Volume 6 • Issue 4554

Research Article

Gating Of the Channel Pore of Ionotropic Glutamate Receptors with
Bacterial Substrate Binding Proteins: Functional Coupling of the Ectoine
Binding Protein Ehub to Glur0
Max Bernhard1 and Bodo Laube1,2*Abstract
Mammalian neuronal tetrameric ionotropic glutamate receptors(iGluRs) are thought to have originally arisen from the fusion of a bacterial
substrate binding protein (SBP) with an inverted potassium channel. Thishypothesis is based on structural and sequential similarities between theligand binding and channel domains of iGluR subunits with SBPs andpotassium channels. Ligand binding occurs at the interface between two
lobed domains in both ligand binding domains (LBDs) and leads to closureof the shell-like structure, which is considered to be a key element in thetransition from ligand recognition to ion channel gating in iGluRs. Here we
report the functional coupling of the ectoine-binding protein EhuB to thechannel pore of the GluR0 receptor. Fusion of an unmodified EhuB-binding
protein to the transmembrane domain of GluR0 did not result in activationof the channel pore. Only by stabilizing the inserted EhuB-binding
domain with a dimerization interface the resulting chimera was activatedby ectoine, resembling the activation properties of other iGluRs. These
results demonstrate the functional compatibility of SBPs to the gate of thechannel pore of an iGluR and highlight the role of LBD dimerization in thefunctional evolution of iGluRs. Based on the high specificity and affinityof SBPs for an incredible variety of substrates, our results demonstrate
the competence of SBP/ion channel chimeras for the development of new
Biosensors for specific recognition of analytes by functionally linking abacterial binding protein to the channel pore of an iGluR.

Abbreviations:AMPA:
α-amino-3-hydroxy-5-methyl-4isoaxazolepropionic acid; CTD: C-terminal domain; iGluR: Ionotropic
glutamate receptor; LBD: Ligand binding domain; NTD: N-terminal domain;
RMSD: Root-mean-square deviation; SBD: Substrate binding domain; SBP:
Substrate binding protein; TMD: Transmembrane domain

Introduction
Ionotropic glutamate receptors (iGluRs) mediate the majority of rapid
excitatory neurotransmission in the vertebrate central nervous system
by converting a chemical signal (the release of the neurotransmitter
glutamate) into the opening of a cation- permeable ion channel [1]. Basedon their pharmacological properties, these iGluRs can be classified into
four subfamilies: α-amino-3-hydroxy-5- methyl-4-isoaxazolepropionic acid
(AMPA), kainate (KA), N-methyl-D-aspartate (NMDA), and δ-receptors.
Other members of the iGluR family are found throughout the animal kingdom[2], in plants [3], and bacteria [4]. Despite their different physiological
functions, all eukaryotic iGluRs share the same modular architecture

Affiliation:
1
Department of Biology, Neurophysiology and
Neurosensory Systems, Technical Universityof Darmstadt, Schnittspahnstrasse 3, 64287
Darmstadt, Germany
Centre for Synthetic Biology, Technical Universityof Darmstadt, 64283 Darmstadt, Germany

2*Corresponding author:
Bodo Laube, Department of Biology,
Neurophysiology and Neurosensory
Systems, Technical University of Darmstadt,
Schnittspahnstrasse 3, 64287 Darmstadt, Germany.
Citation: Max Bernhard, Bodo Laube. Gating
Of the Channel Pore of Ionotropic Glutamate
Receptors with Bacterial Substrate Binding
Proteins: Functional Coupling of the Ectoine
Binding Protein Ehub to Glur0. Journal of
Biotechnology and Biomedicine. 6 (2023):
554-564.
Received: October 07, 2023
Accepted: October 25, 2023
Published: November 01, 2023

Bernhard M and Laube B, J Biotechnol Biomed 2023
DOI:10.26502/jbb.2642-91280117(Fig. 1a). They consist of an extracellular N- terminal
domain (NTD), an extracellular ligand-binding domain(LBD), a transmembrane domain (TMD; TM1, TM2, TM3)
including the pore loop (P), and an intracellular C-terminal
domain (CTD). The origin of this modular architecture islargely unknown. The common complex architecture ofeukaryotic iGluRs suggests an ancient separation of theprotein family [3,5] with a common ancestor that can betraced back to bacteria [4]. Prokaryotic iGluR subunits, such
as GluR0 [4], have a simplified architecture, lacking the third
transmembrane segment TM3 and an NTD, while exhibiting
unique functional and pharmacological properties, such asa potassium selectivity filter and the ability to be activatedby a wide range of amino acids. Interestingly, individual
domain segments of iGluRs share structural similarities withother prokaryotic protein families. For example, the LBD ofiGluRs is structurally homologous to bacterial class II solutebinding proteins (SBP) [6] and the TMD exhibits an inverted
architecture of tetrameric K+ channels [7,8], leading to the
proposal that iGluRs may have arisen from the fusion of a
SBP and potassium channels [9]. Recent findings confirm
the general relationship between K+ channels and iGluRs by
coupling an iGluR LBD to a small viral K+ channel [10],suggesting a conserved activation mechanism of the channelpore in both protein families.
However, the sequence identities between the LBDs ofiGluRs and SBPs are low [11] and the evolutionary link
between the two remains unclear. SBPs mediate the uptakeof a variety of substances across the cell membrane [12]and are also involved in chemotaxis and DNA regulation[13]. Despite low sequence similarity, SBPs, like iGluR
LBDs, display a highly conserved three-dimensional shelllike structure consisting of two α/β-domains (D1 and D2)
connected by a small hinge of connecting strands. Ligand
binding occurs between the interface of the two-subdomains
and initiates closure in a Venus fly trap mechanism [14], a key
element of ligand recognition and signal transduction in all
SBP-associated protein families. SBPs sometimes recognize
even small targets in a highly specific manner through noncovalent protein-ligand interactions that result in KD values
in the micromolar to nanomolar range. This specificity andthe ability to bind even targets that are difficult for antibodybased detection have made these proteins attractive for use
in sensing applications [15]. Based on their topologicalarrangement, SBPs can be classified into two classes [16] ormore recently into seven clusters [17,18]. The LBD of iGluRs
also forms a binding pocket in the cleft between two lobes(D1 and D2) of a shell-like conformation consisting of two
extracellular regions, where two polypeptide segments on
the amino- terminal side of TM1 (called S1) and between
TM2 and TM3 (called S2) largely form domains D1 andD2, respectively. Many experimental works have shown thatindeed the agonist binding site of soluble LBD constructs are

Volume 6 • Issue 4555

similar to the binding sites and properties in intact receptors[19,20]. The LBDs, which are arranged as two dimers "backto-back" in the tetrameric iGluR, are induced to close the twoshell-like domains after ligand binding, which is directed to
the TMD and leads to the permeation of Na+, K+, and Ca2+
ions across the cell membrane [20-22].
Although iGluR LBDs and SBPs share similarities intheir overall structure and ligand recognition, the functional
compatibility of SBPs to gate the channel pore of an iGluR
has not been demonstrated. We selected the TMDs of GluR0
as a candidate for the functional compatibility of a bacterial
binding protein with a channel pore of an iGluR. Here, wethen used a proof-of-principle approach to couple the bacterial
ectoine binding protein EhuB to the channel pore of GluR0
to construct a functional ectoine-activated receptor. With thisapproach, we provide the first experimental evidence that
iGluRs may have arisen from the fusion of an amino acid
SBP to an ion channel. Furthermore, our results highlight a
conserved ligand binding mechanism in both protein families
and the role of LBD dimerization in the functional evolutionof iGluRs from SBPs. Furthermore, our results support thepossibility of fusion of SBPs, a huge protein family withdiverse pharmacological properties and binding ligands, forthe targeted design of hybrid receptors as the basic module ofa new generation of Biosensors.

Results
Identification of a potential SBP candidate forcoupling to the channel pore of GluR0.
The functional coupling of a bacterial SBP with the ionchannel of an iGluR has not been demonstrated to date,
and thus the requirements of SBPs for successful couplingof ligand binding to the channel gating of iGluRs are still
unclear. To investigate the structural basis of the couplingof SBPs with iGluR pore domains, we aimed to generate
a functional chimeric receptor by replacing the ligandbinding domain (LBD) of the bacterial GluR0 receptor
with a bacterial SBP. The bacterial GluR0 receptor waschosen because of its less complex architecture comparedwith eukaryotic iGluRs, characterized by the absenceof the NTD, the TM3, and a CTD, and may represent theevolutionary link between potassium channels, SBPs, andiGluRs [4]. Because of the high number of SBPs and their
low sequence identity, which is usually less than 20% [17],we decided to identify a suitable SBP based on its structuralsimilarity to the LBD of iGluRs using the Vast (+) algorithm[23]. We used the structure of the glutamate-bound closed
LBD conformation [24] (PDB ID: 1ii5) as a search template
to allow for similar domain closure in ligand binding forsufficient receptor activation [25,26] (see also Materials andMethods section). The 391 identified structures with a total
RMSD value of up to 4 Å were further manually inspected

Citation: Max Bernhard, Bodo Laube. Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins:
Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.

Bernhard M and Laube B, J Biotechnol Biomed 2023
DOI:10.26502/jbb.2642-91280117

to remove duplicates and open or unliganded structures,
as well as structures of enzymes and gene regulators. The
remaining 94 structures included structures that were mostly
part of the TRAP (tripartite ATP-independent periplasmictransporter family), TT- transporter (tripartite tricarboxylatetransporter), and iGluR LBDs and SBPs for amino acids,
sugars, ions, inorganic salts, and various organic compounds.
To analyze the structural relationship between SBPs andiGluR LBDs, all remaining RMSD values were used toconstruct a phylogenetic tree using the Kitsch program(Figure 1b). The structures can be divided into 3 clusters
according to Berntsson et al., [17]. Cluster F includes SBPs
with two hinge segments (8-10 amino acids each) that bind avariety of substrates, including amino acids, trigonal planaranions, compatible solutes, and iron, as well as all iGluR
LBDs (Figure 1b). Almost all SBPs closely related to iGluRs
bind amino acids or derivatives. Structural feature analysis
revealed that the amino acid-binding proteins have a short
C-terminus similar to the length of the GluR0 LBD comparedwith nonamino acid-binding proteins (p < 0.0001, Student's
two-tailed, unpaired t-test with Welch's correction; Figure1c). Furthermore, all iGluR LBDs form a small subclusterwith a branched order, supporting the current assumption that
GluR0 may represent a bacterial iGluR archetype. Cluster
D, which has only SBPs, is characterized by two short hinge
segments (4-5 amino acids each) and binds various substancessuch as sugars, phosphates, iron, and molybdate (Figure 1b).
Cluster E is located between clusters D and F and features
exclusively SBDs of TRAP and TT transporters. Within thecluster, the ectoine-binding protein EhuB turned out to be
the non-amino acid SBP most phylogenetically related to
the GluR0 LBD. EhuB is part of the Ehu ABC transporter
system from Sinorhizobium meliloti, which mediates uptakeof the compatible solutes ectoin and hydroxyectoin with highaffinity (Kd = 1.6 µM) under osmotic stress [27]. Despite asequence identity of only 17%, EhuB shows a nearly similar
overall structure as the GluR0 LBD with an RMSD of 2.2
Å and 216 of 258 overlapping amino acids (Figure 1d). In
addition, both binding domains possess strong rotation of
the two domains during the transition to the closed, ligatedconformation. These favorable features formed the basisof a simple approach for us to combine the ectoine binding
protein EhuB (PDB ID 2Q88) with the channel pore of
GluR0 to prove, in a proof-of-concept approach, the generalmechanistic and evolutionary ability of SBPs to gate an ion
channel by converting chemical information into an electricalsignal.

Replacement of the GluR0 LBD and optimization of
the ectoine binding protein with stabilization of the
dimer interface
In the first step, we wondered whether a simple fusion of
the wt EhuB binding protein with the GluR0 TMD (referred
to as GluR0EhuB) would be sufficient to open the channel

Volume 6 • Issue 4556

pore. After superimposing the crystal structures of bothbinding domains (Figure 2a), the ends of the GluR0 LBD
lobes (P139, A255) [24] were set as reference points and
EhuB was fused to the TMD at positions K132 and N135
with the GluR0 S1-M1 and M2-S2 linkers in the form of two
subdomains (Figure 2b). To retain the original GluR0 linker
length, positions K133, G134 in EhuB were deleted in the
GluR0EhuB receptor. In addition, the construct was equippedwith a c-Myc tag, to allow efficient detection of the protein(see Materials and Methods). After heterologous expression,
the GluR0Ehub construct unfortunately showed no function
in electrophysiological measurements after application of
ectoine (up to 1 mM) (data not shown). Analysis of total
and surface expression of GluR0EhuB protein by surface
biotinylation and Western blot (Figure 2c) revealed a single
band with a molecular weight of approximately 40 kDa.
We concluded that the GluR0EhuB chimera is efficiently
expressed at the cell surface, suggesting that folding,assembly, and transport of the receptor to the cell membrane
are unaffected in principle. Extensive intermolecularinteractions within the dimerization interface of adjacent
LBDs are crucial for the conversion of ligand binding into achannel opening in iGluRs [19, 28,29], interactions that may
not be formed due to the monomeric nature between EhuBSBPs. Furthermore, compared with the GluR0 LBD, EhuB has
an elongated C-terminus consisting of three helices (10-12).
We therefore speculated that a lack of LBD dimer interface
and the large C-terminus in GluR0EhuB might prevent LBD
dimerization, resulting in the nonfunctional receptor chimera
GluR0EhuB. In GluR0, the dimerization interface between
LBDs is characterized by multiple hydrogen bonds and vander Waals contacts formed mainly by 9 amino acids (V113,
A118, E348, N349, Q353, K354, L361, N362, Y365) [24].
Therefore, we decided to design a receptor chimera containing
the GluR0 LBD dimer interface in the ectoine binding domain(hereafter referred to as GluR0EhuBInt (Int for interface)).
To reconstruct the GluR0 LBD dimerization interface,
we identified by structural superposition the amino acidpositions in EhuB that mediate LBD dimerization in GluR0(Figure 2a,b) and substituted the corresponding amino acidsin the GluR0EhuB LBD (positions M108V, C113A, K235E,
E238N, R241Q, D242K, A249L, K250N, and E254Y).
In addition, C-terminal helices 10-12 of the original EhuB
binding protein were deleted in the GluR0EhuBInt receptor
chimera. GluR0EhuBInt was expressed like the original
GluR0EhuB construct and localized to the cell surface, as
shown by western blot and surface biotinylation (Figure 2c).
Even application of 10 µM ectoine (Figure 2d) resulted inligand-dependent inward currents for GluR0EhuBInt, incontrast to the unmodified initial GluR0EhuB construct, after
expression in both HEK293 cells and oocytes (Figure 2d anddata not shown). These measurements clearly demonstrated
that the ectoine- binding protein EhuB is able to open thechannel pore of GluR0 with a maximal amplitude comparable

Citation: Max Bernhard, Bodo Laube. Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins:
Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.

Bernhard M and Laube B, J Biotechnol Biomed 2023
DOI:10.26502/jbb.2642-91280117

to wt GluR0. However, the ectoin-induced currents showedmarked desensitization with greatly reduced receptor currents
after re-application of ectoin (run down), which prevented
the generation of ectoin dose-response curves (Figure 2d).
However, these results indicate that i) Ehub is able to forma functional LBD that can gate the ion channel of GluR0 andii) that the formation of a GluR0-analog LBD dimerizationinterface is important to couple ectoin binding within the
LBD with channel gating.
Our GluR0EhuBInt resulted in a chimera receptor
that showed strong desensitization. We hypothesized that
our modified dimerization interface in GluR0EhuBInt
is not yet able to reorganize after activation. Although
the cα-backbones of GluR0 LBD and EhuB are highly
congruent, there are some minor structural differences atthe exchanged positions, especially at positions C113A (1.9
Å), D242K (1.6 Å), A249L (1.6 Å), and E226Y (1.7 Å).
We hypothesized that these side chains are unable to form
intermolecular interactions, weakening the dimerizationinterface. To test this hypothesis, we decided to strengthen
the LBD dimerization interface by introducing a disulfide
bond at positions P119C and L376C to covalently couple
the two LBD domains (GluR0EhuBInt,P119C,L376C).
These positions (Figure 2a,b), when substituted with Cys in
AMPA receptors, prevent desensitization of the receptor bystabilizing the LBD dimers [22,30,31]. Application of ectoine(Figure 2d) induced concentration-dependent activation after
expression of GluR0EhuBInt,P119C,L376C with an EC50
value of 14.1 ± 7.3 nM (mean ± SEM, n= 4; Figure 2e).
Receptor currents showed no desensitization upon prolonged
application of ectoine, which is in good agreement withprevious studies that stabilized LBD interactions throughdisulfide bonds within other members of the iGluR family.
Apparently, by further stabilizing the dimerization interface,
we were able to maintain receptor activity across multiple
ectoine reapplications. Thus, our results demonstrate thefunctional coupling of an SBP to an iGluR channel porethrough the exchange of specific amino acids within the
LBD by mimicking an LBD dimerization interface of GluR0and by introducing an additional disulfide bridge leading toligand- and concentration-dependent currents that resemble
general features of non-desensitizing iGluRs.

Discussion
Although it has long been suspected that the LBDs ofiGluRs are derived from bacterial SBPs (6), the evolutionary
and functional compatibility of SBPs as a module for ligand
recognition in iGluRs has not yet been confirmed. In thisstudy, we characterized the basic molecular requirements
for functional coupling of an SBP to the channel pore of an
iGluR, thereby demonstrating functional compatibility andstrengthening a possible evolutionary relationship between
the two protein families. All iGluRs consist of at least two

Volume 6 • Issue 4557

domains: the LBD and the TMD forming an ion channel. The
common molecular architecture is most likely the result ofdifferent protein precursors that fused during the evolutionof iGluRs [9]. It has been clearly shown that potassiumchannels share similarities in overall structure, topology, andsequence with the pore domain of iGluRs [4,32,33] and share
a common gating mechanism [10], suggesting a common
origin. Felder and colleagues first proposed over 20 years ago[6] that SBPs represent a blueprint for modern iGluR LBDs
because they share a common ligand-binding mechanism.
However, due to their high functional diversity and low
sequence identity, it is difficult to draw conclusions about acommon origin of the two protein domains. By coupling theectoine-binding protein EhuB to the channel pore of GluR0
in a semi-rational approach, we support the assumption of
a common origin and ligand-binding mechanism of SBPs
and iGluRs. For iGluRs, ligand binding can result in either
partial closure of the LBD, as found in the preactive state, orcomplete closure associated with the active state. The extent
of receptor activation depends on both the ability to effectcomplete closure of the LBD and the fraction of time that the
LBD remains in this fully closed conformation. In contrast,incomplete closure of the domain results in conformations
with reduced activity, as has been observed with partial
agonists [34-39]. Interestingly, SBPs also exhibit a wide
range of conformations of partially closed structures that are
actively involved in substrate transport [40-42]. Therefore,
it stands to reason that both the GluR0 LBD and EhuB must
adopt a similar, fully closed conformation in the presence of
the TMD to open the pore of the GluR0 channel. Furthermore,
the functionality of our chimeric receptors suggests that thegeneral mechanism of ligand binding and domain closure in
EhuB and GluR0 must be similar to adopt the fully closed
conformation. These results highlight the general modular
architecture of iGluRs and support the common hypothesisthat the LBD is derived from a bacterial SBP. They further
suggest that the underlying mechanism of complete andpartial agonism as well as competitive inhibition in iGluRs
could be traced back to its bacterial precursors.
In addition, our data shed light on the minimal molecularrequirements to preserve receptor function after a potential
fusion event and to determine the physiological propertiesof modern iGluR LBDs. The formation and maintenance ofthe back-to-back dimer arrangement by the D1-D1 interface
is critical for iGluR gating. During activation, the interface
is responsible for keeping two adjacent LBDs attached toeach other. This allows the D2 lobes within the LBD dimers
to separate from each other to transfer the conformationalchange to the ion channel via the linkers [20-22]. In contrast,
the same force that causes elongation of the connecting
linker during ligand binding can lead to partial or complete
rupture of the dimerization interface, which is referred
to as desensitization. Breakage of the LBD dimerization

Citation: Max Bernhard, Bodo Laube. Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins:
Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.

Bernhard M and Laube B, J Biotechnol Biomed 2023
DOI:10.26502/jbb.2642-91280117

interface leads to realignment of the LBD dimer, allowing
the D2 lobes to adopt a conformation similar to the closed
state, which uncouples the closed LBD conformation fromchannel activation [30,43-45]. Our data suggest that the mere
fusion of an SBP with an ion channel pore is not sufficient
to form a functional receptor. We suggest that the TMD of
the GluR0EhuB receptor assembles as a tetramer because it
is displayed at the plasma membrane and forms a putative
functional channel pore. Because EhuB has not been reported
to assemble as dimers or oligomers in solution [27], it isunlikely that functional dimer interactions form within
the LBD layer of the GluR0EhuB receptor. Therefore, wehypothesize that ligand binding to the EhuB-binding domain
is uncoupled from channel gating because the force generated
by clam-shell closure cannot be used to separate the D2lobes and be transmitted by the linkers. This is consistent
with previous studies showing that complete disruption of
the D1 dimerization interface by covalently linking the D2lobes leads to uncoupling of agonist binding from ion channel
gating [21]. Remarkably, alteration of a few amino acids
within the putative LBD dimerization interface is sufficient toachieve basic receptor activity, as seen with GluR0EhuBInt.
However, the GluR0 LBD and the EhuB protein are notcompletely homologous in their spatial structure. This isconsistent with the fact that even minor perturbations of the
dimerization interface by single point mutations enhance
desensitization [30,46-48]. By covalently stabilizing the
LBD dimerization interface in GluR0EhuBIntP119CL376C,
we demonstrated that desensitization can be overcome.
Moreover, the activation and deactivation kinetics of
GluR0EhuBIntP119CL376C resemble the slow activationof GluR0 and constitutive activity upon prolonged ligand
application observed in LBD-stabilized AMPA and kainate
receptors. In summary, these results confirm that the
dimerization interface is a key element in the molecular
evolution of iGluRs that is critical for coupling the clamshellclosure of the binding domain with ion channel gating. The
fact that only minor adjustments are required to form an LBD
dimerization interface to obtain a functional iGluR archetypeis likely an advantage in functional evolution.
In conjunction with coupling to ion channels, the future of
SBP-based sensing offers significant opportunities. SBPs canenable detection of molecules or ions that is not possible with
antibodies, and can be tailored to specific applications throughtargeted mutagenesis of protein specificity and affinity. SBPs
have consequently been used in a variety of platforms,
e.g., those based on enzyme-linked immunosorbent assays,
surface plasmon resonance, immunomagnetic separation, andfluorescence resonance energy transfer (reviewed in Edwards[15]). The tremendous intrinsic amplification of the signal ofligand-gated ion channels together with the unusual extreme
sensitivity and selectivity of SBPs represents another major
potential for the application of SBP-based biosensors. Due

Volume 6 • Issue 4558

to their specific response, SBP-gated ion channels represent
a new class of biosensors that can be used to perform
sophisticated analytical measurements and will certainly
attract interest in medicine, pharmaceuticals, biochemistry,and environmental analysis. Ion channels are also integral
membrane proteins that play a special role in neuronal
excitability and synaptic transmission. Using chimeric SBPgated ion channels to modulate neuronal activity would also
be an attractive possibility. There would be the possibilityof developing SBP-derived ion channels that could beselectively introduced into neurons by genetic engineering
methods and influence the activity of neuronal populations by
tailoring ligands. Our work also allows a perspective on theevolutionary origin of iGluR LBDs. It is worth highlightingthat the most closely structurally related SBPs in cluster F
all bind amino acids or amino acid derivatives. This findingis less surprising because GluR0 can be activated by polaramino acids as a putative link between the evolution ofmodern metazoan iGluRs from SBPs and potassium channels[4]. Moreover, the LBD of AMPA receptors and GluR0 share
weak amino acid sequence homology with glutamine-binding
protein from Escherichia coli [4]. For this reason, it is very
likely that the ancient SBP precursor belongs to class F SBPs.
It is noteworthy that all of the identified amino acid andamino acid derivative class F SBPs have a relatively short
C terminus compared with all of the nonamino acid- binding
proteins of the more distant classes D and E. This is becauseof the length of the C terminus. These elongated C termini
often pack onto the D1 lobe and obscure the same helicesthat mediate LBD dimerization in iGluRs. Presumably, in theevolution of iGluRs, it was advantageous that the putative
LBD-dimer interface in amino acid-binding proteins was
freely accessible, allowing rapid functional adaptation of
dimer-dimer interactions without extensive changes inprotein structure. Moreover, the short C-termini of aminoacid-binding proteins could potentially serve as putative S2TM3 linkers for coupling the additional TM3 helix in later
evolutionary iGluR stages.
In summary, by functionally coupling the ectoine-binding
protein EhuB to the GluR0 channel pore, we provide evidence
for compatibility between the two protein families and shedlight on the functional and molecular evolution of iGluRsfrom bacterial SBPs and potassium channels. Our results
may contribute to the mechanistic understanding of ligand
recognition in both protein families and to drug development.
Finally, in combination with the diversity of recognized
ligands and the structural adaptability of SBPs, our approach
could be used as a versatile tool for the development ofbiosensors with high specificity and efficiency.

Materials and Methods
Identification of homologous SBP and structural
distance tree

Citation: Max Bernhard, Bodo Laube. Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins:
Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.

Bernhard M and Laube B, J Biotechnol Biomed 2023
DOI:10.26502/jbb.2642-91280117

For structure identification and building a structural
distance tree, we used the previously described method from
Scheepers et al., [18] with some modifications. To identify
SBPs that are structurally homologous to the LBD of GluR0,
we used the ligand-bound LBD conformation (1ii5) as asearch template against the Molecular Modeling Database(MMDB) using the Vector Alignment Search Tool (VAST+)[23]. The resulting 354 PDB codes were matched against
the UniProt IDs to filter out multiple structures of the same
protein. Additionally, unliganded structures, enzymes andgene regulators were filtered out by manual inspection. Thissearch resulted in about 94 SBP- and iGluR LBD-structures
similar to the GluR0 LBD. The remaining structures were
pairwise aligned using the PDBeFold server [49]. To build
the structural distance tree, the RMSD values obtained fromPBDeFold were loaded into the KITSCH program of the
PHYLIPS package with default parameters [50]. Protein
clusters were verified by visual inspection using UCFS
Chimera [51] according to Berntsson et al., 2010 [17] and thestructural distance tree was visualized using iTOL (https://itol.embl.de/). For the determining the C-termini lengths ofamino acid and non-amino acid SBP, structures were pairwisealigned to the GluR0 LBD (1ii5) and inspected using UCSF
Chimera. The relative C-terminus was specified relative toposition Ser366 in the GluR0 LBD. Statistical significancewas determined at the p ≤0.05 (*), p ≤ 0.01 (**), p ≤ 0.001(***) and p ≤ 0.01 (****), levels using a Student’s twotailed, unpaired t-test with Welch’s correction. Equality ofvariances was tested using a F-test.

Protein engineering
In this study, we used the sequences from GluR0

Volume 6 • Issue 4

559(UniProt ID P73797) and EhuB (UniProt ID Q92WC8).
Signal peptide cleavage site was predicted using SignalP v.
4.1[52]. The intrinsic GluR0 (residues 1-19) or EhuB signal
peptides (residues 1 – 27) were replaced by the GluR6 signal
peptide (MRIICRQIVLLFSGFWGLAMG), followed by ac-myc tag (EQKLISEED) and a short linker (SGTPT). For
GluR0EhuB, EhuB D1 domain (residues 28 – 132) was fusedto the N-terminal and the D2 domain (residues 135 – 283) tothe C-terminal end of the GluR0 TMD, including the linker(residues 139 – 255). For GluR0EhuBInt, the positions M81V,
C86A, K208E, E211N, R214Q, D215K, A222L, K223N and
E226Y were substituted and residues 266 – 283 were deleted(positions corresponding to the original EhuB protein). For
receptor expression in eukaryotic expression systems, thesequences of all constructs were codon-optimized for Xenopus
laevis. GluR0, GluR0EhuB and GluR0EhuBInt DNA strings
were synthesized (GeneArt, Thermo Fisher Scientific,
Regensburg, Germany) and subsequently cloned into thevector pCDNA3.1(+) via included NheI and XhoI restriction
sites. For GluR0EhuBInt,P119C,L376C, the primers P119C
forward (ATTGTTTGTCAAGTGTGAGAGAGCCGCT) andreverse(AGCGGCTCTCTCACACTTGACAAACAAT),
as
well
as
L376C
forward(TGATGTTGAACTCTGTAATCTGAAATACTCCG)
andreverse(CGGAGTATTTCAGATTACAGAGTTCAACATCA)
were used for mutagenesis polymerase chain reaction (PCR).
PCR reaction parameters were as follows: initial denaturation
at 95 °C for 60 s; 30 cycles at 95 °C and 30 s, 56 °C for 15s and 72 °C for 150 s; and one cycle at 72 °C for 180 s. Allconstructs were confirmed by sequencing (Seqlab, Göttingen,Germany).

Sequences
GluR0EhuB
MRIICRQIVLLFSGFWGLAMGEQKLISEEDLSGTPTDENKLEELKEQGFARIAIANEPPFTAVGADGKVSGAAPDVAREIFKRLGVADVVASISEYGAMIPGLQAGRHDAITAGLFMKPERCAAVAYSQPILCDAEAFALKGKPVSLWERFSPFFGIAALSSAGVLTLLLFLVGNLIWLAEHRKNPEQFSPHYPEGVQNGMWFALVTLTTVGYGDRSPRTKLGQLVAGVWMLVALLSFSSITAGLASAFSTALSEASNPLGLKSYKDIADNPDAKIGAPGGGTEEKLALEAGVPRDRVIVVPDGQSGLKMLQDGRIDVYSLPVLSINDLVSKANDPNVEVLAPVEGAPVYCDGAAFRKGDEALRDAFDVELAKLKESGEFAKIIEPYGFSAKAAMSTTR EKLCAAK

GluR0EhuBInt
MRIICRQIVLLFSGFWGLAMGEQKLISEEDLSGTPTDENKLEELKEQGFARIAIANEPPFTAVGADGKVSGAAPDVAREIFKRLGVADVVASISEYGAMIPGLQAGRHDAITAGLFVKPERAAAVAYSQPILCDAEAFALKGKPVSLWERFSPFFGIAALSSAGVLTLLLFLVGNLIWLAEHRKNPEQFSPHYPEGVQNGMWFALVTLTTVGYGDRSPRTKLGQLVAGVWMLVALLSFSSITAGLASAFSTALSEASNPLGLKSYKDIADNPDAKIGAPGGGTEEKLALEAGVPRDRVIVVPDGQSGLKMLQDGRIDVYSLPVLSINDLVSKANDPNVEVLAPVEGAPVYCDGAAFREGDNALQKAFDVELLNLKYSGEFAKIIEPYG

Citation: Max Bernhard, Bodo Laube. Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins:
Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.

Bernhard M and Laube B, J Biotechnol Biomed 2023
DOI:10.26502/jbb.2642-91280117

Heterologous expression in Xenopus laevis oocytes
Constructs were expressed in X. laevis oocytes as described
previously [10]. In brief, all constructs in pCDNA3.1(+)
were linearized with NotI and cRNA was synthesized using
the AmpliCap-Max™ T7 High Yield Message Maker Kit(Cellscript, Madison, WI, USA). Surgically obtained oocytes
from female X. laevis were enzymatically separated anddefoliculated using 0.8 mg collagenase in ringer solution(96mM NaCl, 2 mM KCl, 1mM CaCl2, 1mM MgCl2, 5mM
HEPES; pH 7.4) for 12 h and reaction was stopped with Ca2+free ringer solution. 50 ng of capped and polyadenylated RNA
with 50 nl water was microinjected in defoliculated stage
IV – V oocytes and incubated 3 – 7 days in ND-96 solution(96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES,
50-mg ml-1 gentamycin; pH 7.5) at 18 °C. Related oocyte
experiments (for example total and surface expression) were
performed with the same oocyte batches on the same day.

Heterologous expression in HEK293 cells
HEK293 cells were cultured in minimum essential
medium (MEM) supplemented with 10 % (v/v) FCS, 2
mM L-glutamine and streptomycin (100 μg/ml). Before
transfection, 5x105 cells were reseeded into T25 flask andtransfected using TurboFect (Thermo Fisher Scientific,
Waltham, MA, USA) and 4 µg plasmid DNA per flask of
the respective construct in pCDNA3.1(+) and pEGFP-N1 asa transfection control with a ratio of 4:1. After transfection,
cells were incubated for 48-72 h at 37 °C and 5% CO2.

Surface biotinylation
For surface biotinylation, 20 oocytes per construct were
injected as described above and incubated for 3 days in
ND-96 solution at 18 °C. Oocytes were washed three times
with PBS (100 mM phosphate buffer, 150 mM NaCl; pH
7.2) and surface proteins were biotinylated using 0.5 mg/
ml Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) in
PBS for 40 min at room temperature. Reaction was stopped
with 50 mM Tris/HCl; pH 8.0. Oocytes were mechanicallyhomogenized and membrane proteins were extracted using
DDM (100 mM phosphate buffer, 150 mM NaCl 0.5 %n-dodecyl-β-D-maltoside, SIGMAFAST Protease Inhibitor
Cocktail (Sigma-Aldrich, St. Louis, MO, USA); pH 8.0) for
15 min at 4°C. Biotinylated membrane proteins were purified
using Streptavidin High Performance Spintrap columns(GE Healthcare, Chicago, IL, USA).

Western blotting
For total protein expression, 10 oocytes per construct were
injected and incubated for 3 days. Oocytes were washed with
0.1 M sodium phosphate buffer, pH 8.0 and mechanicallyhomogenized. Membrane proteins were solubilized with
25 µl lysis buffer (0.1 M sodium phosphate buffer pH 8.0,
0.5 % DDM, 0.01% pefa block) for 10 min at 4 °C andVolume 6 • Issue 4560

subsequently centrifuged with 1000 x g at 4°C. HEK293
cells were transfected by electroporation as described above
and incubated 48 h. Cells were scraped, lysed with 200µl lysis buffer and subsequently centrifuged 20 min with
16000 xg at 4°C. For SDS-PAGE and western blot, total andsurface labeled proteins were separated using a 10% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and transferred to a PVDF membrane (Bio-Rad,
Feldkirchen, Germany). The membrane was blocked for 1 h
in PBS-T supplemented with 5% skim milk and afterward
incubated with 1:600 primary c-Myc-tag Polyclonal antibody(sc-789, Santa Cruz Biotechnology, Dallas, TX, USA) in
PBS-T containing 1% skim milk over night at 4 °C. Afterwashing with PBS-T, the secondary goat anti-rabbit IgGHRP (1:10000) (sc-2054, Santa Cruz Biotechnology) was
incubated in TBS-T containing 1% skim milk for 1 h atroom temperature. The membrane was washed three times
in TBS-T and the signal was visualized using Pierce Western
Blotting Substrate (Thermo Fisher Scientific) and detectedwith a CCD camera.
Electrophysiological recordings of Xenopus laevis oocytes
using two-electrode voltage clamp 3-7 days after injection,
whole-cell currents were recorded by two-electrode voltageclamp at -80 mV using microelectrodes filled with 3M KCl(resistance 0.8–2.5 MΩ) as described previously [53]. Data
were sampled at 5 kHz after low-pass filtering at 200 Hz
using an Axoclamp 900A amplifier connected to a Digidata
1550A digitizer and recorded with Clampex 10.7 (Molecular
Devices, San Jose, USA). Oocytes were placed in a perfusion
chamber and rinsed with high potassium Ringer’s solution(100 mM KCl, 1 mM CaCl2, 5 mM HEPES, pH 7.4 with
KOH). Ectoine (10 µM to 0.1 nM) or L-glutamate (1 mM to 1µM) was applied to the oocytes in external solution. For doseresponse analysis, normalized current responses were plottedagainst the agonist concentration and fitted with a sigmoidal
Hill equation
𝐼⁄𝐼𝑚𝑎𝑥 = 100 × 𝑥ℎ + 𝐸𝐶50ℎ in GraphPad Prism 9 (GraphPad
Software Inc., La Jolla, USA) where I/Imax is the normalized
current, x the concentration, h the Hill coefficient andEC50 the agonist concentration resulting in a half-maximal
response. Data and graphs presented in mean ± SEM.

Electrophysiological recordings using Port-a-patch
Electrophysiological recordings in HEK293 cells
were performed using the Port-a-Patch system (Nanion
Technologies, München, Germany). Adherent HEK293 cells
were harvested 48-72 h after transfection using 1% accutase
solution. The reaction was stopped with MEM and the cellsuspension was centrifuged at 100 xg for 3 min. Cells were
resuspended in extracellular solution (140 mM KCl, 1 mM
MgCl2, 2 mM CaCl2, 5 mM D-glucose, 10 mM HEPES; pH
7.4; osmolarity 298 mOsmol) with a final concentration of

Citation: Max Bernhard, Bodo Laube. Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins:
Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.

Volume 6 • Issue 4

Bernhard M and Laube B, J Biotechnol Biomed 2023
DOI:10.26502/jbb.2642-91280117

2x106 - 3x106 cells/ml. Measurements were performed usinga Port-a-Patch system with external perfusion system (Nanion
Technologies) and NPC-1 chips (3-5 MΩ) according to the
manufacturer instructions, connected to an EPC10 amplifier(HEKA, Ludwigshafen, Germany), PatchControl (Nanion
Technologies) and Patchmaster software (HEKA). External
solution and internal solution (50 mM KCl, 10 mM NaCl, 60
mM KF, 20 mM EGTA, 1 mM ATP, 10 mM HEPES; pH 7.2;
osmolarity 285 mOsmol) were used for experiments and seal
enhancer (80 mM NaCl, 3 mM KCl, 10 mM MgCl2, 35 mM
CaCl2, 10 mM HEPES; pH 7.4; osmolarity 298 mOsmol)
was used for sealing cells. For recording receptor currents,
cells were clamped at -60 mV and continuously perfused withexternal solution during recording. Ectoine dilution series (10µM – 0.1 nM) was dissolved in external solution. Agonist
was applied for 3 s, followed by 10 s of wash. Dose-response
analysis was performed as described above. Data and graphspresented in mean ± SEM.

561

Author contributions
M.B. designed and performed the experiments andanalyzed the data. M.B. and B.L. wrote the paper.

Competing interests
The authors declare no competing interests.

Acknowledgements
This work has been supported in the frame of the LOEWE
project iNAPO by the Hessen State Ministry of Higher
Education, Research and the Arts. The authors acknowledge
support by the German Research Foundation and theOpen Access Publishing Fund of Technische Universität
Darmstadt.

Data availability
All data supporting the findings of this study are available

Figure 1: Structural relationship between iGluRs and SBPs. a Modular organization of SBPs, K+-channels and prokaryotic and eukaryotic
iGluRs (left) and tetrameric organization of metazoan iGluRs (right) including the amino-terminal domain (ATD), the ligand binding domain(LBD) with the dimerization interface (red star) and the transmembrane domain (TMD). b Structural distance tree of iGluR LBDs and
SBPs structural homologous to the GluR0 LBD (1ii5). Clusters were categorized following the nomenclature of Berntsson et al. [17]. The
structural distance tree is subdivided into the following clusters: cluster F: two hinged SBPs including all iGluR LBDs (orange; GluR0:1ii5,
AvGluR1:4io3, GluR5:2f36, GluR4:3epe, GluR2:5fth) and the ectoine binding protein EhuB (2q88), cluster E: SBDs from TRAP-transports
cluster D: short hinged SBPs. c Comparison of the C-terminus lengths between amino acid (aa) or aa derivate SBPs and non-amino acid
SBPs relatively measured to position Ser366 in the GluR0 LBD (p < 0.0001, Student’s two-tailed, unpaired t-test with Welch’s correction).
d Structural superposition between the ligand-bound, closed conformations of the GluR0 LBD (purple, 1ii5) and the ectoine binding protein
EhuB (green, 2q88).
Citation: Max Bernhard, Bodo Laube. Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins:
Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.

Bernhard M and Laube B, J Biotechnol Biomed 2023
DOI:10.26502/jbb.2642-91280117

Volume 6 • Issue 4562

Figure 2: Design and expression of the chimeric GluR0EhuB and GluR0EhuBInt receptors. a Local RMSD of cα- atoms between the
GluR0 LBD and EhuB (left) and structural overview (right) of the exchanged amino acid positions within the EhuB LBD in GluR0EhuB and
GluR0EhuBInt. The connection points to the LBD-TMD linkers (orange) and amino acid positions that are responsible for LBD dimerizationin GluR0 and are exchanged in GluR0EhuBInt (red) are highlighted. b Structural sequence alignment of EhuB and the GluR0 LBD, including
the linker positions (orange a,b), the substituted amino acid positions of the dimerization interface (red 1-9) and the deleted helices 10-12 (darkred) in GluR0EhuBInt. Previously published amino acid positions substituted with Cys to covalently couple the D1 domain of each LBDdimer in AMPA- and kainate recepors and stabilizing the LBD dimerization interface in GluR0EhuBInt,P119C,L376C by disulfide bonds arehighlighted with asterisks. c Western blot of total and surface expression of GluR0EhuB (left) and GluR0EhuBInt (right) with a calculated
molecular weight of 40 kDa and 38 kDa, respectively. Proteins were detected using a c-Myc tag located at the N-terminus after the signal
peptide. d Representative traces of whole-cell currents of GluR0EhuBInt (left) and GluR0EhuBInt,P119C,L376C (right) in response from 1
nM to 10 µM ectoine in HEK293 cells. e Dose-response analysis of GluR0EhuBInt,P119C,L376C revealed an EC50 value of 14.1 ± 7.3 nM(mean ± SEM, n = 4).

within the article or are available from the corresponding
author upon reasonable request. All cDNA constructs areavailable from the corresponding author based on reasonable
request.

Ethical approval
All procedures involving animals were in accordancewith the guidelines and regulations of the local animal careand use committee. Methods were approved by the Technical
University of Darmstadt (II25.3-19c20/15, RP Darmstadt,
Germany).

Competing interests

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Citation: Max Bernhard, Bodo Laube. Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins:
Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.

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DOI:10.26502/jbb.2642-91280117

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Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.

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Citation: Max Bernhard, Bodo Laube. Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins:
Functional Coupling of the Ectoine Binding Protein Ehub to Glur0. Journal of Biotechnology and Biomedicine. 6 (2023): 554-564.