Journal of Insect Physiology 95 (2016) 51–65

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Journal of Insect Physiology

journal homepage: www.elsevier.com/locate/jinsphys

Review

A look inside odorant-binding proteins in insect chemoreception

Nathália F. Brito a, Monica F. Moreira a,b, Ana C.A. Melo a,b,⇑
a
Universidade Federal do Rio de Janeiro, Instituto de Química, 21941-909 Rio de Janeiro, RJ, Brazil
b
Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Detection of chemical signals from the environment through olfaction is an indispensable mechanism for
Received 13 May 2016 maintaining an insect’s life, evoking critical behavioral responses. Among several proteins involved in the
Received in revised form 13 September 2016 olfactory perception process, the odorant binding protein (OBP) has been shown to be essential for a nor-
Accepted 14 September 2016
mally functioning olfactory system. This paper discusses the role of OBPs in insect chemoreception. Here,
Available online 14 September 2016
structural aspects, mechanisms of action and binding affinity of such proteins are reviewed, as well as
their promising application as molecular targets for the development of new strategies for insect popu-
Keywords:
lation management and other technological purposes.
Odorant-binding protein
Insect
Ó 2016 Elsevier Ltd. All rights reserved.
Review
Olfactory system
Chemical communication

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2. Molecular organization of insect peripheral olfactory system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3. Detection of semiochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4. Expression in non-sensory tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
5. Physicochemical properties and structural aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
6. Three-dimensional structure-based mechanism of action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
7. Activation of ORs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
8. Binding affinity and selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
9. Applied research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
10. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

1. Introduction

Interpretation of chemical signals from the environment is an

essential mechanism for maintaining insect life. It is well
Abbreviations: OBP, odorant-binding protein; OR, odorant-receptor; PBP, established that insects use a variety of semiochemicals – such
pheromone-binding protein; PR, pheromone receptor; GOBP, general odorant- as pheromones, plant volatiles and animal odorants – to find one
binding protein; SNMP, sensory neuron membrane protein; IR, ionotropic receptor;
CSP, chemosensory protein; ODE, odorant-degrading enzyme; GPCR, G protein-
another, besides locating food sources, oviposition sites, mating
coupled receptor; ORCo, odorant receptor co-receptor; cVA, cis-vaccenyl acetate; partners and suitable hosts, in addition to identifying predators,
DEET, diethyltoluamide; AMA, 1-aminoanthracene; 1-NPN, N-phenyl-1- among other functions (Zwiebel and Takken, 2004). Semiochemi-
naphthylamine; ANS, 1-anilino-8-naphthalenesulfonic acid. cals are perceived through olfaction, the sensory modality which
⇑ Corresponding author at: Universidade Federal do Rio de Janeiro, Instituto de
is responsible for the transduction of the olfactory signal, evoking
Química, 21941-909, Rio de Janeiro, RJ, Brazil.
E-mail address: anamelo@iq.ufrj.br (A.C.A. Melo).
behavioral responses that are critical for survival and reproduction

http://dx.doi.org/10.1016/j.jinsphys.2016.09.008
0022-1910/Ó 2016 Elsevier Ltd. All rights reserved.
52 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65

of insects. In order to initiate signal transduction, it is necessary
that these hydrophobic molecules get across the hydrophilic sen-
sillum lymph surrounding sensory neurons and stimulate specific
dendrites (Fan et al., 2011), where olfactory receptors (ORs) are
located on the membrane surface (Breer, 2003). Inside olfactory
sensilla (delicate hair-like porous cuticular structures located on
antennae), odorant-binding proteins are synthesized by special-
ized accessory cells and secreted in the sensillar lymph, where they
are found in high concentrations (Klein, 1987; Vogt et al., 1985).
Although olfaction is not yet completely understood at molecular
level, many studies indicate these proteins participate in the chem-
ical communication process and constitute the first line of olfac-
tory proteins involved in odorant recognition. Several functions
have been proposed for odorant-binding proteins, among which
protection of odorant molecules from the action of odorant-
degrading enzymes and transport of hydrophobic semiochemicals
through the sensillum lymph to the odorant receptors are the most
studied. Odorant-binding proteins are also found in vertebrates;
these, however, belong to the lipocalin family and show no struc-
tural similarity to the insect OBPs (Bianchet et al., 1996).
Even though information regarding insect odorant-binding
proteins have been summarized in excellent reviews over the last
years (Fan et al., 2011; Leal, 2013; Pelosi et al., 2006, 2014; Venthur
et al., 2014; Vieira and Rozas, 2011; Zhou, 2010), knowledge about
this subject has been constantly increasing. Therefore, this review
aims to provide the reader an up-to-date integrated scenario on
what has been described for insect OBPs so far. Also, this paper
dedicates an entire section to discussing current ideas and perspec-
tives for applied research involving insect OBPs, an important topic
which seems not to be taken into account in the majority of
published reviews.
2. Molecular organization of insect peripheral olfactory system
Upon encountering the antennae, odorants are able to penetrate
the cuticle wall through multiple pores present at the surface of
sensilla and reach the aqueous environment of the sensillar lymph,
rich in soluble proteins. Odorant-binding proteins (OBPs), odorant
receptors (ORs), ionotropic receptors (IRs), sensory neuron
membrane proteins (SNMPs), chemosensory proteins (CSPs) and
odorant-degrading enzymes (ODEs) are the main proteins of the
peripheral olfactory system involved in the odorant reception pro-
cess (Leal, 2012).
OBPs are responsible for the connection between the external
environment and ORs (Leal, 2005). Once odorants penetrate the
pore tubules of sensilla, they are bound and solubilized by OBPs,
transported through the sensillar lymph and finally reach sensory
dendrites, where they activate membrane-bound ORs (Fig. 1).
The number of genes encoding putative OBPs is very variable
across different insect orders, with some species having up to sev-
eral tens of them (Gong et al., 2009a; Hekmat-Scafe et al., 2002;
Manoharan et al., 2013). The OBP-family also includes proteins that
specifically bind and transport pheromones to pheromone recep-
tors (PRs), called pheromone-binding proteins (PBPs). Regarding
OBPs mode of action, two not necessarily mutually exclusive
hypothesis stand out: although there is evidence in moths and,
mainly, in mosquitoes, that OBPs act as passive carriers and the
ligand per se activates its OR (Damberger et al., 2007; Horst et al.,
2001; Sandler et al., 2000; Wojtasek and Leal, 1999), in some cases
OBPs seem to play a more direct role, where the formation of a
specific OBP-ligand complex is necessary for receptor activation
(Laughlin et al., 2008; Ronderos and Smith, 2010; Xu et al., 2005).
CSPs belong to another family of small soluble proteins identi-
fied across multiple insect orders. They are smaller than OBPs

Fig. 1. Schematic view of the odorant perception process in insects. Once odorants penetrate pore tubules of the sensillum, they are bound and solubilized by OBPs,
transported through the sensillum lymph and finally reach sensory dendrites, where they activate membrane-bound ORs. Signal transduction evokes a response behavior to
the detected stimulus and stray odorants are rapidly degraded. For further information, see Sanchez-Gracia et al. (2009). Details regarding different mechanisms of ligand
binding and release by OBPs are not represented in this scheme and are discussed in another section.
 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65
and can bind a wide range of chemicals. The immunocytochemical
localization of these proteins in the hemolymph of olfactory sen-
silla suggests they could be involved in insect chemical signaling.
In ants, CSPs are highly expressed in antennae, where they might
play a role in nest mate recognition signals (Gonzalez et al.,
2009; Ozaki et al., 2008). However, CSPs have a larger tissue distri-
bution when compared with OBPs and several members of this
group are expressed in non-sensory organs where they seem to
be involved in different functions such as development and differ-
entiation (Jacquin-Joly et al., 2001; Maleszka et al., 2007; Nomura
et al., 1992; Zhou et al., 2013). In cockroaches, for instance, p10, a
protein of this class, was reported to promote the regeneration of
legs (Kitabayashi et al., 1998; Nomura et al., 1992).
In contrast to soluble OBPs and CSPs, SNMPs genes encode a
transmembrane protein which represents a subclade of the CD36
gene family in insects (Nichols and Vogt, 2008) and several studies
support a role for SNMPs in pheromone-based chemical communi-
cation, where an PBP passes the pheromone to the SNMP, which
then delivers it to the receptor. SNMP1 from Drosophila melanoga-
ster was demonstrated to be essential for detecting the sex phero-
mone Z11-18OAc (also known as cis-vaccenyl acetate or simply
cVA) in T1 sensilla (Benton et al., 2007; Jin et al., 2008), while, in
moths, SNMP1s are associated with sex pheromone-detecting neu-
rons in antennae (Forstner et al., 2008; Rogers et al., 2001; Thode
et al., 2008).
The OR consists of a seven transmembrane domain protein
which is not homologous to vertebrate GPCR receptors. Insect
ORs adopt an inverse membrane topology when compared to clas-
sical GPCRs, with the N-terminal region located intracellularly
(Benton et al., 2006; Buck and Axel, 1991). ORs are usually part
of a heteromeric complex comprising at least two subunits: a
ligand-specific OR and a highly conserved co-receptor (ORCo)
(Vosshall and Hansson, 2011). Molecules specifically bind the OR,
so the ORCo is not directly involved in odorant recognition
(Guidobaldi et al., 2014; Larsson et al., 2004). Heteromeric ORCo
complexes form ion channels induced by odorant binding to the
OR (Sato et al., 2008; Wicher et al., 2009), which leads to signal
transduction.
IRs are related to ionotropic glutamate receptors, even though
both families are divergent given the fact that important residues
for glutamate binding are not present in insect IRs. These receptors
are expressed along with a co-receptor in sensory neurons that do
not express ORs or ORCo and, in contrast to ORs, there are different
co-receptors for IRs (Rytz et al., 2013). Although functional proper-
ties of IRs have not yet been studied in heterologous expression
systems, their localization, and structural features suggest they
are chemosensory receptors that might function as ligand-
induced ion channels (Benton et al., 2009).
For insects whose navigation is guided by odorants, kinetics of
the olfactory system requires stray odorant molecules to be quickly
inactivated, on a millisecond timescale (Ishida and Leal, 2005).
There are two competing hypotheses currently trying to explain
signal termination. The first one indicates ODEs participate in
ligand degradation in a way so fast that it terminates the signal
(Ishida and Leal, 2005, 2008). The second hypothesis defends the
existence of still unknown ‘‘scavengers” (Kaissling, 2009; Leal
and Kaissling, 2004) responsible for signal termination.
3. Detection of semiochemicals
OBPs role in the transport of molecules in insect antennae was
described for the first time in Lepidoptera, using male Antheraea
polyphemus antennae (Vogt and Riddiford, 1981). A few years later,
a study with D. melanogaster mutants provided evidence of the par-
ticipation of OBPs in the presentation of odorants to ORs (Kim
53
et al., 1998). It had already been demonstrated three years before
that, in Phormia regina, the electrophysiological answer of gusta-
tive cells was completely abolished in the presence of anti-OBP
antibody (Ozaki et al., 1995).
Although OBPs binding affinity for different ligands has been
determined for several species since 1981, evidence that these pro-
teins are essential for a normally functioning olfactory system was
only obtained much more recently. Knockdown studies have
demonstrated that OBP DmelOBP76a (or LUSH) is necessary in
the olfactory process of D. melanogaster (Laughlin et al., 2008; Xu
et al., 2005). In experiments where pheromones were solubilized
with PBPs, threshold responses from corresponding receptors were
two to three orders of magnitude lower than when pheromones
were solubilized with DMSO (Forstner et al., 2009; Grosse-Wilde
et al., 2006, 2007). Similar results were obtained in vivo by express-
ing the bombykol receptor from Bombyx mori, BmorOR1, in the
‘‘empty neuron” of D. melanogaster: sensitivity to bombykol was
significantly enhanced when BmorOR1 was co-expressed with a
silkworm PBP, BmorPBP1 (Syed et al., 2010). Furthermore, behav-
ioral assays with Drosophila mutants (Matsuo et al., 2007;
Swarup et al., 2011) and aphids (Qiao et al., 2009; Sun et al.,
2012a) have also indicated OBPs are, indeed, engaged in the semio-
chemical detection process. Results of a study involving different
combinations of PBPs and pheromone receptors (PRs) from Chilo
suppressalis implicate PBPs are crucial for the olfactory response
and that PRs sensitivity to pheromones is enhanced by up to four
orders of magnitude in the presence of PBPs. The authors suggest
the correct combination between OBP and OR is fundamental for
the detection of a specific odorant (Chang et al., 2015).
Functions assigned to OBPs have been described by biochemi-
cal, biophysical, structural biology and kinetic studies (Horst
et al., 2001; Leal et al., 2005a,b; Mao et al., 2010; Sandler et al.,
2000; Wogulis et al., 2006; Wojtasek and Leal, 1999; Xu and
Leal, 2008), as well as using interference RNA and electrophysiol-
ogy approaches (Biessmann et al., 2010; Pelletier et al., 2010). It
is currently accepted that OBPs assist the passage of odorants
through the aqueous environment of the sensillum lymph to the
ORs, aside from contributing to the sensitivity of the olfactory sys-
tem (Leal, 2012).
4. Expression in non-sensory tissues
Although OBPs fundamental role in olfaction is supported by
several studies demonstrating that the majority of these proteins
is specifically expressed in antennae (Pelosi et al., 2006;
Shanbhag et al., 2001), as the list of identified members of the
OBP-gene family grows, it has become clear that not all of them
are associated with sensory organs, indicating OBPs might exhibit
different functions.
In Phormia regina, an OBP is responsible for solubilization of
dietary fatty acids (Ishida et al., 2013). Bloodsucking insects such
as Culex nigripalpus and Rhodnius prolixus had OBPs found in their
gut transcriptome (Ribeiro et al., 2014; Smartt and Erickson,
2009) which indicates these proteins could be associated with
the transport of nutrients or other molecules involved in intestinal
function. OBPs have also been observed in pheromone producing
glands and reproductive organs, as in the case of OBP10 from Heli-
coverpa armigera (Sun et al., 2012b) and Aedes aegypti OBP22 (Li
et al., 2008), where they might participate in the controlled release
of semiochemicals into the environment.
Nevertheless, it is highly unlikely that all OBPs predicted from
an insect’s genome are indeed olfactory proteins. In D. melanoga-
ster, for instance, the OBP-gene family comprises as many as 51
putative OBPs, but only seven of them have been demonstrated
to be expressed specifically in olfactory organs of adults (Galindo
54 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65

and Smith, 2001). The genome of the silkmoth B. mori contains 44
genes encoding OBPs, but to date only one pheromone-binding
protein and a few general odorant-binding proteins have been
identified (Dani et al., 2011; Krieger et al., 1996; Maida et al.,
1997). The Anopheles gambiae genome has 66 genes encoding pro-
teins that have been classified as OBPs based on sequence similar-
ity (Biessmann et al., 2005; Zhou et al., 2008). However, studies
have already demonstrated that several of these genes encode pro-
teins that can actually be found in non-sensory organs (Li et al.,
2005; Biessmann et al., 2005; Pitts et al., 2011) and in association
with other structures, such as the mosquito eggshell (Amenya
et al., 2010). A proteomic investigation of soluble olfactory proteins
suggests that female antennae express a larger number and higher
quantities of OBPs than males, with 24 OBPs being found in female
antennae while only 19 were identified in male antennae
(Mastrobuoni et al., 2013).
Olfactory and non-olfactory proteins from the OBP-gene family
present the same structural properties. Their helix-rich structures
suggest these proteins encapsulate hydrophobic odorants and
other ligands, with the ability to transport them through aqueous
environments. Hence, it is reasonable to presume that such stable
proteins could be used in a variety of tissues where there is need
for transportation of hydrophobic molecules in aqueous media or
protection of chemicals from degradation, as well as to assure a
gradual release of semiochemicals in the environment. For this rea-
son, Leal (2006) mentions these proteins should be named ‘‘encap-
sulins”, to imply the common role of encapsulating small ligands.
The labeling ‘‘odorant-binding protein” then should be restricted
to olfactory proteins of this family. It is possible that a large
number of genes annotated from insect genomes as putative OBPs
are merely encapsulins (Leal, 2006).
However, the majority of insect OBPs studied so far is highly
expressed in chemosensory structures and not found in non-
olfactory tissues (such as legs and wings, for instance), thus being
presumably associated with the detection of chemical signals.
5. Physicochemical properties and structural aspects
Insect OBPs are small (13–17 kDa) water-soluble proteins, gen-
erally consisting of chains of 130–150 amino acids (Leal, 2012;
Vieira and Rozas, 2011). These proteins are expressed with a signal
peptide that is removed during processing and is no longer found
in their mature form. OBPs usually present acidic features,
however, it has already been discussed that, in Diptera, OBPs have
a wider range of isoelectric points than the acidic pIs reported for
lepidopteran OBPs. Thus, OBPs in dipteran species can be positively
or negatively charged at the physiological pH in insect antenna
(Zhou et al., 2008). A large range of pIs has also been found for
OBP candidates in the genome of the hemipteran R. prolixus
(Mesquita et al., 2015).
According to the number of amino acid residues found in their
primary structure, odorant-binding proteins are classified as long-
chain OBPs (
160 residues), medium-chain OBPs (
120 residues)

Fig. 2. Representative alignment of OBPs from different insect species. NvitOBP78 (CCD17847.1); AlinOBP13 (ACZ58084.1); BmorPBP1 (2FJY_A); BmorGOBP2 (2WC5_A);
AlinOBP10 (ACZ58081.1); TcasOBP25 (EFA04747.2); LmigOBP1 (4PT1_A); AaegOBP1 (3K1E_A); CquiOBP1 (3OGN_A); AgamOBP1 (2ERB_A); RproOBP17 (VectorBase ID:
RPRC000118); AlinOBP4 (ACZ58030.1); LlinLAP (AAC43033.1); AlinOBP6 (ACZ58032.1); AlucOBP5 (AEP95759.1); AlinOBP1 (ACZ58027.1); ApisOBP1 (CAR85628.1); ApisOBP8
(CAR85635.1); DmelLUSH (NP_524162.1); AmelOBP5 (3R72_A). The pattern of six cysteines is well conserved. The conserved cysteines are marked with asterisks. Nvit –
Nasonia vitripennis; Alin – Adelphocoris lineolatus; Bmor – Bombyx mori; Aluc – Apolygus lucorum; Apis – Acyrthosiphon pisum; Dmel – Drosophila melanogaster; Tcas – Tribolium
castaneum; Lmig – Locusta migratoria; Aaeg – Aedes aegypti; Cqui – Culex quinquefasciatus; Agam – Anopheles gambiae; Rpro – Rhodnius prolixus; Llin – Lygus lineolaris; Amel –
Apis mellifera.
 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65
and short-chain OBPs (
100 residues). Primary sequences of this
type of proteins are highly divergent and amino acid identity
between OBPs from the same species might be lower than 10%.
The correct assignment of a protein into the OBP-family is mainly
based on a pattern of six cysteines whose relative positions are
well-conserved across all insect orders (Fig. 2) (Briand et al.,
2001a; Leal et al., 1999).
OBPs are usually comprised of six a-helical domains folded into
very compact and stable globular structures (Sandler et al., 2000;
Tegoni et al., 2004). Experiments have demonstrated that the six
cysteines form three interlocked disulfide bridges in an arrange-
ment that provides great stability to the protein’s three-
dimensional structure and also seems to be a conserved feature
among OBPs. This cysteine pattern has become a ‘‘signature” for
insect classical OBPs (Pelosi et al., 2006).
Based on their amino acid sequences, OBPs initially described in
Lepidoptera were subdivided into five classes: PBPs (pheromone-
binding proteins), GOBP1s (type 1 general odorant-binding pro-
teins), GOBP2s (type 2 general odorant-binding proteins), ABP1s
(type 1 antennal binding proteins) and ABP2s (type 2 antennal
binding proteins), previously known as ABPX (antennal binding
proteins) (Gong et al., 2009a; Krieger et al., 1996). However, since
this classification is not well defined for other insect orders, OBPs
were then categorized according to their conserved cysteine pat-
terns. The so-called classical OBPs present six conserved cysteines
in their sequences (Fig. 2), while C-Minus OBPs exhibit four or five.
OBP14 from Apis mellifera was the first C-Minus OBP to have its
structure determined and the study demonstrated the presence
of only two disulfide bonds (Spinelli et al., 2012). The Plus-C type
is distinguished by the presence of at least two additional cysteines
and a conserved proline after the sixth cysteine (Xu et al., 2003;
Zhou et al., 2004b). Crystal structure of AgamOBP47, a Plus-C
OBP from A. gambiae, revealed the existence of as many as six
disulfide bridges (Lagarde et al., 2011a). The family referred to as
dimer OBPs, described in Drosophila (Hekmat-Scafe et al., 2002;
Zhou et al., 2004b), seems to be formed by two classical six-
cysteine motifs fused together (Zhou et al., 2004a). Finally, the
atypical subfamily was first described as displaying a classical
domain in the N-terminal region elongated by a not well character-
ized extended C-terminal region. It has more recently been demon-
strated that atypical OBPs, which have so far been identified only in
mosquitoes (Xu et al., 2003; Zhou et al., 2008), actually present two
domains that are homologous to those of classical OBPs, and then
were renamed to two-domain OBPs and considered as belonging to
the dimer OBPs subfamily (Vieira and Rozas, 2011).
6. Three-dimensional structure-based mechanism of action
Crystal X-ray diffraction analysis and solution nuclear magnetic
resonance (NMR) are powerful tools for determining the three-
dimensional structure of a protein, whether in its free form or in
a complex with its ligand. To date, several insect OBP structures
have been solved and further details can be found in Table 1.
The first OBP to have its structure determined both alone and
bound to its ligand bombykol, was BmorPBP1 from B. mori. The
structure of a BmorPBP1-bombykol crystal grown at pH 8.2 was
initially resolved by X-ray crystallography (Fig. 3a). In this com-
plex, BmorPBP1 consists of a compact structure formed by a
roughly conical arrangement of six a-helices knitted together by
three disulfide bridges. Bombykol is bound in a completely
enclosed hydrophobic cavity, allowing its transport through hydro-
philic environments. The surface of BmorPBP1, however, is covered
with charged residues, which is not surprising for such a soluble
protein (Sandler et al., 2000).
55
Later, the structure of unligated BmorPBP1 was determined by
two studies involving solution NMR. The first one, attempted at
acidic pH (4.5), revealed the existence of a seventh helix occupying
a position corresponding to the binding pocket observed in the
crystal structure of the BmorPBP1-bombykol complex (Horst
et al., 2001) (Fig. 3b), providing a reasonable explanation for the
observation that PBPs generally do not bind ligands below pH 5.0
(Briand et al., 2001b; Prestwich, 1993). The next study, performed
at the physiological pH of the sensillum lymph (6.5), reported a
quite similar structure to the one obtained for the complex by
X-ray crystallography that exhibits a hydrophobic cavity with suf-
ficient volume to accommodate a bombykol molecule, concluding
this should be the reactive conformation (Lee et al., 2002).
The occurrence of pH-dependent conformational changes was
evidenced for the first time in Lepidoptera. Studies involving
BmorPBP1 had already described two distinct pH-dependent
conformational states (Damberger et al., 2007) and also that the
protein binds bombykol with high affinity at the sensillum lymph
pH, but shows no affinity for the pheromone at low pH, so it was
postulated that a transition from the high or near-neutral pH
(B-form, also called open form) to the low pH (A-form, also called
closed form) of the protein would be physiologically relevant
(Wojtasek and Leal, 1999). In such context, the comparison
between structures BmorPBP1A and BmorPBP1B, resolved by
NMR, and the structure of the complex BmorPBP1-bombykol,
determined by X-ray crystallography, suggests a biological func-
tion for the observed conformational change. The pheromone
would bind the active form of the protein, BmorPBP1B, found at
the physiological pH of the sensilla lymph and be transported
through the aqueous environment. When the BmorPBP1-
bombykol complex approaches the OR, located on the membrane
of sensory neurons, where the local pH is reduced, the OBP
undergoes a conformational change to its acidic form, BmorPBP1A,
triggering ligand release by the formation of a seventh a-helix that
occupies the binding pocket.
The C-terminus of BmorPBP1 is composed mostly of nonpolar
amino acid residues, except for three acid residues, Asp132,
Glu137, and Glu141, which lie on the surface of the helix and are
well-conserved in Lepidoptera. Of these, Asp132 (or Glu132 in
Amyelois transitella) and Glu141 form salt bridges with protonated
histidine residues: His70 in A. polyphemus and B. mori (Damberger
et al., 2007; Xu and Leal, 2008) or His80 in A. transitella (Xu et al.,
2010c), and His95, respectively, at low pH. Xu et al. (2010c)
demonstrated that in AtraPBP1, two salt bridges induced by acidic
conditions promote the formation of a seventh helix at the C-
terminal region. This mechanism of salt bridges formation was
named histidine protonation switch. The importance of histidine
residues protonation had already been demonstrated in previous
studies (Damberger et al., 2007; Zubkov et al., 2005) and it was
proposed that such mechanism provides stabilizing forces for the
insertion of the new helix into the binding cavity, taking into con-
sideration that both ends of the helix form salt links (His80-Glu132
and His95-Glu141) that position the C-terminal helix inside the
pheromone binding site. Deprotonation of histidine residues at
neutral pH disrupts the salt bridges, promoting the ejection of
the seventh helix outward from the binding pocket. Extrusion of
the now unstructured C-terminus allows exposure of hydrophobic
residues in the protein core which then can interact with the pher-
omone to enable binding (Xu et al., 2010c). Mutation of charged
residues involved in the switch (His95Ala, Asp132Asn, and
Glu141Ala) dramatically affect the pH-dependent binding of bom-
bykol to BmorPBP1 (Xu and Leal, 2008). In AtraPBP1, mutation of
His80 and His95 residues inhibit the formation of salt bridges at
both ends of the C-terminus helix, allowing the OBP to bound the
pheromone at low pH (Xu et al., 2010b).
56 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65

Table 1
List of published odorant-binding protein three-dimensional structures.

OBP Name Species Determined by Complexed with Reference

OBP1 Aedes aegypti X-ray (1.85 Å) Serendipitous ligand (consistent with PEG) Leite et al. (2009)
PBP1 Amyelois transitella NMR Free Xu et al. (2010)
OBP1 Anopheles gambiae X-ray PEG Wogulis et al. (2006)
(1.5 Å)
OBP7 Anopheles gambiae X-ray Serendipitous ligand (consistent with a 16-carbon-long fatty Lagarde et al. (2011b)
(1.85 Å, 2.19 Å) acid), AZO
OBP47 Anopheles gambiae X-ray Free Lagarde et al. (2011a)
(1.8 Å)
OBP1 Anopheles gambiae X-ray DEET, PEG Tsitsanou et al. (2012)
(1.60 Å)
OBP20 Anopheles gambiae X-ray (1.80 Å) Free, indole, heptene-2-one, DEET, others Ziemba et al. (2013)
NMR
OBP1 Anopheles gambiae X-ray 6-Methyl-5-hepten-2-one Murphy et al. (2013)
(2.20 Å)
OBP48 Anopheles gambiae X-ray PEG Tsitsanou et al. (2013)
(3.30 Å)
PBP1 Antheraea NMR Free Mohanty et al. (2004)
polyphemus
PBP1 Antheraea NMR Free Zubkov et al. (2005)
polyphemus
PBP1A Antheraea NMR Free Damberger et al. (2007)
polyphemus
ASP2 Apis mellifera NMR Free Lescop et al. (2001)
ASP1 Apis mellifera X-ray Serendipitous ligand (n-butyl-benzene-sulfonamide) Lartigue et al. (2004)
(1.6 Å)
ASP1 Apis mellifera X-ray Free, serendipitous ligand (nBBSA), 9-ODA, QMP blend, HDOA Pesenti et al. (2008)
(2.6 Å, 1.60 Å, 2.15 Å, 2.25 Å,
2.00 Å)
ASP1 Apis mellifera X-ray Free, serendipitous ligand (nBBSA), 9-ODA Pesenti et al. (2009)
(2.56 Å, 1.54 Å, 1.85 Å)
OBP14 Apis mellifera X-ray Free, 1-NPN, citralva, eugenol Spinelli et al. (2012)
(1.88 Å, 1.22 Å, 1.24 Å, 1.6 Å)
PBP1 Bombyx mori X-ray Bombykol Sandler et al. (2000)
(1.8 Å)
PBP1A Bombyx mori NMR Free Horst et al. (2001)
PBP1B Bombyx mori NMR Free Lee et al. (2002)
PBP1 Bombyx mori X-ray Iodohexadecane, bell pepper odorant Lautenschlager et al.
(1.9 Å, 2.0 Å) (2007)
GOBP2 Bombyx mori X-ray Free, bombykol, bombykal, other structural analogues Zhou et al. (2009)
(1.60 Å , 1.95 Å, 1.74 Å)
OBP1 Culex X-ray (1.30 Å) MOP Mao et al. (2010)
quinquefasciatus NMR
OBP76a Drosophila X-ray Ethanol, n-propanol, n-butanol Kruse et al. (2003)
(LUSH) melanogaster (1.49 Å, 1.45 Å, 1.25 Å)
OBP76a Drosophila X-ray Ethanol, butanol Thode et al. (2008)
(LUSH) melanogaster (1.8 – 2.0 Å)
OBP76a Drosophila X-ray cVA Laughlin et al. (2008)
(LUSH) melanogaster (1.4 Å)
PBP Leucophaea maderae X-ray Free, ANS, H3B2 Lartigue et al. (2003)
(1.7 Å, 1.6 Å, 1.7 Å)
OBP1 Locusta migratoria X-ray Free Zheng et al. (2015)
(1.65 Å)
OBP3 Megoura viciae X-ray Free Northey et al. (2016)
(1.3 Å)
OBP3 Nasovonia ribisnigri X-ray Free Northey et al. (2016)
(2.02 Å)

Abbreviations used are: PEG, polyethylene glycol; DEET, N,N-diethyl-m-toluamide; AZO, 4-hydroxy-40 isopropyl-azobenzene; 1-NPN, N-phenyl-1-naphthylamine; nBBSA, n-
butyl benzenesulfonamide; 9-ODA, 9-keto-2(E)-decenoic acid; QMP, queen mandibular pheromone; HDOA, hexadecanoic acid; MOP, mosquito oviposition pheromone
((5R,6S)-6-acetoxy-5-hexadecanolide); cVA, 11-cis-vaccenyl acetate; ANS, 1-anilino-8-naphthalenesulfonic acid; H3B2, 3-hydroxy-butan-2-one.

Even though BmorPBP1 is reported to bind equally well to both
bombykol and bombykal (Grater et al., 2006; He et al., 2010; Zhou
et al., 2009) by the mechanism described above, BmorGOBP2
seems to be able to discriminate between these two compounds.
Zhou et al. (2009) demonstrated that bombykol adopts different
conformations when it binds to each one of these proteins. The
hydroxyl group of bombykol forms a hydrogen bond with the side-
chain of Ser56 when bound to BmorPBP1, while binding to Bmor-
GOBP2 involves a hydrogen bond with Arg110 via a water
molecule. The extended interaction via a water molecule makes
the formation of an additional hydrogen bond to Glu98 possible.
This seems to be a good explanation for the fact that BmorGOBP2
presents greater affinity towards bombykol than bombykal. The
latter is not able to form the additional hydrogen bond to Glu98
since its polar group is an aldehyde and, as such, it is only able
to accept hydrogen bonds, in contrast to the hydroxyl group from
bombykol, which is able to both accept and donate, explaining the
ligand discrimination by BmorGOBP2 at the structural level. These
authors also reported that in the ligand-free structure of Bmor-
GOBP2, the C-terminus does not occupy the binding site as seen
in BmorPBP1 and that there is no conformational change among
the protein upon binding different sex pheromone components
 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65 57

Fig. 3. Three-dimensional structures of OBPs. a-Helices are shown in red, loops in green and disulfide linkages are highlighted in blue. (a) Bombyx mori pheromone-binding
protein, BmorPBP1, complexed with its specific ligand, the pheromone bombykol, at pH 6.5. The C-terminus is not the structure at this pH (Sandler et al., 2000). PDB ID Code:
1DQE. (b) The same protein at pH 4.5. The C-terminus is folded into a seventh a-helix that occupies the binding pocket, triggering ligand release (Horst et al., 2001). PDB ID
Code: 1GM0. (c) Aedes aegypti odorant-binding protein, AaegOBP1. The C-terminus is pulled to the core of the protein to form part of the binding pocket wall, which can
function as a ‘‘lid” for the release of ligands (Leite et al., 2009). PDB ID Code: 3K1E. (d) Culex quinquefasciatus odorant-binding protein, CquiOBP1 complexed with a mosquito
oviposition pheromone (MOP). In contrast to previously published ligand-bound OBP structures, a large part of MOP is not located inside the central cavity. Instead, MOP has
its long lipid ‘‘tail” bound to a hydrophobic tunnel formed by helices 4 and 5 and only its lactone/acetyl ester ‘‘head” sticks into the central cavity (Mao et al., 2010). PDB ID
Code: 3OGN. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and analogues. However, it is important to notice that the seventh
helix formed by the C-terminus was only observed in BmorPBP1 at
low pH and, in this study, BmorGOBP2 crystals were grown at pH
8.5, underlining the need for further studies in order to fully eluci-
date the binding and release mechanism of BmorGOBP2 (Zhou
et al., 2009).
Nonetheless, internalization of the C-terminus in form of a helix
into the binding cavity at acidic pH has already been shown not to
be a general feature of all lepidopteran PBPs. Zubkov and col-
leagues in 2005 observed that PBP1 from Antheraea polyphemus
undergoes a reorientation of helices a1, a3, and a4 at acidic pH
caused by protonation of His69, His70 and His95, which provides
the driving force behind the opening of the ligand binding cavity
and the release of the pheromone molecule from its carrier protein
near the membrane (Zubkov et al., 2005). The authors postulate
that these structural differences are likely caused by the difference
in the position of the residue which forms a hydrogen bond with
the ligand (Ser56 for BmoPBP1 and Asn53 for ApolPBP1). In the
neutral pH structure of ApolPBP1, there is a kink in the middle of
a3 that causes the side-chain of Asn53 to be a part of the phero-
mone binding pocket. At acidic pH, the kink disappears and the
helix straightens at this point with Asn53 moving away from the
protein core. Once helix a3 loses the kink at Asn53, the interaction
between a1b and a3 is also disrupted and helix a1 straightens out,
opening the cavity entrance and allowing the ligand to be released.
In the case of BmorPBP1, helix a3 is straight and the residue
responsible for pheromone headgroup interaction (Ser56) is found
inside the cavity at both neutral and acidic pH. Therefore, it is
possible that at low pH, the cavity in B. mori PBP opens due to
unfolding of a1a, similar to ApolPBP, but since Ser56 does not
move away, helix a7 can form and competitively displace the
ligand from the open binding cavity, Details of the pH-dependent
conformational switch in PBPs seem to vary for different moth spe-
cies and appear to depend on the structure of the binding site and,
in particular, the amino acid residues responsible for the direct
interaction with the ligands. Studies regarding ligand-interaction
kinetics of a pheromone-binding protein from the gypsy moth
Lymantria dispar provided evidence for the existence of yet another
mechanism of ligand binding for lepidopteran OBPs. LdisPBP2
seems to bind hydrophobic ligands in two steps: one rapid, the
other slow (Gong et al., 2009b). Authors suggest that the rapid
phase involves the uptake of the ligand by the C-terminus and
binding to an external site located near this region, where the ini-
tial interactions between ligand and OBP occur. By contrast, the
slow step corresponds to the specific reorientation and embedding
of the ligand to the internal binding pocket. It appears that both
steps are necessary in order to obtain a final complex. First, LdisPBP
and the ligand form an intermediate complex through diffusion-
controlled collision. Then, the ligand is relocated from an external
binding site to a different internal one, resulting in a more stable
ligand-OBP complex (Gong et al., 2009c).
Unlike what has been described for Lepidoptera, most dipteran
OBPs do not possess a C-terminal region long enough to form a
new helix that would compete with the ligand for the binding
cavity. However, AgamOBP1, AaegOBP1 and CquiOBP1 also
undergo pH-dependent conformational changes associated with
loss of binding affinity (Leite et al., 2009; Mao et al., 2010;
Wogulis et al., 2006), suggesting the molecular mechanism
58 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65
underlying pH-dependent affinity, in this case, would be different
from the observed in BmorPBP1. Leite et al. (2009) described that
the C-terminal region of these proteins acts as a ‘‘lid” over the
binding cavity (Fig. 3c), in a feature that is observed only in Diptera
(Wogulis et al., 2006). This ‘‘lid” is held in place by a triad of well-
conserved hydrogen bonds (Mao et al., 2010) that seems to work as
a pH-sensitive hinge. C-terminal ‘‘lids” of these OBPs differ only in
the last residue, where Val125 is found in AgamOBP1 and
CquiOBP1, and Ile125 in AaegOBP1. Carboxylate oxygens of such
residues form hydrogen bonds with the hydroxyl of Tyr54 and
the d-nitrogen of His23 in AgamOBP1 and CquiOBP1 (Mao et al.,
2010; Wogulis et al., 2006), and Arg23 in AaegOBP1 (Leite et al.,
2009). It is likely that when the OBP-odorant complex reaches
the proximity of dendritic membrane, where the pH is reduced,
some of these interactions are disrupted due to carboxylate proto-
nation, leading the C-terminus to move away from the binding
cavity, thus ‘‘opening the lid’’ and promoting ligand release.
Contrary to other OBPs described in the literature, CquiOBP1
from Culex quinquefasciatus was observed to bind the oviposition
pheromone MOP in a different fashion than the usual, where the
ligand is completely ‘‘buried” in the binding cavity. In this case,
the pheromone long lipid ‘‘tail” binds to a hydrophobic tunnel
formed between helices 4 and 5, with only its lactone/acetate head
sticking into the central cavity (Mao et al., 2010) (Fig. 3d). The
authors hypothesize that CquiOBP1 does not recognize the specific
functional group of MOP but rather recognizes the length of lipid
chains that fit into its hydrophobic tunnel. The same hydrophobic
channel has been reported in AgamOBP1 (Wogulis et al., 2006) and
AaegOBP1 (Leite et al., 2009), and it was described later that
AgamOBP1 binds DEET through this hydrophobic region
(Tsitsanou et al., 2012).
For the ASP1, from Apis mellifera, ligand affinity is pH-
dependent in an opposite way to the observed for other OBPs.
Experiments have shown that, at pH 4.0, the protein exhibits high
binding affinity for 9-keto-2-(E)-decenoic acid (9-ODA), the main
component of the queen bee pheromone, while at pH 7.0 the
OBP presents itself as a domain-swapped dimer with ligand affinity
ten times lower (Pesenti et al., 2008). Authors postulate that in
solution at pH 7.0, the loose C-terminus can recruit the
N-terminus of another ASP1 molecule to form a more stable
domain-swapped dimeric form. This form is still able to bind
ligands, but with lower affinity when compared to the acidic
ASP1 monomeric form. Dimerization seems to be strongly related
to residue Asp35, giving that mutants Asp35Asn and Asp35Ala,
which favor the formation of hydrogen bonds at neutral pH, exhibit
exactly the same monomeric structure in every pH tested and
increased binding affinity at pH 7.0 (Pesenti et al., 2009). Although
their findings were not conclusive, authors postulate that different
OBPs very likely exhibit different mechanisms of ligand release,
which might occur at low pH or at neutral or high pH, such as
the case of AmelASP1, indicating there might be a factor triggering
ligand release other than the pH drop at the vicinity of the mem-
brane, as reported for most OBPs. In this context, the discovery that
OBPs may interact with SNMPs (Jin et al., 2008) offers an appealing
alternative explanation. The trigger for ligand release could be the
ionization state of the SNMP ectodomain, which should emerge out
of the membrane surface and impose its own electrostatic influ-
ence on PBPs to capture them and lead to pheromone release at
the vicinity of the OR. This ionization state may create a local pH,
in some cases acidic and in other cases neutral or basic, triggering
the conformational changes of PBPs and the passage of ligands to
SNMPs. At least for olfactory neurons of T1 trichoid sensilla of D.
melanogaster, SNMPs are reported to be necessary for normal func-
tioning detection of 11-cis-vaccenyl acetate, a pheromone which
mediates social behavior in this species (Jin et al., 2008). SNMPs
were also shown to be associated with sex pheromone detecting
neurons in moths (Forstner et al., 2008; Rogers et al., 2001) and
genes encoding such proteins have already been identified in mos-
quitoes, however, their function remains intriguing since no sex
pheromone communication has been characterized yet for these
insects. It is possible that pheromone signaling is more specialized
compared with general odor detection, requiring additional factors
such as SNMPs, supporting the idea that a SNMP with local pH
might be involved in pheromone release by the domain-swapped
dimer of AmelASP1 at neutral pH. In 2013, AgamOBP48 was
reported as the first example of a domain-swapped dimer OBP in
dipteran species (Tsitsanou et al., 2013).
Lartigue et al. (2004) demonstrated that LmaPBP binding cavity,
although containing a majority of hydrophobic side-chains, con-
tains also a significant amount of polar or charged residues. Such
information is in contrast with the cavities of other OBPs, which
are almost exclusively formed by hydrophobic residues. However,
the author highlights the existence of these polar/charged residues
is consistent with the hydrophilic nature of Leucophaea madere
pheromone, so the protein is able to bind small hydrophilic ligands
such as H3B2 (3-hydroxy-butan-2-one), the main component of
this species pheromonal blend. The absence of the seventh helix
together with the evidence of a hydrophilic cavity binding a hydro-
philic ligand suggests that a more classical mechanism of direct
ligand release might be used. Also, the fact that LmaPBP binds to
a compound highly soluble in water raises doubt about the role
of OBPs as passive transport proteins and reinforces the hypothesis
of a mechanism in which the OBP-odorant complex would activate
the receptors. OBP conformational changes induced by pheromone
binding have been reported to be required for the activation of T1
neurons in D. melanogaster and details regarding this mechanism
are discussed in the next section.
Even though there are over 300 hemipteran OBPs annotated in
NCBI GenBank, crystal structures of aphid OBPs were reported only
very recently (Northey et al., 2016). Analysis of OBPs from the
vetch aphid Megoura viciae and from the currant-lettuce aphid
Nasonovia ribisnigri revealed similar structures to other classical
OBPs: six helices connected by extended loops, with three disulfide
bridges formed by the six conserved cysteine residues. However,
the two OBPs also have some unique features which are different
from other classical OBPs. There is a significant groove in the sur-
face of aphid OBPs formed primarily from the N-terminal region of
the chains and parts of helices a1 and a2, with no significant inter-
nal binding pockets being predicted. Besides the absence of an
internal binding cavity, OBP3 also differs from the vast majority
of OBPs with respect to the location of the N-terminus. In aphid
OBP3, this region is in an unusual place close to the center of the
molecule, where the C-terminus is normally found. Molecular
docking studies indicated that known ligands were mainly docked
in the groove and Tyr30, a residue which lies close to the predicted
surface binding site, may play an important role in ligand binding.
Molecular dynamics showed that Tyr30 is displaced towards the
protein in MvicOBP3, while in NribOBP3, Tyr30 is not significantly
displaced, located towards the binding site. Authors discuss that
the most likely explanation is that Tyr30 is involved in the crystal
packing/dimerization in MvicOBP3 and is relaxing towards its
probable monomeric conformation, similar to what is seen in
NribOBP3. However, a detail that could easily go unnoticed might
provide valuable information: NribOBP3 has been crystallized at
pH 5.0 whereas MvicOBP3 at pH 7.5. Different conformations
adopted by Tyr30 in such structures may be directly related to a
pH-dependent binding and release mechanism, a vastly reported
feature among insect OBPs, indicating that the unique features
described for the two aphid OBP3s (no internal binding pocket, sur-
face binding site and involvement of the N-terminus in forming the
binding pocket) need further evaluation in order to fully elucidate
their role in OBPs biological function.
 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65
On the whole, ligand binding and release by OBPs seems to pre-
sent specific features depending on the organism in question,
which is not exactly surprising given the fact that a unique
mechanism would hardly be consistent with insect diversity. It is
not very likely that selective pressures that led moths, for instance,
to have a very sensitive and dynamic olfactory system have acted
in the same way in the fruit fly. Analyses of OBPs from 12
Drosophila genomes suggest that OBPs play an important role in
ecological diversification (Vieira et al., 2007), therefore, it is
possible that behavioral and ecological differences between insect
species may affect not only the types of semiochemicals they are
able to perceive, but also how they detect and process these chem-
ical signals.
7. Activation of ORs
Up to this point, two models have been proposed in order to
explain ORs activation mechanism: the first one suggests the odor-
ant per se activates the receptor while the second model supports
the idea that the OR is activated by a specific OBP-odorant
complex.
Considerations mentioned above regarding moths and mosqui-
toes support the hypothesis that odorants are solubilized upon
being encapsulated in the binding cavity of OBPs and transported
through sensillar lymph, therefore being protected from degrada-
tion by ODEs. Eventually, when the complex reaches the dendrite
region, the decrease in local pH triggers ligand release, which then
activates the OR. In such cases, OBP-ligand complexes have a short
life at low pH values, corroborating the idea that odorants stimu-
late olfactory receptors in an independent fashion. This model is
consistent with the experimental observation that receptors are
activated by their specific ligands in the absence of OBPs
(Nakagawa et al., 2005). However, contrary to this direct ligand
hypothesis, a whole different way of signal transduction has been
suggested for the odorant-binding protein LUSH, from D. melanoga-
ster (Xu et al., 2005).
In Drosophila antennae, there are three morphologically distinct
types of sensilla that contain odor and pheromone-sensitive recep-
tor neurons: basiconic and coeloconic sensilla house neurons that
detect general odorants, while trichoid sensilla are thought to be
specialized for pheromone reception (Benton, 2007; Smith,
2007). A subset of trichoid sensilla, the T1 sensilla, contains neu-
rons that are activated specifically by the cVA pheromone
(Kurtovic et al., 2007; Xu et al., 2005). The perception of cVA is
described as complex and requires the participation of a specific
odorant receptor, OR67d, and, at least two more factors: a SNMP
and the OBP LUSH. OR67d is exclusively expressed in T1 neurons
and participates in cVA response (Ha and Smith, 2006; Kurtovic
et al., 2007). The SNMP is located along with the olfactory receptor
complex in dendrites of T1 neurons (Benton et al., 2007) and infu-
sion of a SNMP antiserum in T1 sensillar lymph results in loss-of-
function mutants (Jin et al., 2008), suggesting SNMP is directly
required in pheromone-sensitive neurons for cVA sensitivity.
A study conducted with LUSH-deficient mutants revealed this
protein is necessary for typical cVA-induced behavior and normal
sensitivity of T1 neurons (Xu et al., 2005). Structural studies point
out unique conformational changes mediated by residue Phe121
are induced upon binding of LUSH with cVA pheromone
(Laughlin et al., 2008). Interaction of cVA with residue Phe121 from
LUSH seems to mediate the conformational change into the active
form of the OBP through disruption of a salt bridge between resi-
dues Asp118 and Lys87. Experiments in which Asp118 was substi-
tuted by Ala were able to generate an active conformation of the
protein. Disruption of the salt bridge by mutagenesis results in a
conformational shift at the C-terminus virtually indistinguishable
59
from the LUSH-cVA structure, indicating the salt bridge possibly
maintains LUSH in the inactive state. Mutant Asp118Ala is able
to stimulate T1 neurons in the absence of cVA. In fact, T1 sensilla
infused with the mutant show no additional increase in activity
when stimulated with cVA, possibly indicating that the protein
has adopted an activated conformation that is not further activated
by cVA (Laughlin et al., 2008). Data obtained in these experiments
indicates that conformational changes in LUSH mediate T1 neuron
activation through OR67d and SNMP and that LUSH is not a passive
carrier of cVA. The pheromone, in turn, stimulates the conversion
from the inactive form of LUSH to an active conformation that is
capable of triggering the activation of T1 neurons. These observa-
tions are consistent with the model in which an OBP-odorant com-
plex is required for the activation of the OR. However, it should not
go unmentioned that this model was contested by a study indicat-
ing that cVA directly activates ORs, where authors conclude that
neurons can be activated in the absence of LUSH, but not SNMP
or OR67d (Gomez-Diaz et al., 2013).
8. Binding affinity and selectivity
pH-dependent conformational change associated with change
in binding affinity is reported to be common to OBPs from a variety
of insects. Moreover, it also seems to show a special character to
different groups. Binding activity of OBPs to small organic
compounds, such as pheromones and other semiochemicals, is
the key feature of these proteins and provides essential informa-
tion for understanding their physiological function (Fan et al.,
2011; Pelosi et al., 2006).
The ligand specificity of insect OBPs has not yet been demon-
strated conclusively. PBPs (at least from Lepidoptera) present great
binding specificity towards pheromones, with the pheromone
bombykol (IUPAC name (10E,12Z)-hexadeca-10,12-dien-1-ol)
being described as the specific ligand for the B. mori PBP
(BmorPBP1) (Sandler et al., 2000). Also, the male moth A. polyphe-
mus has three different PBPs which interact with components of
the sex pheromone of this species: ApolPBP1 exhibits higher affin-
ity towards (E,Z)-6,11-hexadecadienal, ApolPBP3 preferentially
binds (E,Z)-4,9-tetradecadienyl acetate and ApolPBP2 binds only
(E,Z)-6,11-hexadecadienal (Maida et al., 2003). However, OBPs
may also bind to a wide range of odorant molecules and GOBPs
seem to have a rather broad ligand-binding affinity in comparison
with specificity. A good example is the LstiGOBP2, a general
odorant-binding protein from the meadow moth Loxostege
sticticalis. This OBPs has been shown to have high affinity to plant
volatiles from essential oils such as (E)-2-hexenal and (Z)-3-hexen-
1-ol, and to the pheromone component (E)-11-tetradecen-1-yl
acetate (Yin et al., 2012). Nonetheless, there is also evidence
showing that PBPs can bind to general odorants in addition to
sex pheromone components and analogues (Jin et al., 2014; Song
et al., 2014) and that, in some cases, GOBPs exhibit preference to
special sex pheromone components besides host-plant odorants
(Gong et al., 2009c; He et al., 2010).
Insect OBPs were analyzed for the first time by native gel
electrophoresis of an antenna extract previously incubated with
the tritium-labeled pheromone (E,Z)-6,11-hexadecadienyl acetate,
followed by autoradiography (Vogt et al., 1985). Later, this method
was widely used either as described in the original paper or with
modifications. In some studies, radioactive photoaffinity labels with
structural similarity to the pheromone were used (Feng and
Prestwich, 1997), however, the preparation of radioactive pho-
toaffinity labels is often complicated and expensive. In early work
to identify pheromone-binding proteins, radiolabeled pheromones
were employed in qualitative binding assays (Plettner et al., 2000).
With the availability of recombinant proteins, radiolabeled
60 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65
pheromones can be used in quantitative assays in which free ligands
are separated by gel filtration from bound ligands. Although a valu-
able tool for studies of pheromone-PBP interactions, this type of
binding assay has limited application in screening programs because
radioactive test ligands are required. Leal et al. has described a bind-
ing protocol for OBPs that does not require radioactive ligands
named ‘‘the cold binding assay” (Leal et al., 2005b). After incubation
of test compounds with an OBP, the free ligand is removed by filtra-
tion, whereas the protein-bound ligand is retained in the centrifugal
device and extracted with an organic solvent containing an internal
standard. Binding is quantified by gas chromatography and the iden-
tity of the recovered ligand is confirmed by mass spectrometry. This
method presents the advantage that it can be applied to mixtures of
organic compounds, all incubated at the same time with the protein,
allowing the identification of the best ligands in a single experiment.
Nowadays, fluorescence binding assays represent the most used
technique in studies regarding binding properties of OBP to
putative ligands. Such method uses a fluorescent probe that, upon
protein binding, has its emission spectrum modified due to the less
polar environment inside the binding cavity, resulting in a blue shift
accompanied by a marked increase in intensity. Competitive bind-
ing experiments using a fluorescent probe (Fig. 4) are widely used
to investigate OBPs selectivity by comparing their binding affinities
to physiologically relevant compounds, surveying their ability to
preferentially transport some ligands over others. One limitation
of the method is the need of a fluorescent compound that exhibits
some affinity for the protein of interest. However, when applicable,
it offers the unique advantage of allowing binding measurements at
the equilibrium, besides being simple, fast and safe.
Fluorescence assays were used for the first time in insects to
demonstrate the unspecific binding capacity of OBPs from
Antheraea polyphemus and Mamestra brassicae to a series of ligands
in a competitive experiment using the probe 1-aminoanthracene
(AMA) (Campanacci et al., 2001). This kind of experiment also
revealed that, in contrast with other OBPs described so far, the
PBP of the cockroach L. maderae, LmaPBP, seems to be the only
Fig. 4. Schematic representation of binding assays using a fluorescent probe. (a) This technique uses probes that fluoresce weakly in aqueous media, but become very strongly
fluorescent in non-polar or hydrophobic environments, such as the binding cavity of an OBP. Modified from Zhuang et al. (2013). (b) In order to investigate binding affinities
of OBPs to different compounds, test ligands are added to the OBP-probe complex. Competition for the binding site displaces probe molecules from the hydrophobic cavity,
resulting in a decrease in overall fluorescence proportional to the amount of bound ligand.
one which binds a hydrophilic ligand, 3-hydroxy-butan-2-one, a
component of the pheromonal blend for this species (Riviere
et al., 2003). The specific binding of A. mellifera OBP3 to (E)-b-
farnesene was also discovered by competitive binding experi-
ments, this time using N-phenyl-1-naphthylamine (1-NPN), which
is also extensively used as a fluorescent probe (Qiao et al., 2009).
The binding affinity of AfunOBP1 from Anopheles funestus was
tested against different compounds that elicited significant elec-
troantennographic (EAG) responses, and 2-undecanone was identi-
fied as the best ligand (Xu et al., 2010a). In a similar study, A.
gambiae AgamOBP1 was demonstrated to specifically bind indole
(Biessmann et al., 2010). ANS (1-anilino-8-naphthalenesulfonic
acid) is too a fluorescent probe widely used in this type of assays
and was employed by Wojtasek and Leal (1999) in a remarkable
study regarding BmorPBP1 pH-dependent structural flexibility.
In 2014, a possible involvement of the protein CcapOBP83a-2,
from Ceratitis capitata, in olfactory processes and its specificity to
(E,E)-a-farnesene were exposed by this kind of assay (Siciliano
et al., 2014). More recently, using a fluorescent probe and a panel
of 34 physiologically relevant compounds, Yin and collaborators
investigated binding affinities of three OBPs from C. quinquefascia-
tus, concluding CquiOBP2 is a carrier for the oviposition attractant
skatole, and CquiOBP1 and CquiOBP5 might transport the oviposi-
tion pheromone MOP, a human-derived attractant nonanal, and
the insect repellent picaridin (Yin et al., 2015). Not long ago, fluores-
cence ligand-binding assays revealed that, in the oriental fruit moth
Grapholita molesta, GOBP1 has dual functions in recognition of host
plant volatiles and sex pheromone components, while GOBP2 is
mainly involved in the perception of dodecanol, a minor sex phero-
mone (Li et al., 2016). Fluorescence competition assays with OBP11
from the alfalfa plant bug Adelphocoris lineolatus provided insights
into understanding the physiological role of an OBP highly
expressed in mouthparts of this hemipteran species (Sun et al.,
2016). Results clearly showed that AlinOBP11 displayed preferential
binding abilities to non-volatile host plant secondary metabolites
than to volatile compounds, suggesting this protein could act as a
 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65
carrier in gustatory system for non-volatile compounds detection
when plant bugs begin to search suitable food substrates by using
their mouthparts to rub or to tap plant surfaces.
In some cases, when a proper probe cannot be found, it is pos-
sible to measure the intrinsic fluorescence of a tryptophan residue
in order to monitor the presence of a ligand, since it has been
proposed that Trp37 is positioned in the binding pocket and that
its interaction with the ligand appreciably affects its fluorescence
properties (Campanacci et al., 2001; Leal et al., 2005b). Observation
of intrinsic fluorescence changes revealed, for instance, the exis-
tence of three different kinds of interactions between ApolPBP1,
from A. polyphemus, and the components of its pheromonal blend,
(E6,Z11)-hexadecadienal, (E6,Z11)-hexadecadienyl-1-acetate and
(E4,Z9)-tetradecadienyl-1-acetate (Bette et al., 2002).
The binding potential between a characterized OBP and differ-
ent ligands can also be estimated by computational methods.
Although most studies assess binding affinity using structural
information and/or biochemical measurements, the use of molecu-
lar docking and molecular dynamics simulations as virtual meth-
ods to predict best ligands for insect OBPs is gaining eminence
(Venthur et al., 2014), in an approach that has already been widely
used for drug design research (Okimoto et al., 2009). Docking stud-
ies include the search for the most favorable conformation for the
protein-ligand complex in three-dimensional space and the score
of resulting geometries is reflected in the binding energy. Thermo-
dynamically, a ligand binds tightly to the active site of the protein
when the free binding energy is low. Therefore, compounds with
lower free binding energy are predicted to be better ligands
(Pagadala Damodaram et al., 2014).
Jiang et al. (2009) used molecular docking simulations to eval-
uate the strong binding affinity between LmigOBP1 and pentade-
canol, which suggested that Asn74 and is a key residue in the
binding site of this particular OBP (Jiang et al., 2009). This result
was then validated by site-directed mutagenesis and fluorescence
assays. A recent study has provided a methodology for rapid
screening of behaviorally active semiochemicals for the oriental
fruit fly Bactrocera dorsalis based on computational techniques. A
general odorant-binding protein of the insect was analyzed for
its binding potential against a panel of different semiochemicals.
The compounds predicted to be best ligands were then subjected
to behavioral bioassays and were found to be highly attractive to
insects (Pagadala Damodaram et al., 2014). Molecular docking
studies performed between OBPs from the scarab beetle Hylamor-
pha elegans and volatiles from Nothofagus obliqua revealed better
interaction energy values with sesquiterpenic compounds and
molecular dynamics reported that hydrophobic amino acids would
take part of these relationships (Gonzalez-Gonzalez et al., 2016).
Fig. 5. Updated concept of the reverse chemical ecology approach for the prospection of behaviorally active ligands. The first screening happens in silico, where computational
methods are able to rapidly select compounds from extensive libraries. In the next step, the binding affinity of the selected compounds for olfactory proteins (in this case,
OBPs) is evaluated in vitro by biochemical techniques such as fluorescence assays or NMR. Finally, the final stage involves bioassays in olfactometers and field tests to
determine if the affinity observed in vitro is actually capable of evoking a behavioral response in vivo and, in the positive case, the nature of the observed effect (attraction or
repellency).
61
It is reasonable to assume that computational methods will be
increasingly used in screenings for potential semiochemicals. In sil-
ico studies are able to provide a fine selection of ligands from
extensive libraries and their association with in vitro biochemical
analysis, such as the widely used fluorescence assays described
above, is undoubtedly becoming a very important tool in the field
of chemical ecology, with the purpose of providing valuable infor-
mation for the development of new pest control strategies based
on the manipulation of insect behavior and the prevention of
human diseases transmitted by insect vectors.
9. Applied research
The comprehension of behaviors of various agricultural pests
and pathogenic insects strongly depends on a better understanding
of the molecular mechanism underlying the olfactory system.
Moreover, such information is crucial for the development of
new strategies of vector control capable of interfering in
olfaction-mediated behavioral responses. Prospecting for novel
insect attractants or repellents can be based on molecular interac-
tions of candidate compounds with olfactory proteins.
In this context, since no odorant comes to ORs except through
OBPs, functional OBPs can be utilized as molecular targets for the
screening of insect behaviorally active compounds (attractants or
repellents) in an approach similar to receptor-based drug discovery
named ‘‘reverse chemical ecology”. Besides, when compared to ORs,
OBPs are more accessible targets for research, considering they are
small, soluble, stable and easier to manipulate and modify. The
reverse chemical ecology approach narrows down the number of
odorant candidate compounds based on their binding affinity to
olfactory proteins, saving time and lowering research costs in rela-
tion to the conventional trial-and-error screening performed in the
field.
This strategy was successfully employed for the first time in the
development of better lures for the Navel Orangeworm moth, Amye-
lois transitella (Leal et al., 2005c). Later, using CquiOBP1 as a molec-
ular target in binding assays, a study combining reverse and
conventional chemical ecology approaches to identify unnatural
ligands has contributed to the development of the commercially
available oviposition attractant for C. quinquefasciatus (Leal et al.,
2008).
The concept of reverse chemical ecology has been recently
updated and computational methods are starting to be incorpo-
rated to this approach in order to reduce even more the number
of candidate compounds and accelerate the discovery of new
semiochemicals (Fig. 5). The combination of molecular docking
and molecular dynamics was shown to be reliable for the
62 N.F. Brito et al. / Journal of Insect Physiology 95 (2016) 51–65
prediction of behaviorally active compounds for the agricultural
pest B. dorsalis (Pagadala Damodaram et al., 2014). Thus, the very
first screening is now performed in silico, followed by the in vitro
assessment of binding affinities between OBPs and the preselected
ligands by different techniques such as fluorescence, calorimetry,
NMR or even by measuring the amount of bound ligand, for
instance. Finally, insects’ behavioral response to the presence of
the best ligands is verified in vivo by bioassays and field tests.
Even though their use as molecular targets for the development
of new products for insect population management is the most
commonly discussed biotechnological application of insect OBPs,
a few research groups have already discussed their potential for
other technological purposes. A few studies have used odorant-
binding proteins in biosensors for the detection of odorants. By
mimicking biological olfaction, these artificial devices can be used
for the detection of important ligands in complex environments.
Larisika et al. (2015) have designed an olfactory biosensor based
on a reduced graphene oxide transistor functionalized with the
OBP14 from the honeybee A. mellifera for the in situ and
real-time monitoring of a broad spectrum of odorants in aqueous
solutions known to be attractants for bees (Larisika et al., 2015).
Current oscillations are reproducible and seem to linearly vary
with odorant concentration. Despite the different specificities
towards several organic compounds that make OBPs interesting
tools for building devices for odor discrimination, not many studies
have used such proteins with this intent.
Another interesting property of insect OBPs that can be exploited
for analytical purposes is their affinity and specificity for small
organic molecules. Margaryan et al. (2006) reported the synthesis
and characterization of affinity columns derivatised with PBP1 from
B. mori and OBP1 from C. quinquefasciatus (Margaryan et al., 2006).
Binding capacity was not affected by immobilization and OBPs also
retained their pH-dependent ligand affinity, which can be used for
gradual release of bound ligand from the column. Also, proteins
would be ideal substrates to differentiate enantiomers, but their
usually poor stability to temperature and solvents makes their
application in analytical columns very difficult. However, it has
already been suggested that OBPs exceptional stability to thermal
denaturation and proteolytic degradation could provide the solution
for this matter (Pelosi et al., 2014). In fact, it is reasonable to imagine
that, if properly employed as active chromatographic phases, OBPs
could, indeed, be directly used for resolving racemic mixtures of
pheromones and/or other natural compounds.
10. Concluding remarks
Although the progress in the field of insect olfaction in the past
decade has been remarkable, further knowledge of insect olfactory
perception is still lacking in order to unravel the molecular mecha-
nism whereby OBPs participate in chemoreception. How OBPs
release odorants to ORs and whether they play a role in the selection
of these molecules are still a matter of debate. However, taking into
consideration the substantial diversity in insect species, it is not
exactly surprising that available information regarding the roles
and modes of action of olfactory proteins sometimes seems to go
in different directions. When it comes to the physiological function
of OBPs, current understanding designates a role to these proteins as
carriers for odorants and pheromones across the sensillar lymph,
keeping them protected from degradation. Such concept was first
suggested about 30 years ago for OBPs of both vertebrates (Pelosi
and Tirindelli, 1989; Pevsner et al., 1990) and insects (Vogt et al.,
1985) and remains the most widely accepted hypothesis. Neverthe-
less, odorant-binding proteins appear to be feasible molecular tar-
gets to disrupt important behaviors in novel strategies for insect
population management, as well as other biotechnological
applications.
Funding
This study was supported by grants from the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq), Instituto
Nacional de Ciência e Tecnologia em Entomologia Molecular
(INCT-EM/CNPq), Fundação Carlos Chagas Filho de Amparo à Pes-
quisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES). No addi-
tional external funding was received for this study.
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