defined pattern that
distinguishes the b-defensins from the a-defensins. None-
theless, rearranging the disulfide pattern in HBD-3 or
reducing them does not alter the antimicrobial activity,
nor does it abolish the overall formation of the native
secondary structure elements [38,39]. Although antimicro-
bial activity may be unaffected, chemotactic activities are
decreased for the analogs with non-native disulfide lin-
kages.
Interestingly, reducing the disulfides in HBD-1 creates
a flexible unstructured peptide with improved activities
against Candida albicans and some Gram-positive bacte-
ria [40]. The presence of reduced HBD-1 could be of great
physiological relevance in the anaerobic environment of
the colon.
Extended AMPs
Extended AMPs, which do not fold into regular secondary
structure elements, often contain high proportions of cer-
tain amino acids, specifically Arg, Trp or Pro residues. The
His-rich salivary histatin peptides are sometimes consid-
ered to be extended AMPs, however they are not bacterio-
static nor bactericidal despite being fungicidal and anti-
parasitic [41]. Many of the extended peptides are not
membrane active. The Pro-rich insect-derived pyrrhocor-
icin, drosocin and apidaecin peptides penetrate across the
membrane and exert their antimicrobial activities by mak-
ing interactions with intracellular proteins such as the
heat-shock protein DnaK and GroEL to inhibit the DnaK
ATPase activity and chaperone-assisted protein folding,
respectively [10].
Trp and Arg side chains are prominent in peptides with
less than 15 residues [42]. Two of the shortest active
peptides include RRWQWR and RAWVAWR, which have
been identified as the active antimicrobial fragments from
bovine lactoferricin and human lysozyme, respectively. In
addition, combinatorial peptide library screening has iden-
tified the hexameric sequence RRWWRF as having potent
broad spectrum activity [4]. Although these peptides can
fold into defined amphipathic molecules in the presence of
a membrane, they are not membrane active and, instead,
468
Trends in Biotechnology September 2011, Vol. 29, No. 9
they accumulate in the cytoplasm. The hexapeptide
WRWYCR, which was identified as a DNA-repair inhibitor
that binds to Holliday junctions, was subsequently shown
to have broad bactericidal activity [43]. Given the sequence
similarity of this peptide to those mentioned above, their
efficacy may be due to this mechanism, where trapping of
the replication fork prevents recombination and DNA
repair.
The 13-residue Arg- and Trp-rich tritrpticin and indo-
licidin peptides from porcine and bovine leukocytes, re-
spectively, can cause membrane leakage [42]. In a
membranous environment, the structures of both peptides
feature multiple Pro-induced turns that give them amphi-
pathic boat-like conformations. These peptides are suffi-
ciently small that simple residue substitutions can have
large consequences for their structural and functional
properties. Replacing both Pro residues with Ala in tritrp-
ticin transforms it into an a-helical peptide with slightly
improved membrane leakage and antimicrobial activity,
but also higher cytotoxicity [44]. The role of the two Pro
residues is distinct, with Pro5 having more of a structural
role preventing the induction of a-helical structure. Ironi-
cally, the characteristic Arg and Trp side chains do not
seem an optimal choice of residues for tritrpticin. In ana-
logs with Arg-to-Lys and Trp-to-Phe substitutions, the
antimicrobial activity is maintained and the slight hemo-
lytic activity of tritrpticin is abolished. The reduced hydro-
gen bonding capabilities of Lys compared to the Arg
guanidinium group seem to increase membrane selectivity
for the peptide. Replacement of the three Trp residues with
Phe created a tritrpticin variant that was no longer able to
adopt a single well-defined conformation in a membrane
[44]. Interestingly, this peptide required a few hours to
take effect whereas native tritrpticin completes bacterial
killing in less than one hour [45]. The fact that multiple
mechanisms are in play is further substantiated by the
synergistic effects observed between the Trp-to-Phe and
membrane active Pro-to-Ala peptides [45]. The Pro- and
Arg-rich porcine PR-39 peptide interacts with cytoplasmic
proteins such as the 20S proteasome and SH3-domain
proteins, and it is believed that this contributes to its
antimicrobial mode of action [46,47].
Indolicidin has been a template for many analogs with
improved biological properties, the most successful one
being omiganan, which was developed by Migenix
(renamed to Biowest Therapeutics) and has entered phase
III clinical trials [48]. A number of contributing mecha-
nisms may explain the efficiency of indolicidin. This
includes the activation of phospholipase A2 secreted by
human lacrimal fluid to hydrolyze anionic membranes [10]
or a direct membrane action either through lipid segrega-
tion, the targeting of oxidized phospholipids or acting as a
small anion carrier across the membrane [27,49]. More-
over, at the intracellular level, both covalent and non-
covalent DNA–indolicidin interactions can interfere with
DNA-binding enzymes and induce filamentation in Escher-
ichia coli [50]. It is unclear how the short and unique
sequence of indolicidin, which includes five Trp residues
and three Pro-induced turns, can exhibit all these activities
and which of these proposed mechanisms contributes most
to its activity in vivo.
 Review
Protein-derived antimicrobial fragments and
antimicrobial proteins
AMPs can be derived from large proteins. In addition,
several cationic proteins have been discovered to possess
direct nonenzymatic antimicrobial activity themselves,
some of which are shown in Figure 3. The primary function
of these antimicrobial proteins is usually immunity-relat-
ed, therefore they show significant functional overlap with
AMPs (Table 2).
Lactoferrin (Lfin) is a multifunctional 80 kDa glycopro-
tein present in biofluids, most notably in milk and neutro-
phil granules. The protein is involved in several
immunoregulatory processes that primarily take advan-
tage of its strong antimicrobial properties. This activity is
due in part to its ability to sequester iron, which prevents
bacterial growth. However, Lfin also has an iron-indepen-
dent mechanism arising from direct interactions with
bacterial cell components [51]. Three cationic antimicrobi-
al fragments have been identified in different parts of the
Lfin sequence: lactoferricin, which is a naturally occurring
pepsin digestion product, lactoferrampin and kaliocin
(Figure 3a) [18,32,52]. The structural differences for the
isolated peptides and the corresponding regions in Lfin are
significant; in particular, bovine lactoferricin and kaliocin
form amphipathic b-hairpin peptides, whereas lactofer-
rampin requires a membraneous environment to adopt
its native a-helical structure. The intact protein and the
peptides probably kill bacteria in a different manner,
especially lactoferricin, which is substantially more active
than intact Lfin.
[(Figure_3)TD$IG]
(a)
(b)
Figure 3. The structures of immunity-related proteins with direct antimicrobial activity and antimicrobial fragments. (a) Human lactoferrin (PDB ID 1FCK) with segments
corresponding to its antimicrobial fragments: lactoferricin in blue, kaliocin in red and lactoferrampin in yellow (left panel). The electrostatic potential surface plot of
lactoferrin is shown in the right panel. (b) Macrophage inflammatory protein-3a (PDB ID 2JYO) with the MIP-3a51–70 peptide segment colored in blue (left panel). The
electrostatic potential surface plot of the monomeric unit of MIP-3a is shown in the middle panel and the same plot for the MIP-3a dimer is in the right panel, showing the
stronger cationic surface around the self-associating a-helices.
Trends in Biotechnology September 2011, Vol. 29, No. 9
Whether they are naturally released proteolysis pro-
ducts or designed synthetic peptides, many AMPs have
been derived from proteins such as casein [53], lysozyme
and ovotransferrin [54], myoglobin and hemoglobin [55],
histones [9], growth factors [56], and thrombin [57]. Thus,
scanning the proteome for related sequences with appro-
priate combinations of cationic and hydrophobic residues
may uncover additional AMPs. Some of these proteins such
as lysozyme and histones already have intrinsic antimi-
crobial activity and, as is the case for Lfin, their peptide
fragments are expected to have different modes of action
[58].
Recently, more than 20 human chemokine proteins have
been shown to be directly antimicrobial, with macrophage-
inflammatory protein-3a (MIP-3a) possessing the most
potent and broadest spectrum of activity (Figure 3b)
[59]. In the case of the chemokine CCL28, the antimicrobial
mechanism has been identified as membrane permeation
[60]. Conversely, a few defensins are now known to possess
chemotactic activity [61]. A common and unifying feature
of the antimicrobial chemokines is the presence of a large
uninterrupted cationic surface. Some chemokines tend to
dimerize under physiological conditions, further extending
the cationic surface [62]. For example, HBD-3 has a stron-
ger tendency to dimerize, which may contribute to its high
salt-insensitive activity compared to other b-defensins
[63]. The C-terminal a-helices of several antimicrobial
chemokines have multiple positively charged residues
and when synthetic peptides of this region are studied,
they behave like membrane-active a-helical AMPs [64].
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Table 2. A selection of human AMPs and antimicrobial proteins highlighting their multifunctionality in the immune system
Main sources Indirect antimicrobial and additional functions a Refs
AMPs
LL-37 Leukocytes, epithelia Endotoxin neutralization, chemotaxis, reactive [61]
oxygen species (ROS) formation, wound healing,
mast cell degranulation
Defensins Leukocytes, epithelia Endotoxin neutralization, opsonization, chemotaxis, [61]
ROS formation, wound healing, complement inhibition
Dermcidin Skin Chemotaxis [7]
Histatins Saliva Wound healing [75]
Lactoferricin Digested milk protein Endotoxin neutralization, complement [32]
inhibition, transcription factor
Antimicrobial proteins
Chemokines Leukocytes Chemotaxis [59]
Neutrophil serprocidins Neutrophils Protease, ROS formation, opsonization, [61]
(azurocidin, cathepsin G, elastase) macrophage activation
Bactericidal/permeability Neutrophils Endotoxin neutralization [61]
increasing protein
Lipocalins Neutrophils, secretory fluids Siderophore sequestering [76]
Lysozyme Secretory fluids, neutrophils Peptidoglycan digestion [61]
Lactoferrin Secretory fluids, neutrophils Iron sequestering, endotoxin neutralization, [61]
phagocytosis stimulation
RNAse 7 Skin Ribonuclease [77]
Psoriasin Skin Neutrophil activation, ROS formation [77]
Histones Nuclei Chromatin regulation [78]
Prion protein Brain, other tissues Unknown [79]
a
These are usually secondary functions for AMPs and primary functions for antimicrobial proteins.

This property may explain the bactericidal properties of
several intact chemokines; however, some of the a-helical
peptides do not retain activity and, furthermore, some
chemokines do not have cationic C-terminal regions de-
spite displaying activity. For the platelet chemokine neu-
trophil activating protein-2 (NAP-2), the proteolytic
removal of the two C-terminal Ala-Asp residues leads to
a drastic increase in the antimicrobial potency. This was
attributed to the negatively charged Asp side chain inter-
fering with a positive patch on the protein surface that
includes side chains from the C-terminal a-helix as well as
the b-sheet loops [65].
Concluding remarks
Widely different structural scaffolds seem to support the
activity of AMPs and proteins, with amphipathicity remain-
ing as the only overarching physical property. A combina-
tion of a well-defined cationic region together with an
imperfect hydrophobic surface area frequently seems to
result in broad spectrum activity. In addition, the dynamic
properties of these peptides can affect their biological prop-
erties, with structural rigidity generally hindering activity.
Initially, AMPs were considered as a uniform group of
molecules that ‘punch holes’ in bacterial membranes. In
recent years, it has become apparent that their mechanism
of action is much more complex and diverse. Current re-
search efforts now also include antimicrobial fragments
from intact proteins and proteins with direct antimicrobial
activity, forcing structural biology researchers to reevaluate
the common properties within this expanding family. Clear-
ly, host defense systems have developed a broad arsenal and
use a multi-pronged strategy to combat invading pathogens.
Many AMPs seem to be capable of killing bacterial cells in
multiple ways, making it a difficult task to unravel all the
molecular events that occur when a peptide encounters a
bacterial cell. Consequently, the design and prediction of
peptide analog characteristics is not straightforward and all
peptides and analogs need to be studied on a case-by-case
basis. Apart from their biological activities, other aspects
need to be considered when developing these peptides for
systemic use, including in vivo stability [66], side effects and
production costs. Based on our current understanding of the
structure–function relationships for different classes of
AMPs, some non-peptide and peptidomimetic compounds
have been developed and have advantageous properties
because they combine stability with high antimicrobial
potency [67,68]. Overall, detailed structure–function and
biophysical studies can rationalize why a certain peptide
is more active or behaves differently from a related peptide
or an analog. Such comprehensive understanding will be
essential to guide the design of better AMPs.
Acknowledgments
Research on AMPs in the H.J. Vogel laboratory is supported by a grant
from the Novel Alternatives for Antibiotics Program of the Canadian
Institute for Health Research. H.J.V. is the holder of a Scientist award
from the Alberta Heritage Foundation for Medical Research.
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