The GlpF residue Trp219 is part of an amino-acid cluster crucial for aquaglyceroporin oligomerization and function

Accepted Manuscript The GlpF residue Trp219 is part of an amino-acid cluster crucial for aquaglyceroporin oligomerization and function

Margareta Trefz, Rebecca Keller, Miriam Vogt, Dirk Schneider PII: DOI: Reference:

S0005-2736(17)30336-X doi:10.1016/j.bbamem.2017.10.018 BBAMEM 82618

To appear in: Received date: Revised date: Accepted date:

30 June 2017 10 October 2017 15 October 2017

Please cite this article as: Margareta Trefz, Rebecca Keller, Miriam Vogt, Dirk Schneider , The GlpF residue Trp219 is part of an amino-acid cluster crucial for aquaglyceroporin oligomerization and function. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbamem(2017), doi:10.1016/ j.bbamem.2017.10.018

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ACCEPTED MANUSCRIPT The GlpF residue Trp219 is part of an amino-acid cluster crucial for aquaglyceroporin oligomerization and function Margareta Trefz, Rebecca Keller, Miriam Vogt and Dirk Schneider*

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Johannes Gutenberg University, Johann-Joachim-Becher-Weg 30, 55128 Mainz, Germany

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*Corresponding author:

Dirk Schneider, Johannes Gutenberg-University Mainz, Institute of Pharmacy and Biochemistry,

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Johann-Joachim-Becher-Weg 30, 55128 Mainz, Germany

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Phone: (+49) 6131 39-25833, Fax: (+49) 6131 39-25348, E-mail: [email protected]

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Abbreviations used: AQP, aquaporin; DDM, n-dodecyl β-D-maltoside; E. coli, Escherichia coli; GlpF, glycerol uptake facilitator; LB, lysogenic broth; LmAQP, Leishmania major aquaglyceroporin; MD, molecular dynamics; OD600, optical density at 600 nm; OG, n-octyl β-D glucopyranoside; PAGE, polyacrylamide gel electrophoresis; PfAQP, Plasmodium falciparum aquaglyceroporin; SDS, sodium dodecyl sulfate; TM, transmembrane; wt, wild type; χSDS, micellar mole fraction of SDS.

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ACCEPTED MANUSCRIPT Abstract The vestibule loop regions of aquaglyceroporins are involved in accumulation of glycerol inside the channel pore. Even though most loop regions do not show high sequence similarity among aquaglyceroporins, loop E is highly conserved in aquaglyceroporins, but not in members of the

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homologous aquaporins. Specifically, a tryptophan residue is extremely conserved within this

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loop. We have investigated the role of this residue (Trp219) that deeply protrudes into the protein

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and potentially interacts with adjacent loops, using the E. coli aqualgyeroporin GlpF as a model.

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Replacement of Trp219 affects the activity of GlpF and impairs the stability of the tetrameric protein. Furthermore, we have identified an amino acid cluster involving Trp219 that stabilizes

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the GlpF tetramer. Based on our results we propose that Trp219 is key for formation of a defined

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vestibule structure, which is crucial for glycerol accumulation as well as for the stability of the

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active GlpF tetramer.

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ACCEPTED MANUSCRIPT

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Graphical abstract

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Keywords:

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Aquaporin; protein folding; membrane protein; monomerization; stopped flow; E. coli

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ACCEPTED MANUSCRIPT 1.

Introduction

From bacteria to men, aquaporins, a family of water-conducting transmembrane channels, mediate the cellular uptake of water, an essential prerequisite for life [1]. A subgroup of aquaporins, the aquaglyeroporins, can additionally facilitate the flux of linear polyalcohols, such

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as glycerol, across cellular membranes [1-5]. One characteristic feature of members of the

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aquaporin family is its highly conserved structure and all family members form tetramers [6-12]. The monomers of canonical aquaporins as well as of aquaglyceroporins consist of six

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transmembrane (TM) helices and two half-spanning helices that are connected by five loop

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regions (A-E) [13]. It is assumed that the monomer structure emerged from tandem intragenic duplication of three and a half helices, explaining the occurrence of an inverted repeat within the

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monomer [14, 15]. The arrangement of the right-handed GlpF α-helix bundle gives the monomer

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an hourglass-like shape, and the active unit of the tetrameric protein is represented by an amphipathic pore that lies within each monomer (compare Fig. 1). Even though aquaporins form

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tetramers under in vivo and in vitro conditions, the benefits of protein tetramerization as well as

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the potential function of the pore formed within the center of a tetramer are enigmatic and topics of current research [16].

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During the course of evolution, the divergence of aquaporins and aquaglyceroporins likely occurred rather early [17]. This separation can already be found in E. coli that contains the classical aquaporin AqpZ as well as the aquaglyceroporin GlpF. These two membrane proteins are good representatives of their respective classes and have often been used as architypes. Whereas AqpZ only facilitates the flux of water and small solutes across a membrane, GlpF reveals a highly shifted permeation towards glycerol, however also permeating water in smaller amounts [18]. The main structural differences between these two proteins lie within the pore 4

ACCEPTED MANUSCRIPT width as well as in the selectivity filter, which allows passage of glycerol in aquaglyceroporins [19]. Intriguingly, there are also structural differences within the periplasmic vestibule regions: The extended periplasmic loops of aquaglyceroporins are a striking feature that clearly distinguishes aquaglyceroporins from classical aquaporins. In several cases, this vestibular

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regions have been found to directly influence the activity and selectivity of aquaglyceroporins, as

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e.g. observed for Plasmodium falciparum [20] or Leishmania major aquaglyceroporins [21]. In

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case of the E. coli GlpF it is assumed that the asymmetric structure of the monomer caused by these loops results in an attractive energy well that functions as a funnel for glycerol [22]. This

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free energy minimum might increase the chance of glycerol-binding at the periplasmic side of the

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bacterial membrane, a feature that is of great importance in times of higher cellular glycerol

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demand [19].

Based on a sequence alignment, in the present study we have identified one Trp residue in the

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loop E region that is highly conserved among aquaglyceroporins and that is exposed to a variety

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of potential interaction partners. We have assumed that this Trp residue is of general importance for the stability of aquaglyceroporins and key for structuring the extended aquaglyceroporin-

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specific loop C and E regions. Thereby, this Trp potentially indirectly influence the activity of

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aquaglyceroporins. In fact, the loop E region is on average about ten amino acids longer in aquaglyceroporins than in aquaporins and interacts together with loop C with the substrate conducting pore [23]. Thus, the structure and interactions of soluble loop regions potentially directly affect substrate conductance of aquaglyceroporins. For this reason, we have investigated the role of the conserved loop E residue Trp219 on glycerol conduction, stability and oligomerization of the E. coli GlpF. Previous analyses have shown that GlpF forms stable tetramers that unfold via a dimeric unfolding intermediate [24, 25]. Furthermore, a correlation between the oligomeric state of the protein and its activity has been observed [26]. Our results 5

ACCEPTED MANUSCRIPT show that Trp219 is crucial for the GlpF activity, and this residue is also necessary for formation and/or maintenance of the tetrameric GlpF structure. When Trp is replaced by Phe, the activity and tetrameric structure is largely retained, mainly due to the replacement of Trp by another aromatic residue. Furthermore, we have identified Trp219 being part of a cluster of aromatic

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residues. Interactions of these residues stabilize the GlpF tetramer and influence the activity of

Materials and Methods

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2.

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importance for the structure and activity of all aquaglyceroporins.

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GlpF. As Trp219 is highly conserved in aquaglyceroporins, this Trp residue likely is of general

2.1 Expression and purification of GlpF

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Construction of the pRSET and pMal-based plasmids for the expression of GlpF in BL21(DE3)

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and SK46 E. coli cells, respectively, is described in [26]. After site-directed mutagenesis, the

Ebersberg, Germany).

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sequence of the glpF gene was confirmed via DNA sequencing (Eurofins MWG Operon,

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For expression of GlpF, BL21(DE3) cells were transformed with pRSET-His-GlpF and plated on

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LB agar plates containing 100 µg/mL ampicillin. A 50 mL overnight culture was inoculated with a single colony, and 1 L of fresh LB medium was inoculated with the overnight culture the next morning. For induction of protein expression, isopropyl-β-D-thiogalactopyranosid (IPTG) with a final concentration of 0.5 mM was added at an OD600 = 1.2. After 3-4 h, cells were harvested by centrifugation, cell pellets were resuspended in 50 mM phosphate buffer (pH 8.0), 300 mM NaCl, 30% glycerol (v/v), and cells were lyzed by tip sonification. The GlpF protein was purified from isolated membranes via affinity chromatography, as described in detail in [24]. The protein concentration was determined by measuring the absorbance at 280 nm using the following 6

ACCEPTED MANUSCRIPT calculated extinction coefficients: εwt =37930 M-1 cm-1, εWA/F = 32430 M-1 cm-1, εWY = 33920 M-1 cm-1.

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2.2 SDS-induced equilibrium unfolding and semi-native polyacrylamide gel electrophoresis

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Increasing amounts of SDS were added to 8 µM GlpF dissolved in 10 mM phosphate buffer (pH

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8), 5 mM n-dodecyl β-D-maltoside (DDM). The SDS mole fraction was successively stepwise increased from SDS = 0 to 0.95. Samples were incubated for 1 h at room temperature prior to

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further analyses.

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To maintain the SDS that was added for successive GlpF unfolding, no additional SDS was

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present in the SDS-PAGE sample buffer (50 mM Tris-HCl (pH 6.8), 10% (v/v) glycerol and 0.04% (w/v) bromphenol blue). Omitting SDS from the sample buffer preserves the tetrameric

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state of GlpF. The SDS present in the running buffer (25 mM Tris-HCl, 192 mM Glycin, 0.1%

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SDS (w/v) corresponding to a χSDS = 0.41) was considered in the course of data analysis. Proteins

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electrophoresis.

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were separated on 10% SDS-PAGE gels and stained with Coomassie brilliant blue R250 after

2.3 GlpF activity measurements The activity of GlpF was determined by measuring the flux of the linear polyalcohol ribitol across the E. coli inner membrane, using a SX20 stopped flow spectrometer (Applied Photophysics, Leatherhead U.K.), as described in [26]. SK46 E. coli cells (glpF and aqpZ deficient) were a kind gift of E. Bremer (University of Marburg, Marburg, Germany). SK46 cells were transformed with the plasmid pMalp2 (empty plasmid, also negative control), pGlpF and 7

ACCEPTED MANUSCRIPT pGlpF encoding the mutated glpF genes, respectively. Ribitol was chosen instead of the physiological substrate glycerol due to its lower tendency to permeate through the E. coli inner membrane while sustaining proper conductance [13]. The recorded decrease of the light scattering signal due to the GlpF-mediated ribitol influx was fitted using a single exponential

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decay function to determine the rate constant k.

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Determined rate constants were normalized to the respective expression level. To quantify the

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GlpF expression level, SK46 cells transformed with the respective plasmids were incubated in

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LB medium until an OD600 of 0.8 was reached. Thereafter, cells were treated as described in Klein et al. [27]. GlpF expression was detected by Western blot analysis using an antibody

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directed against the GlpF C-terminus (VVEEKETTTPSEQKASL) [26]. Based on the blot signal

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intensity, the relative expression level was quantified using ImageJ [23] and the rate constants

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determined in the activity assay were normalized to the respective expression level.

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ACCEPTED MANUSCRIPT Results

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Figure 1: Structure of the E. coli aquaglyceroporin GlpF monomer.

Side view (A) and view from the periplasmic side of the membrane (B). The loop regions that are present in GlpF,

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proximity to other loop regions. (PDB-ID: 1FX8).

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but not in AqpZ [28], are depicted in yellow. Trp219 (red) faces inside the GlpF monomer and is located in

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Based on structural alignments and MD simulations, Wang et al. [28] have identified four

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extensions of periplasmic loops within GlpF that do not exist in the classical E. coli aquaporin AqpZ (Fig. 1, yellow). Based on a sequence alignment, part of the loop E region (LAGW) is

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extremely conserved in all human organisms and other vertebrates (Fig. S1). Especially, one Trp

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residue within this region (Trp219 in GlpF) was noticeable, as it is highly conserved even in distantly related groups of pathogens, such as Leishmaniae [29] and Pseudomonae. In the rare cases where this residue is absent, this Trp is replaced by another aromatic amino acid, as e.g. observed in Plasmodium or Caenorhabditis, where this Trp is replaced by Tyr. The Trp219containing part of loop E is in the proximity of a loop C region that is also present in GlpF, but not in AqpZ (Fig. 1B). Often, Trp residues accumulate in membrane proteins at the membranewater interface, where they are supposed to fulfill anchoring functions to prevent hydrophobic mismatch [30-32]. However, the here investigated conserved Trp is deeply protruding inside the 9

ACCEPTED MANUSCRIPT protein and is therefore exposed to a variety of potential interaction partners. Thus, Trp219 likely is of general importance for the stability and/or activity of aquaglyceroporins and is key for structuring the extended aquaglyceroporin-specific loop C and E regions.

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3.1 Trp219 influences the activity of GlpF

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To determine the importance of Trp219 for the activity of GlpF, Trp219 was mutated to Ala. Ala was used since it has no statistical preference for any position within membrane integral regions

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[33] and due to its average hydrophobic character [34]. However, compared to other amino acids,

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Trp is unique in its chemistry and size, but might be replaced by Phe to preserve the hydrophobic and aromatic nature of Trp, a trick often used in spectroscopic analyses (e.g. [35]). Nevertheless,

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the Trp indole NH-group allows the formation of specific H-bonds, which is not at all mimicked

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by Phe. To partly circumvent this problem, Trp can also be replaced by Tyr that contains a highly reactive hydroxy group. Nevertheless, one has to keep in mind that the H-bonding potential of

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Trp is not always properly mimicked by Tyr, and a Trp-to-Tyr mutation might significantly

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influence the stability of proteins [36, 37]. To assess the activity of the modified proteins, an in vivo stopped flow activity assay was

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performed using E. coli cells expressing wt and modified GlpF, respectively. After mixing an E. coli cell suspension with a hypertonic solution of the linear polyalcohol ribitol, E. coli cells started shrinking owing to osmosis, resulting in an increase in light scattering (Fig. 2A). In our experiments, ribitol was used instead of glycerol due to its lower natural permeability through the E. coli inner membrane [13]. When ribitol flux across the E. coli inner membrane was mediated by expression of an active GlpF protein, E. coli cells re-swelled, resulting in a decreased lightscattering signal (Fig. 2A). From these light-scattering curves, rate constants were determined, 10

ACCEPTED MANUSCRIPT using a single exponential fit to describe the progression of the ribitol influx. Fig. 2B depicts the normalized ribitol influx determined for the wt and mutated GlpF proteins, respectively. As the expression levels of the respective proteins differed in some cases (Fig. 2C), the determined rate

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constants were normalized to the individual protein levels, as described in the Methods section.

Figure 2: Activities of GlpF wt and Trp219 mutated proteins as determined using an in vivo stopped flow

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activity assay.

(A) Representative light-scattering signals for GlpF wt (black), GlpF W219A (red) and GlpF-free control cells (negative control, green). Light scattering decreases slower for GlpF W219A, indicating an impaired ribitol flux

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compared to the wt. Curves are an average of ten measurements. (B) Rate constants were determined relative to the GlpF wt as shown in A (n = 3 ± SD). Rate constants were determined by an approximation of the light scattering curves, using a single exponential decay function. Expression levels of GlpF wt and the mutant proteins were assessed via Western blot analyses and included in the calculation. W219A and W219Y showed a decreased activity. *: p<0.05. n.s.: not significant. (C) Representative Western blot of SDS-denatured GlpF wt and mutant proteins. 2.4 µg of total membrane protein was separated on a 10% SDS-PAGE gel, and GlpF was detected using an anti-GlpF antibody to determine the relative expression levels. Both image sections were taken from the same blot.

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ACCEPTED MANUSCRIPT Replacement of Trp219 by Ala resulted in a diminished activity as visible in the decreased slope of the light scattering signal in comparison to the wt (Fig. 2A). A decreased slope indicates a slower ribitol influx into the E. coli cell and results in a lower rate constant. In fact, replacement of Trp219 by Ala resulted in an about 50% reduced ribitol flux compared to the wt (Fig. 2B).

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Thus, Trp219 is crucial for the activity of GlpF. However, whether this is purely due to an impact

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of Trp219 on the protein structure or whether the periplasmic loop region is directly involved in

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substrate conduction, cannot be deduced from the current measurements. Interestingly, for the W219F mutant, the measurement confirmed a wt-like activity, indicating that an exchange of Trp

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to Phe at this position preserves the GlpF activity (Fig. 2B). In contrast, replacement of Trp219

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by Tyr resulted in a GlpF variant with a ~30% reduced activity (Fig. 2B), which is, however, statistically not significant. Nevertheless, this indicates that a minor change in the aromatic side-

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chain chemistry (from Phe to Tyr) can already have an impact on the GlpF activity. It is worth

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mentioning that, compared to the other mutants, GlpF W219A was expressed only in very low amounts (Fig. 2C), indicating a special relevance of an aromatic residue at this position.

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However, as the reduced expression level was included in our activity estimation, this does not

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explain the low glycerol flux through GlpF W219A.

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To reveal whether the observed changes in the activity are accompanied by changes in the GlpF structure, we have next analyzed the oligomeric state of the GlpF Trp219 mutant proteins, as the GlpF activity appears to be connected to the proteins´ oligomeric state [26].

3.2 Trp219 is crucial for formation of a stable GlpF tetramer The structure, activity and assembly of GlpF has already been analyzed and is understood to some extent [4, 11, 13, 16, 38-40]. The tetrameric structure of GlpF is strongly preserved during 12

ACCEPTED MANUSCRIPT purification, and can even be resolved by semi-native SDS-PAGE analyses (Fig. 3) [11, 24, 41]. Thus, we have applied this method to determine the tetramer stability of the modified GlpF variants. As can be seen in Fig. 3, the isolated GlpF wt tetramer is visible on an SDS gel as a prominent band with an apparent molecular mass of ~100 kDa. The discrepancy between the

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calculated (121 kDa) and the actual molecular mass on SDS gels occurs due to gel shifting [42].

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Apart from the wt, pronounced tetramer bands were also visible in case of the purified GlpF

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mutant protein W219F, indicating that an exchange of Trp to Phe at this position does not significantly disturb the formation and stability of the GlpF tetramer, even though a small

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monomer fraction at ~30 kDa is visible in the SDS gel. However, the tetrameric structure of GlpF

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W219A and W219Y is clearly destabilized and the monomeric form was favored, at least in

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mixed DDM/SDS micelles (Fig. 3).

Figure 3: Stability of GlpF wt tetramers and the Trp219 mutated proteins. SDS gel of purified GlpF wt, GlpF W219A, W219F and W219Y. Protein samples were incubated in sample buffer (50 mM Tris-HCl (pH 6.8), 10% (v/v) glycerol, 0.04% bromphenol blue) and separated on a 10% SDS gel. Molecular masses (M) of the protein standards are given in kDa on the left. GlpF W219F shows a distinct tetramer band at ~ 100 kDa, whereas GlpF W219A and W219Y are mainly monomeric (~ 30 kDa). The GlpF dimer is visible as a band at ~ 50 kDa.

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ACCEPTED MANUSCRIPT To investigate the stability of the GlpF mutants in greater detail, unfolding of the proteins was next monitored by adding increasing amounts of SDS to the protein dissolved in 5 mM DDM, resulting in the formation of mixed SDS/DDM micelles with increasing SDS contents (Fig. 4A). To circumvent the distinction between SDS in bulk solution and SDS inserted into mixed

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micelles, SDS mole fractions (χSDS) from total detergent concentrations were calculated instead of

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the estimated concentrations in micelles [43]. At each SDS concentration, the oligomeric state of

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GlpF was monitored via semi-native SDS-PAGE analysis (Fig. 4A). SDS-induced deoligomerization of GlpF is shown in Fig. 4A for the wt protein, demonstrating a continuous

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dissociation of the tetramer coupled with an increase of the GlpF monomer. The GlpF wt

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tetramer is clearly visible up to an average SDS mole fraction of SDS = 0.76, and a light tetramer band can still be observed at SDS = 0.79 and 0.82. At SDS = 0.88 the GlpF tetramer band is not

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visible anymore. In stark contrast, SDS-induced unfolding of GlpF W219A and W219Y did not

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affect the amount of monomeric vs. oligomeric protein, rather the distribution of tetra-, di- and

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W219Y in Fig. S2).

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monomeric GlpF remained essentially constant at all SDS mole fractions (as shown for GlpF

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ACCEPTED MANUSCRIPT Figure 4: Stability of GlpF wt and GlpF W219F. (A) SDS-induced monomerization of GlpF wt. The GlpF tetramer stability was assessed by unfolding GlpF in presence of increasing SDS amounts followed by SDS-PAGE analysis. Molecular masses (M) of the protein standards are given in kDa on the left. 6.3 µM of GlpF were incubated with increasing concentrations of SDS, treated with sample buffer (50 mM Tris-HCl (pH 6.8), 10% (v/v) glycerol, 0.04% (w/v) bromphenol blue) and separated on a 10% SDS gel. The amount of SDS in the running buffer was constantly included in the determination of the

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micellar mole fraction of SDS (χSDS). The intensity of the band corresponding to the GlpF tetramer (T; ~ 100 kDa)

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constantly decreases with increasing χ SDS, and at χSDS = 0.88 the band of the GlpF tetramer is no longer detectable. The intensity of the band representing the monomer (M) is constantly increasing. (B) The intensities of the GlpF

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tetramer bands were quantified from the SDS gels (see A). Intensities were normalized and plotted against the SDS micellar mole fractions (χSDS). The initial tetramer band intensity of GlpF W219F (red) is similar to the GlpF wt (black) tetramer band intensity but rapidly decreases already at low SDS concentrations, indicating a significantly

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decreased tetramer stability (n = 4 ± SD).

While retaining a tetrameric structure on SDS-gels (Fig. 3), also the W219F mutated protein was

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destabilized when compared to the wt protein (Fig. S3). In Fig. 4B the relative tetramer band

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intensities are shown for GlpF wt and the W219F protein at increasing SDS mole fractions. In contrast to the wt, the GlpF W219F tetramer band intensity decreased already at low SDS mole

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fractions, and the protein reached a fully monomeric state already at SDS = 0.6 (Fig. 4B). Taken

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together, these data suggest that Phe can replace the Trp residue at position 219, but does not

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fully retain the wt-like stability, even though the mutant remains tetrameric at low SDS.

3.3 The Trp219 environment has an effect on the stability and activity of GlpF The above described observations suggest that Trp219 is important for GlpF tetramer stabilization, and an exchange of Trp219 to Ala highly destabilizes the GlpF tetramer. Interestingly, Trp219 is located at the bottom of a periplasmic region of loop E, at the periphery of the GlpF tetramer where it is not in direct contact with any adjacent GlpF monomer (Fig. 1B). 15

ACCEPTED MANUSCRIPT In fact, the Trp219 side-chain is oriented towards the inside of the monomer, facing a variety of potential interaction partners (Fig. 5). Most aromatic interactions occur within a 7 Å range [4447], and within a centroid-centroid distance of 3-7 Å we have identified the aromatic residues Tyr105, Phe112 and Phe135 to potentially interact with Trp219 (Fig. 5). Furthermore, Arg123 is

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located in the direct proximity of Trp219 and potentially interacts with Trp219 via cation-π

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interactions. Just as Trp219, these residues also lie within the vestibule region of GlpF, close to

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other extended loops (compare Fig. 1), and thus structure and/or stabilize this unique region together with Trp219. Consequently, we next studied a potential involvement of these residues in

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GlpF tetramer stability and activity.

Figure 5: Amino acid residues interacting with Trp219.

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Amino acid residues potentially interacting with Trp219 (red) were selected within a radius of 7 Å. The four amino acids Tyr105 (yellow), Phe112 (green), Arg123 (blue) and Phe135 (pink) were further investigated (PDB-ID: 1FX8).

Replacement of the likely Trp219 interaction partners by Ala highly destabilized the GlpF tetramer in all cases (Fig. 6A). GlpF W219A, R123A, Y105A and F112A migrated mainly as a monomer on SDS-gels and essentially no tetramer bands were visible. Furthermore, increasing the SDS (as shown in Fig. 4A for the wt protein) did not alter the band distribution on SDS gels 16

ACCEPTED MANUSCRIPT (not shown), and thus all of the analyzed amino acids are clearly crucial for stabilizing the GlpF tetramer. In line with the results obtained with the W219A mutant, all of these mutants also had an impaired channel activity (Fig. 6B). As described before (Fig. 2B), the activity of W219A

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reached only ~50% of the activity of the GlpF wt, and when the residues interacting with Trp219

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were replaced by Ala, the activity of the mutants was also decreased by ~50-60%. It is worth

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mentioning that we did not succeed in determining the activity of GlpF F135A, as this mutant

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protein did not express in the E. coli SK46 cells used for the activity measurements (Fig. 6C).

Figure 6: Mutating residues surrounding Trp219 affects the stability and activity of GlpF. (A) SDS gel of GlpF wt, W219A and GlpF proteins with the residues surrounding Trp219 replaced by Ala. Molecular masses (M) of the protein standards are given in kDa on the left. Protein solutions were incubated in sample buffer (50 mM Tris-HCl (pH 6.8), 10% (v/v) glycerol, 0.04% (w/v) bromphenol blue) and separated on a 10% SDS gel. The tetramer band of the GlpF wt is visible at ~ 100 kDa. All mutant proteins have a band distribution comparable to GlpF W219A, i.e. the proteins mainly run as monomers (see monomer band at < 30 kDa). F135A could not be expressed in BL21(DE3) cells. (B) Rate constants (relative to the wt) of the analyzed GlpF variants and of GlpF-free control cells (negative control) (n = 3 ± SD). Rate constants were determined by approximation of light scattering curves using a single exponential decay function. Expression levels of GlpF wt and GlpF mutant proteins were assessed via Western blot analyses. GlpF mutant proteins show a 40-60% decreased activity compared to GlpF

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ACCEPTED MANUSCRIPT wt. F112A shows the highest activity, which is, however still decreased by 40% when compared to the wt. *: p<0.05. ***: p<0.01. (C) A representative Western blot of denatured GlpF wt and its corresponding mutant proteins. 2.4 µg of total membrane protein was separated on a 10% SDS gel prior to determination of the relative expression level of the

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investigated mutants using an anti-GlpF antibody. GlpF F135A did not express in SK46 cells.

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Together, Trp219 is crucial for the activity as well as for the tetramer stability of GlpF, which

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might be due to the hydrophobic nature of Trp and its interactions with surrounding amino acid

Discussion

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residues in its proximity.

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4.1 Trp219 is important for the activity and stability of GlpF tetramers

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In the present study, we have elucidated the function of a Trp residue that is highly conserved in aquaglyceroporins, but not in canonical aquaporins, using the E. coli aquaglyceroporin GlpF as a

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model. Based on our results, Trp219 is essential for both GlpF tetramer stability and polyalcohol flux through the protein. Mutation of Trp219, which is positioned far distant from the channel

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pore, resulted in a highly unstable GlpF variant with low activity (Fig. 2B, Fig. 3), underlining the indispensable role of a Trp at this position. Trp219 is part of a region in loop E that is only present in aquaglyceroporins (Fig. S1), and therefore this residue clearly is not necessary for the activity of classical aquaporins. Structural comparisons between GlpF and the E. coli aquaporin AqpZ have shown that this loop belongs to a pair of loop regions in GlpF that greatly contribute to the asymmetric structure on the periplasmic site of the protein [28]. This asymmetric structure is a feature that is common among most aquaglyceroporins and was suggested to be a major 18

ACCEPTED MANUSCRIPT factor in glycerol attraction [28] and high glycerol permeation rates [22]. As Trp219 is a part of one of these loop regions, it might contribute to formation and stability of the periplasmic vestibule region and therefore to proper aquaglyceroporin activity. This assumption is in perfect agreement with the observed decreased activity of the W219A mutant (Fig. 2B). However,

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besides likely being involved in structuring the substrate attracting vestibule region, our results

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clearly show that the loop E region is also crucial for formation and stability of tetrameric GlpF.

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Based on the GlpF crystal structure, a clearly defined oligomerization interface of GlpF is formed

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by the TM helices 1 and 2 that bind to the helices 5 and 6 of an adjacent monomer, primary via hydrophobic, but also via polar interactions [26, 44, 48]. In case of the human AQP1, a three

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amino acid long motif that is involved in interaction of helices 2 and 5 has recently been

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identified to be relevant for tetramerization of the protein [49]. However, these residues are not conserved within the aquaglyceroporin family, and thus likely represent a more specific

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interaction motif. Nevertheless, residues within the soluble loop regions are also in van der Waals

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contact distances in the GlpF tetramer. Thus, it is well possible that the SDS-resistant GlpF tetramer we observed in our analyses is stabilized via interactions of both, the TM and the soluble

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loop regions, whereas only mutations in the TM region dramatically affect the GlpF channel

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activity (at least under the applied experimental conditions). In fact, an interaction network around loop D stabilizes the AQP4 tetramer, but appears to not influence the activity of the individual water channels [50]. In case of GlpF, the exact connection between tetramerization and activity remains largely unclear, even though the activity of a membrane protein and its oligomeric state go hand in hand in several cases [26, 51-53]. However, mutation of Glu43 of the E. coli GlpF resulted in decreased tetramer stability coupled with loss of activity [26]. Importantly, Glu43 appears not to be involved in formation or stabilization of a GlpF monomer but rather stabilizes the tetrameric protein structure [26]. While not finally resolved, the benefit of 19

ACCEPTED MANUSCRIPT tetramerization might lie within the higher rigidity of the tightly packed tetramer [16]. In line with previous analyses of the E. coli GlpF or the human AQP1 proteins, our experiments also show that mutation of a single residue can be sufficient to monomerize the GlpF tetramer [26, 54]. As monomeric GlpF is degraded faster than the tetramer [26], monomerization of GlpF

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might be a safety valve quickly eliminating inactive or impaired GlpF variants [16]. In fact,

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eliminating channels with disturbed selectivity and/or activity might be crucial for cell survival

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[55].

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4.2 Trp219 is part of a cluster stabilizing the GlpF tetramer

The observed impact of the Trp219 mutations on tetramer assembly indicates that stable

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interactions of Trp219 with surrounding residues are key for the GlpF tetramer stability. In

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contrast to the remaining Trp residues of GlpF, the Trp219 side-chain faces the protein interior without having an evident role at this position. Inside the GlpF loop region, Trp219 likely

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interacts with the aromatic amino acids Phe112, Phe135 and Tyr105.

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Both, Phe112 and Phe135, are in a distance to interact with Trp219 (6.8, and 6.1 Å, respectively), even though they do not show a typical edge-to-face or offset geometry common for aromatic

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interactions [47]. In contrast to Phe135, Phe112 is not strictly conserved in aquaglyceroporins and is often replaced by other hydrophobic amino acids, such as Leu or Ile. Mutation of Phe112 clearly destabilizes the GlpF tetramer, as observed after mutation of several other residues surrounding Trp219 (Fig. 6A). However, the impact of the F112A mutation on the GlpF substrate flux was less pronounced when compared to the remaining mutants (Fig. 6B), suggesting that Phe112 is a less important, although still notable, Trp219 interaction partner.

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ACCEPTED MANUSCRIPT GlpF Y105A had the same tendency to monomerize as the W219A variant and exhibited a comparably weak ribitol-conducting activity (Fig. 6). The observation that Tyr105 is replaced by Phe in several other aquaglyceroporins indicates a crucial aromatic interaction with Trp219. However, Tyr105 can also form a hydrogen bond with Arg123, and Arg123 is potentially also

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involved in a cation-π interaction with Trp219 (Fig. 5). The observation that mutation of Arg123

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and Tyr105 resulted in a protein stability and activity similar to the W219A mutant (Fig. 6A)

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supports such an assumption.

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Unfortunately, we were unable to deduce a potential functional interaction of Trp219 with Phe135, since we did not succeed in expressing the F135A mutated protein in E. coli. However,

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this observation already strongly suggests a vital function of Phe135 in GlpF. Just as Trp219,

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Phe135 is highly conserved in aquaglyceroporins. Only in case of the P. falciparum aquaglyceroporin PfAQP this residue is replaced by a Trp, albeit here a Tyr is located at the

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Trp219 homologous position [56]. Thus, likely an aromatic interaction between a Trp and a

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Phe/Tyr at positions 219 and 135 (positions 208 and 124 in PfAQP, respectively) is crucial to maintain wt stability and activity. The PfAQP residue Trp124 resides on the same position as

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Phe135 in GlpF on the C loop at the pore mouth of the channel. Trp124 is significantly involved

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in water permeation through PfAQP, and it forms a hydrogen bond with Arg196, a key residue of the selectivity filter that regulates the water passage inside the pore [56]. In GlpF, Phe135 is located at the Trp124-equivalent position and is indispensable for the water and glycerol conducting activity [20]. Due to its importance as well as its central position within the selectivity filter, it is not surprising that E. coli failed to stably express the Phe135 mutated protein. Together, Trp219 and its interaction with surrounding amino acids are necessary for formation, stability and activity of the GlpF tetramer.

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ACCEPTED MANUSCRIPT 5.

Conclusion

In this study we have investigated the impact Trp219, an amino acid highly conserved within the loop E region of aquaglyceroporins, has on the stability and activity on the tetrameric aquaglyceroporin GlpF. Based on our results, Trp219 is crucial for formation and/or stabilization

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of the GlpF tetramer. While this suggests a key role of Trp219 and loop E in the activity of

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aquaglyceroproins, we here show that formation of an aromatic cluster around Trp219, involving

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loop C residues, stabilizes tetrameric GlpF. A subtle network of aromatic interactions (potentially

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involving electrostatic interactions as well) around Trp219 likely is relevant for formation and stability of a periplasmic vestibule region crucially involved in glycerol attraction. However, out

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of the four extended loop regions that are present in the E. coli aquaglyceroproin GlpF but not in

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the E. coli classical aquaporin AqpZ [28], solely the Trp219-containing extended loop E is also highly conserved in other aquaglyceroporins. Thus, we propose that the periplasmic vestibule

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aquaglyceroporins in general.

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region formed by loop E containing Trp219 is crucial for stabilizing the oligomeric structure of

Acknowledgements

We thank Hildegard Pearson for critically reading the manuscript. We also thank E. Bremer (University of Marburg, Marburg, Germany) for the kind gift of SK46 cells.

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ACCEPTED MANUSCRIPT Funding This work was supported by a grant from the Stiftung Rheinland-Pfalz für Innovation and the

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Stipendienstiftung Rheinland-Pfalz.

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ACCEPTED MANUSCRIPT Highlights

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A Trp residue is conserved within the loop E region of aquaglyceroporins. Mutation of Trp219 affects the GlpF activity. An aromatic cluster involving Trp219 stabilizes the active GlpF tetramer. A Trp residue likely is key for stability and activity of all aquaglyceroporins.

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27