Stockholm University

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Citation for the published paper:Unnerståle, S., Mäler, L., Draheim, R. (2011)"Structural characterization of AS1-membrane interactions from a subset of HAMPdomains"Biochimica et Biophysica Acta - Biomembranes, 1808(10): 2403-2412

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Structural characterization of AS1-membrane interactions 10 from a subset of HAMP domains 11

12 13 14

Sofia Unnerståle1, Lena Mäler1* and Roger R. Draheim1,2* 15 16 17 18 19 20 21 22 23 24 25 1Department of Biochemistry and Biophysics, Center for Biomembrane Research, The 26 Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 27 Stockholm, Sweden; 2Institute of Biochemistry, Biocenter, Goethe University Frankfurt, 28 D-60438 Frankfurt, Germany 29 30 31 Running Title: Structural characterization of AS1-membrane interactions 32 33 *Corresponding authors (L. M. and R. R. D.): 34 35 Department of Biochemistry and Biophysics Institute of Biochemistry 36 Stockholm University Biocenter (N210 1.08) 37 Svante Arrhenius väg 16C Max-von-Laue Str 9 38 SE-10691 Stockholm D-60438 Frankfurt 39 Sweden Germany 40 Tel: +46 8 16 2448 +49 69798 29468 41 Fax: +46 8 15 5597 +49 69798 29495 42 Email: lena@dbb.su.se draheim@biochem.uni-frankfurt.de 43

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    2

Abstract 1

HAMP domains convert an extracellular sensory input into an intracellular 2

signaling response in a wide variety of membrane-embedded bacterial proteins. These 3

domains are almost invariably found adjacent to the inner leaflet of the cell membrane. 4

We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of 5

four different, well-characterized HAMP domains with several membrane model systems. 6

The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli 7

osmosensor EnvZEc, the E. coli nitrate/nitrite sensor NarXEc, and the aspartate 8

chemoreceptor of E. coli (TarEc). Far-UV CD and NMR spectroscopy were used to 9

monitor the induction of secondary structure upon association with neutral or acidic large 10

unilamellar vesicles (LUVs) and bicelles. We observed significant increases in α-helicity 11

within AS1 from NarXEc and TarEc but not in AS1 from the other proteins. To 12

characterize these interactions further, we determined the solution structure of AS1 from 13

TarEc associated with acidic bicelles. The bulk of AS1 formed an amphipathic α-helix, 14

whereas the N-terminal control cable, the region between TM2 and AS1, remained 15

unstructured. We observed that the conserved prolyl residue found in AS1 of many 16

membrane-adjacent HAMP domains defined the boundary between the unstructured and 17

helical regions. In addition, two positively charged residues that flank the hydrophobic 18

surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We 19

interpret these results within the context of the helix interaction model for HAMP 20

signaling and propose roles for AS1-membrane interactions during the membrane 21

assembly and in transmembrane communication of HAMP-containing receptors. 22

23

    3

Keywords: HAMP domain; signal transduction; transmembrane communication; AS1-1

membrane interactions; helix interaction model 2

3

Abbreviations: HAMP domain (domain present in histidine kinases, adenylyl cyclases, 4

methyl-accepting proteins and phosphatases); SHK, sensor histidine kinase; MCP, 5

methyl-accepting chemotaxis protein; TM1, first transmembrane domain; TM2, second 6

transmembrane domain; AS1, first amphipathic sequence; AS2, second amphipathic 7

sequence; AS1p, AS1 containing peptide; AS2p, AS2 containing peptide; CTR, 8

connector domain; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-9

palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); LUV, large unilamellar 10

vesicle; DHPC, 1,2-dihexanoyl-d22-sn-glycero-3-phosphocholine; DMPC, 1,2-11

dimyristoyl-d54-sn-glycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-d54-sn-glycero-3-12

phospho-(1’-rac-glycerol); Far-UV CD, Far-ultraviolet circular dicroism; PFG-NMR, 13

Pulsed field gradient-nuclear magnetic resonance; TOCSY, Total correlation 14

spectroscopy; NOESY, Nuclear Overhauser effect spectroscopy; PD1, periplasmic 15

domain 1; PD4, periplasmic domain 4. 16

17

Highlights: 18

1. AS1-containing peptides from NarXEc and TarEc become structured in the 19

presence of membrane mimetics. 20

2. AS1 from TarEc forms an amphipathic α-helix upon association with acidic 21

bicelles. 22

3. Residues comprising the control cable of TarEc remain unstructured. 23

    4

4. A conserved prolyl residue separates the unstructured and helical regions. 1

5. The propensity for AS1-membrane interactions correlates with signaling 2

mechanisms and subcellular localization. 3

4

    5

1. Introduction 1

2

HAMP domains (recently reviewed in [1]) are found adjacent to the inner leaflet 3

of the cytoplasmic membrane within many prokaryotic membrane-spanning receptors [2], 4

including well-characterized sensor histidine kinases (SHKs) and methyl-accepting 5

chemotaxis proteins (MCPs) that serve as chemoreceptors. These domains have been 6

identified within more than 5500 different proteins [2]. The first HAMP domain was 7

identified by genetic analysis of the region joining the second transmembrane helix 8

(TM2) of the serine chemoreceptor of Escherichia coli (TsrEc) and the cytoplasmic 9

domain responsible for signal propagation [3]. A subsequent bioinformatic analysis 10

demonstrated that similar linker regions exist within four protein families: histidine 11

kinases, adenlyate cyclases, methyl-accepting chemotaxis proteins and phosphatases [4]. 12

Although HAMP domains are poorly conserved at the sequence level, they remain highly 13

conserved at the level of secondary structure, consisting of two amphipathic sequences 14

(AS1 and AS2) that form α-helices joined by a non-helical connector (CTR) [3, 5, 6]. 15

Two models have been proposed for how HAMP domains receive sensory input 16

from periplasmic or membrane-embedded stimulus-sensing domains. The crankshaft and 17

gearbox model is based on the high-resolution structures of the HAMP domain of the 18

Archaeoglobus fulgidus protein (Af1503) [7, 8], which reveal that AS1 and AS2 of each 19

subunit of a homodimeric protein form a parallel four-helix bundle. This model proposes 20

that the C-terminal transmembrane helix (TM2) undergoes an axial rotation that causes a 21

concerted rotation of AS1. The second model suggests that a small (∼1-3 Å) piston-type 22

    6

displacement of TM2 perpendicular to the plane of the membrane occurs upon binding of 1

a ligand to the periplasmic domain of various SHKs and chemoreceptors [9-17]. 2

The second model has led to three different explanations for how these piston-3

type displacements are coupled to alterations in HAMP domain configuration. The first 4

proposes that a piston-type displacement of a relatively rigid connection between TM2 5

and AS1 [1, 18, 19] facilitates a scissor or pivoting-type motion within the HAMP 6

domain [20]. The second, supported by extensive mutational evidence within an intact 7

chemoreceptor (TsrEc) [18, 21, 22], proposes that displacements of TM2 modulate the 8

overall structural stability of the HAMP domain bundle via changing tension of a 9

sequence connecting TM2 to AS1, referred to as the control cable. The third explanation 10

proposes that two HAMP conformations are in equilibrium: one in which the 11

hydrophobic face of AS1 interacts with the inner leaflet of the membrane, and another in 12

which AS1 interacts with AS2 [6]. 13

None of the previous studies directly assessed the influence of the membrane on 14

the sensor mechanism. The work presented here was undertaken to determine whether 15

AS1 is capable of association with a phospholipid bilayer. We chose four well-16

characterized HAMP domains and used circular dicroism (CD) and solution NMR to 17

monitor the ability of their AS1 or AS2 peptides to interact with various model 18

membrane systems. The Archaeoglobus fulgidus Af1503 protein was chosen because it 19

was used for the high-resolution structures [7, 8]. The E. coli EnvZ osmosensor (EnvZEc) 20

and the nitrate/nitrite-sensing histidine kinase (NarXEc) were selected because their 21

HAMP domains have been extensively characterized [23-26]. Finally, the E. coli 22

aspartate chemoreceptor (TarEc) was analyzed because it is a well-characterized 23

    7

chemoreceptor [9-13, 20] and extensive genetic studies have been performed with the 1

closely related serine chemoreceptor TsrEc, which have formed the basis for the dynamic 2

bundle model of HAMP signaling [1, 18, 21, 22]. 3

4

2. Materials and Methods 5

6

2.1 Materials 7

8

Synthetic peptides corresponding to either AS1 or AS2 (denoted AS1p and AS2p, 9

respectively) were analyzed in the presence of different membrane model systems. The 10

initial high-resolution three-dimensional structure of Af1503 HAMP (S278-V328) [7] 11

served as the template for peptide design. Residues that form the flexible connector 12

(G297-A309) were excluded from AS1p-Af1503 and AS2p-Af1503. For EnvZEc, NarXEc 13

and TarEc, similar peptides were constructed based on previously published [7, 23, 27] 14

residue alignments. The resulting eight peptides are shown in Table 1. They were 15

obtained from PolyPeptide Group (Strasbourg, France) and used without further 16

purification. 17

Large unilamellar vesicles (LUVs) and phospholipid bicelles were used as 18

membrane model systems. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 19

and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) were used to 20

produce LUVs. 1,2-dihexanoyl-d22-sn-glycero-3-phosphocholine (DHPC), 1,2-21

dimyristoyl-d54-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-d54-sn-22

    8

glycero-3-phospho-(1’-rac-glycerol) (DMPG) were used to produce phospholipid 1

bicelles. Phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL, USA). 2

3

2.2 Preparation of samples containing LUVs for CD measurements 4

5

LUVs with a diameter of 100 nm were used for circular dichroism (CD) 6

measurements because their large size and low curvature was deemed appropriate for 7

studies of peptide-membrane interaction [28]. In order to produce neutral and acidic 8

LUVs, POPC or a 4:1 mixture of POPC/POPG, respectively, were initially dissolved in 9

chloroform to produce 20 mM stock solutions. The solutions were subsequently dried 10

under a flow of nitrogen gas and stored under vacuum overnight to ensure that no solvent 11

remained. These dried lipid films were soaked in 100 mM sodium phosphate buffer (pH 12

7.2) and vortexed for 10 minutes to obtain a more defined size distribution multilamellar 13

vesicles. These solutions were then subjected to five freeze-thaw cycles to decrease 14

lamellarity. Finally, to obtain uniform LUVs, the samples were extruded approximately 15

20 times through a polycarbonate microfilter with a pore size of 100 nm. 16

Peptide samples were prepared by dilution from the stock solutions of 20 mM 17

LUVs, 1 mM AS1-, and 1 mM AS2-containing peptides to a final mixture containing 50 18

µM peptide, 50 mM sodium phosphate buffer (pH 7.2), and 1 mM neutral or acidic 19

LUVs. This low concentration of LUVs was chosen to diminish disturbances due to light 20

scattering. Samples containing a mixture of 25 µM AS1 and 25 µM AS2 peptides were 21

also prepared for analysis. To study the nature of the interaction between the peptide and 22

    9

the LUVs, 150 mM KF was added to appropriate samples [29]. KF was used because 1

chloride salts are known to interfere with far-UV CD spectra [30]. 2

3

2.3 Preparation of phospholipid bicelles for CD and NMR measurements 4

5

Small phospholipid bicelles with a q-ratio between 0.25-0.5 form isotropic 6

solutions that are especially suitable for high-resolution membrane-interaction studies by 7

NMR [Sofias new references, see attached file]. The q-ratio is the ratio of long-chained 8

phospholipids [DMPC and DMPG] to short-chained phospholipids [DHPC]. 9

Samples for pulsed-field-gradient NMR (PFG NMR) were prepared in D2O to 10

avoid severe baseline distortions in spectra originating from the water signal. Initially, a 11

solution of 50 mM sodium phosphate (pH 7.2) was dried and subsequently dissolved in 12

D2O. DMPC or a 4:1 mixture DMPC/DMPG was lyophilized in this solution and 13

subsequently mixed with the desired amount of a 1 M stock solution of DHPC in D2O. 14

This mixture was vortexed until a clear solution was obtained. The final concentration of 15

phospholipid was maintained at 150 mM and the q-ratio was maintained at 0.5. AS1 or 16

AS2 peptides were then added to the phospholipid bicelle solution to yield a peptide 17

concentration of 500 µM. For measurement of peptide diffusion in the absence of bicelles 18

(Dfree), AS1 or AS2 peptides were also added to 50 mM sodium phosphate (pH 7.2) in 19

D2O to a final concentration of 500 µM. 20

For far-UV CD measurements, samples were prepared in the same manner except 21

that H2O was used instead of D2O, resulting in solutions containing 500 µM peptide in 50 22

mM sodium phosphate (pH 7.2) or a mixture of 500 µM peptide and 150 mM neutral 23

    10

(DMPC) or acidic (4:1 mixture DMPC/DMPG) bicelles (q = 0.5) in 50 mM sodium 1

phosphate (pH 7.2). 2

To prepare acidic phospholipid bicelles for 2D 1H-1H NMR analysis, similar 3

mixtures were made with slightly altered phospholipid ratios to arrive at a final mixture 4

containing 300 mM [DMPC/DMPG 9:1]/DHPC bicelles with a q-ratio of 0.25 in 50 mM 5

sodium phosphate (pH 7.2) in H2O. These changes in q-ratio, total lipid concentration and 6

charge of the phospholipid bicelle were made to assure collection of usable NMR spectra 7

with acceptable line broadening. 2 mM AS1p-TarEc was dissolved in this bicelle solution. 8

Finally, 10% D2O was added to achieve field/frequency lock stabilization during NMR 9

experiments. 10

11

2.4 Far-UV circular dicroism (Far-UV CD) spectroscopy 12

13

Far-UV CD spectra were acquired on a Chirascan CD spectrometer. A 1 mm 14

quartz cell was used for the samples with 50 µM peptide concentration, and a 0.1 mm 15

quartz cell was used for the samples with 500 µM peptide concentration. The temperature 16

was maintained at 25 ˚C with a TC 125 temperature control. Wavelengths ranging from 17

190 to 260 nm were measured with a 0.5-nm step resolution. Spectra were collected and 18

averaged over ten measurements. Background spectra of the sodium phosphate buffer, 19

LUVs, or bicelles without peptide were subtracted where appropriate. The CD spectra 20

were further analyzed with Dichroweb [31] using the CONTIN method [32] with 21

reference set 7, optimized for the range 190-240 nm. 22

23

    11

2.5 NMR spectroscopy 1

2

All 1H-NMR experiments were performed on a Bruker Avance spectrometer, 3

equipped with a triple resonance probe head and operating at a 1H frequency of 600 MHz. 4

For translational diffusion analysis, a standard sample of 0.01% H2O in D2O and 1 mg/ml 5

GdCl3 was used for gradient calibration at 25 ˚C. Diffusion coefficients were measured 6

using a modified Stejskal-Tanner spin-echo experiment [33-35] with a fixed diffusion 7

time and gradient length with a gradient strength that increased linearly over 32 steps. 8

The attenuation of the signal, as a result of the increasing gradient, is described by the 9

Stejskal-Tanner equation [34]. The intensity of the signal was measured and plotted 10

against the corresponding gradient strength according to this equation and the diffusion 11

coefficient was deduced from the slope of the resulting curve. The linearity of the 12

gradient was calibrated as described previously [36]. The diffusion coefficient for HDO 13

was measured and divided by the standard diffusion of HDO in D2O (1.9 10-9 m2/s) [37]. 14

This factor was then multiplied by all measured diffusion coefficient, to correct for 15

viscosity differences induced by the sample. To estimate the amount of peptide bound to 16

the bicelle, a two-state model was used. In this model, it is assumed that the peptide is 17

either bound to the bicelle, and thus diffuses with the rate of the bicelle (Dbicelle), or the 18

peptide is free, and thus diffuses with the diffusion rate of the peptide in buffer (Dfree). 19

Dcomplex is the diffusion time of the peptide in the presence of bicelles and x is the fraction 20

of peptide molecules bound to the phospholipid bicelle. 21

22

x = (Dcomplex – Dfree) / (Dbicelle – Dfree) Equation 1. 23

    12

1

Two-dimensional TOCSY [38], and NOESY [39] spectra were recorded for 2

AS1p-TarEc in the presence of q=0.25 300 mM acidic phospholipid bicelles. The TOCSY 3

spectra were recorded with mixing times of 30, 60 and 85 ms, and the NOESY spectra 4

were recorded with mixing times of 100, 150, 250 and 300 ms. The 150 ms and the 250 5

ms NOESY spectra were used for assigning distance constraints. Spectra were typically 6

collected as 2048−4096 x 512 data point matrices using 32−64 scans. Water suppression 7

was achieved using excitation sculpting. The spectra were processed with Topspin 8

version 2.1, and spectral analysis was performed with Sparky 3 [40]. All NMR 9

experiments were performed at 25 °C. 10

11

2.6 Structure calculation 12

13

The spectra collected from AS1p-TarEc were partly obscured by large peaks from 14

the phospholipid bicelles. Therefore, cross-peaks from both the 150 and 250 ms mixing 15

time NOESY had to be used to derive distance constraints for calculation of a solution 16

structure for AS1p-TarEc. In all, 133 distance constraints were assigned (59 intraresidue, 17

41 sequential, and 33 medium-range). The cross-peak intensities were initially converted 18

to distances using routines in CYANA 2.0 [41]. These distances were subsequently 19

altered manually in several stages as described previously [42]. Structures were generated 20

using CYANA, applying standard annealing algorithms. Analyses of the structures, 21

including secondary structure and backbone dihedral angles, were performed with 22

PROCHECK NMR [43]. A total of 100 structures were calculated, and a final ensemble 23

    13

of 25 structures was selected, on the basis of the CYANA target function, to represent the 1

final solution structure. The coordinates of the final ensemble of structures and the 2

distance constraints have been deposited in the Protein Data Bank as entry 2l9G. The 3

chemical shift assignments have been deposited within the BMRB as entry 17450. 4

5

3. Results 6

7

3.1 LUVs induce α-helical content in AS1 peptides from the NarXEc and TarEc 8

HAMP domains 9

10

The secondary structure within the various AS1 and AS2 peptides was 11

investigated using far-UV CD spectroscopy. The peptides were analyzed under three 12

different experimental conditions: buffered aqueous solution of sodium phosphate at pH 13

7.2, the same buffered solution with the addition of 1 mM LUVs composed of POPC, and 14

finally, the same buffered solution with the addition of 1 mM LUVs composed of a 4:1 15

mixture of POPC and POPG (Figure 1). These conditions were selected to study whether 16

secondary structure was induced by the presence of LUVs and to investigate whether the 17

charge on the LUV surface is important for structural induction. The CD data were then 18

analyzed with the CONTIN method to estimate the amount of helical secondary structure 19

(Figure 2). 20

In phosphate buffer, random coil features remained dominant for all the AS1 21

peptides. Upon addition of LUVs consisting of POPC, we observed a 5-fold induction of 22

α-helical content in AS1p-TarEc. Addition of LUVs consisting of a 4:1 mixture of 23

    14

POPC/POPG caused a 3.4-fold induction of α-helical content in AS1p-NarXEc and a 1

futher induction of α-helical content in AS1p-TarEc to 8 times the amount observed in 2

phosphate buffer. We observed no significant induction of α-helical charcater in any of 3

the AS2 peptides. However, AS2p-Af1503 exhibited a consistent intrinsic (∼ 16%) α-4

helical character under all three conditions. The amount of helix, as determined from the 5

analysis of CD data, can be interpreted as the peptide possessing this extent of α-helical 6

structure, but it is not possible to discern whether only part of the peptide is structured or 7

if this number represents an average over time. It is also worth nothing that it is much less 8

difficult to quantify differences in structural content than to determine abolsute structural 9

content. Nevertheless, a significant part of this AS2 peptide is on avarage helical, 10

regardless of solvent. 11

Even though the Af1503 HAMP domain possesses extensive secondary structure 12

[7, 8], our results indicate that the AS1p-Af1503 peptide is predominately unordered on 13

its own. We therefore asked whether the AS1 and AS2 peptides when present together 14

would take on increased helical structure. However, the far-UV CD spectra from the 15

mixtures of all four cognate pairs of AS1 and AS2 peptides were simply composites of 16

the average spectral properties of the two peptides measured individually (Figure 1). 17

This result is perhaps not surprising, because the intervening CTR has been implicated in 18

stabilization of the HAMP bundle [22]. Notably, an intact EnvZEc HAMP domain 19

containing a single point mutation (R218K) introduced to aid in purification has also been 20

shown to be unstructured [27]. 21

To investigate the nature of the association between the LUVs and AS1p-NarXEc, 22

or AS1p-TarEc, we monitored the influence of addition of 150 mM KF to the samples 23

    15

(Figure 3). In the peptide samples with neutral LUVs, no difference was seen in the CD 1

spectra, suggesting that AS1p-TarEc interacts with neutral LUVs predominately via 2

hydrophobic interactions. In contrast, addition of KF to the peptide samples with acidic 3

LUVs, resulted in a decrease of the helical content of AS1p-NarXEc and AS1p-TarEc. 4

Raising the ionic strength of the buffer should yield a similar result to lowering the 5

surface charge of the LUV. Thus, the α-helical induction of AS1p-NarXEc and the extra 6

α-helical induction of AS1p-TarEc seen with acidic LUVs is probably caused by 7

electrostatic interactions between the lipids and the peptide. 8

9

3.2 AS1p-NarXEc, AS1p-TarEc, and AS2p-Af1503 interact with phospholipid 10

bicelles 11

12

To study the interaction of the peptides with membranes in further detail, 13

translational-diffusion measurements were used to quantify the extent to which the 14

peptides interact with membrane. Here, isotropic phospholipid bicelles were used as a 15

membrane mimetic, since they are more suitable for NMR measurements [Sofias new 16

number 41]. The peptides were analyzed under three different experimental conditions: 17

buffered aqueous solution of sodium phosphate at pH 7.2, the same buffered solution 18

with the addition of 150 mM DMPC/DHPC bicelles (q=0.5), and finally, the same 19

solution with the addition of 150 mM [DMPC/DMPG 4:1]/DHPC bicelles (q=0.5). All 20

samples were dissolved in D2O. The derived translational-diffusion cofficients were then 21

used to estimate how much peptide that binds to the membrane (Table 2). 22

    16

The results in Table 2 show that AS1p-NarXEc exhibits a diffusion coefficient of 1

1.5⋅10-10 m2/s in phosphate buffer, a value consistent with the translational motion of a 2

peptide of this size [44]. The diffusion coefficient was reduced to 0.40⋅10-10 m2/s in the 3

presence of neutral bicelles and to 0.35⋅10-10 m2/s in the presence of acidic bicelles, 4

values comparable to the coefficients for the bicelles alone (0.38⋅10-10 m2/s and 0.35⋅10-10 5

m2/s, respectively). AS1p-TarEc diffuses at 1.4⋅10-10 m2/s in phosphate buffer, and this 6

value fell to 0.38⋅10-10 m2/s in the presence of neutral bicelles and to 0.36⋅10-10 m2/s in the 7

presence of acidic bicelles. AS2p-Af1503 is also seen to interact strongly with the 8

bicelles. For the other AS1- and AS2-containing peptides examined, all show a slight 9

reduction in the diffusion coefficient upon addition of bicelles, which suggests that a 10

smaller proportion of the peptide is interacting with the bicelles, but none were found to 11

completely associate with the bicelles extensively. 12

13

3.3 Phospholipid bicelles induce α-helical content in AS1p-NarXEc, AS1p-TarEc 14

and AS2p-Af1503 15

16

AS2p-Af1503 was shown to bind completely to the phospholipid bicelles despite 17

showing no additional induction of secondary structure in the presence of LUVs. To 18

examine the efficacy of bicelles in inducing structural changes, far-UV CD measurements 19

were performed in samples of the same composition as in the PFG-NMR experiments, 20

with the only difference being that these samples were dissolved in H2O instead of D2O. 21

From these spectra (Figure 4), the extent of helical secondary structure was estimated 22

(Figure 5). 23

    17

The results were similar to those obtained when LUVs were employed. Of the eight 1

peptides examined, only AS1p-NarXEc, AS1p-TarEc and AS2p-Af1503 showed 2

significant secondary structure in the presence of bicelles. Addition of neutral bicelles 3

caused a 9-fold induction of helical character in AS1p-NarXEc. Thus, AS1p-NarXEc 4

interacted with neutral bicelles but not with neutral LUVs. Although some differences 5

exist between LUVs and bicelles, it is important to note that the concentration of LUVs 6

was only 1 mM, whereas the concentration of bicelles was 150 mM. For AS1p-TarEc, an 7

11-fold induction in helical character was observed with neutral bicelles. In the presence 8

of acidic bicelles, a slight additional induction of helicial character, to 11-fold over 9

phosphate buffer, was observed with AS1p-NarXEc and AS1p-TarEc became essentially 10

completely α-helical. AS2p-Af1503 exhibited a 4-fold induction of helical character in 11

the presence of neutral bicelles and a 5-fold increase of helical character in the presence 12

of acidic bicelles. 13

Although more helical structure was induced by bicelles than by LUVs, the results 14

with both membrane systems agree qualitatively and clearly demostrate that only two of 15

the AS1 peptides, AS1p-NarXEc and AS1p-TarEc adopted helical structure in the presence 16

of a bilayer. The only AS2-containing peptide seen to contain any significant amount of 17

helical structure was AS2p-Af1503. 18

19

3.4 Solution structure of AS1p-TarEc in the presence of acidic bicelles 20

21

To assess the nature of the structural change seen with AS1p-TarEc in the presence 22

of membranes, an NMR structure of the membrane-associated peptide was determined. 23

    18

Two-dimensional TOCSY and NOESY spectra were used to obtain resonance 1

assignments in the presence of 300 mM 10% negatively charged phospholipid bicelles 2

with a q-ratio of 0.25. These bicelles are small, soluble lipid aggregates that have been 3

used extensively for solution studies of membrane-associated peptides [42, 45-48]. A 4

combination of the sequential-assignment strategy [49] and the main chain-directed 5

approach (MCD) [50, 51] was utilized to identify some initial spin systems in the TOCSY 6

spectrum and to identify local NOE connectivity patterns. The assignment proceeded by 7

alternating between the two assignment methods. Assignments for all non-proline HN and 8

Hα-protons were found except for Arg15, which probably underwent rapid exchange with 9

water. Based on this assignment, secondary chemical shifts [52] for the Hα-protons were 10

calculated (Figure 6A). In the representation in Figure 6, an α-helix is defined as at least 11

three sequential amino acids possessing a secondary Hα shift of less than -0.1 [52]. Based 12

on this criterion, the residues comprising the control cable, (i.e. Arg1 - Pro6) possess no 13

secondary structure, whereas chemical shifts representative of α-helical structure were 14

observed for the residues between Leu7 and Gly19, which is the part of the primary 15

structure that corresponds to AS1 proper [21]. 16

In total, 133 NOEs were assigned in the two-dimensional NOESY spectra, with 17

mixing times of 150 ms and 250 ms. Intraresidue-, sequential- and medium-range 18

constraints are found for all residues except Pro6 and Arg15. Many NOEs typical for α-19

helical structure (i.e. Hα-HN(i, i+3), Hα-HN(i, i+4), Hα-HN(i, i+2) and HN-HN(i, i+1)) were 20

identified. The typical pattern of these NOE connectivities (Figure 6B) and the secondary 21

Hα shifts shown in Figure 6A provide strong evidence for the presence of α-helical 22

character. A solution structure for the peptide was calculated on the basis of these 23

    19

distance constraints. From the final round of calculations, an ensemble of 25 structures 1

was selected to represent the solution structure (Figure 7A). These structures give a low 2

target function and modest distance violations in CYANA (Table S1). In Figure 7A, the 3

location of the side-chains of Pro6, Ile11, His13, Arg15, Ile17 and Gly19 from the 4

average structure are shown within the ensemble. The helical portion of the structure 5

starting withLys9, is shown in Figure 7B. 6

7

4. Discussion 8

9

To assess whether AS1 segments from four different HAMP domains are capable 10

of association with a phospholipid bilayer, we monitored their interaction with LUVs and 11

bicelles. We observed that only AS1 from NarXEc and TarEc became structured in the 12

presence of either membrane mimetic. However, differences were seen when the spectra 13

with LUVs were compared to those with bicelles. AS1p-NarXEc was found to associate 14

only with acidic LUVs but with both neutral and acidic bicelles, while AS1p-TarEc 15

became structured in the presence of all membrane models examined. This is likely due 16

to the different concentrations of lipids used and demonstrates why it is important to use 17

complementary membrane models when analyzing lipid-peptide interactions. 18

Nevertheless, these results show that out of the four HAMP domains investigated, only 19

two possess AS1 segments that extensively associate with membranes. 20

To better understand the selectivity of AS1-membrane association, we compared 21

various physiochemical properties of the peptides. We began by calculating the net 22

charge of each peptide at pH 7.2 (Table 1) because electrostatic potential was important 23

    20

for association of the AS1 segments with acidic LUVs. AS1 of EnvZEc, NarXEc and TarEc 1

are all positively charged at pH 7.2, however, AS1 of EnvZEc does not associate with the 2

acidic LUVs to a significant extent, suggesting that the helix-inducing association is more 3

complex than a simple electrostatic interaction. We also compared the hydrophobic 4

moment (µH) according to Eisenberg [53] and did not observe a significant difference 5

between the various AS1-containing peptides (Table 1). Finally, the free energies of 6

partitioning from unfolded peptide in solution to an α-helical peptide associated with a 7

bilayer interface (ΔGwif) were calculated using Membrane Protein Explorer (MPEx) [54]. 8

AS1p-NarXEc and AS1p-TarEc were found to possess the most favorable ΔGwif values, 9

suggesting that a more complex mechanism facilitates helix-inducing associations with 10

phospholipid membranes. Based on these observations, we considered that the 11

positioning of individual residues might be critical for association. We constructed helical 12

wheel projections, which revealed that the amphipathic nature of AS1p-NarXEc and 13

AS1p-TarEc is more pronounced than those of the other peptides (Figure S1). Two 14

positively charged residues are projected to flank the hydrophobic surface within these 15

AS1 segments but not within those that fail to become structured in the presence of 16

membranes. 17

To visualize the nature of these interactions in detail, we calculated an ensemble 18

of 25 structures of AS1p-TarEc associated with acidic bicelles (Figure 7A). Based on α-19

proton chemical shifts (Figure 6A) and the pattern of NOE connectivites (Figure 6B), the 20

residues N-terminal to Pro6 are unstructured while those C-terminal to Pro6 form an 21

extended α-helix. This unstructured region, which correlates with the region recently 22

termed the control cable [1, 18] within full-length receptors, is located between the 23

    21

aliphatic core of TM2 and the first critical packing residues within AS1 [18, 21]. The 1

change in helical register between TM2 and AS1 [5, 20] is supported by sulfhydryl-2

reactivity experiments with TarSt. . This region also tolerates a variety of mutations that 3

do not disrupt TarEc function [19]. When taken together, these results strongly suggest 4

that the region between TM2 and AS1 is not a contiguous helix within these well-5

characterized chemoreceptors. 6

Two physiological roles for AS1-membrane interactions have been previously 7

suggested. First, AS1-membrane interactions have been predicted to participate in 8

transmembrane communication [6]. Therefore, the similar behavior of AS1 peptides from 9

NarXEc and TarEc may not be surprising because of the evidence that they share a 10

common mechanism of transmembrane communication. Within, TarEc and other enteric 11

chemoreceptors, a small downward displacement TM2 relative to the plane of the 12

membrane is induced by binding of an attractant ligand [9-13, 18]. In the case of NarXEc, 13

recent high-resolution structures of a soluble form of the periplasmic domain in the 14

presence and in the absence of nitrate have shown a slight (∼ 1Å) displacement 15

downward of the N-terminal helices (PD1 and PD1') within the periplasmic four-helix 16

bundle relative to the C-terminal helices (PD4 and PD4') [14]. When the intact receptor is 17

embedded within the cytoplasmic membrane, PD4-TM2 and PD4'-TM2' may slide 18

upward relative to the plane of the membrane. The two other HAMP domains are from 19

receptors, Af1503 and EnvZEc, that do not possess a well understood mechanism of 20

transmembrane communication. In addition, based on secondary structure prediction with 21

JPRED3 [29] or biophysical analyses [55], respectively, these receptors do not possess 22

the periplasmic four-helix bundle shared by NarXEc and TarEc. 23

    22

The helix interaction model [6] proposes that the amphipathic nature of AS1 1

should allow it to align parallel to the membrane, with its hydrophobic face in contact 2

with the hydrophobic interior of the phospholipid bilayer and its positively charged 3

residues in association with the polar headgroups (Figures 7B and S1). Displacements of 4

TM2 into the cytoplasm are proposed to destabilize AS1-membrane interactions, which, 5

in turn, would facilitate interaction between AS1 and its cognate helical partner (Figure 6

8). However, it remains important to note that no direct evidence for the helix interaction 7

model has been observed within full-length receptors. 8

A second possibility is that AS1-membrane association could also be required for 9

proper membrane assembly or subcellular localization of SHKs or chemoreceptors. AS1-10

membrane associations have been shown to serve as a critical membrane topology 11

determinant [56-58]. Furthermore, a recent study has shown that prolyl substitutions 12

within the control cable or AS1, resulting in the presence of two prolyl residues within 13

this region, causes TsrEc receptors to be defective in ternary complex and cluster 14

formation [21]. In the structure presented here, Pro6 serves as a critical residue for 15

separating the structured and unstructured regions of AS1p-TarEc. In the case of NarXEc 16

and TarEc, AS1-membrane interactions could be critical for proper membrane integration 17

of monomeric receptors, dimerization of these monomers or formation of higher-order 18

complexes. Further experimentation must be performed within full-length receptors to 19

differentiate these possibilities. 20

In summary, the results of this study underscore the point that not all HAMP 21

domains are created equal. HAMP domains serve as a universal coupler between various 22

classes of domains and, thus, different subclasses of HAMP domains would be expected 23

    23

to exist [2]. These differences may explain some of the discrepancies observed between 1

the bodies of evidence that exist for HAMP signaling within various different membrane-2

spanning receptors. Although only based on four HAMP domains, we find it intriguing 3

that correlations are beginning to emerge between the propensity for AS1-membrane 4

interactions and the mechanisms of transmembrane signaling and subcellular localization 5

of HAMP-containing receptors. 6

7

Acknowledgements 8

9

Members of the von Heijne group (Stockholm University) provided valuable support and 10

discussion during the early stages of this manuscript. We thank an anonymous reviewer 11

for critical and insightful comments that helped improve the final output. L.M. was 12

supported by the Swedish Research Council and the Center for Biomembrane Research 13

(CBR). R. R. D. was supported by a Kirschstein National Research Service Award from 14

the National Institutes of Health (AI075773) and the German Research Foundation 15

(SFB807 – Transport and Communication Across Biological Membranes). 16

17

    24

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15 16

17

    28

Figure legends 1

2

Figure 1. Far-UV CD spectra of the AS1- and AS2-containing peptides in the presence of 3

LUVs. The peptides analyzed are constituents of the HAMP domains from (A) Af1503, 4

(B) EnvZEc, (C) NarXEc, and (D) TarEc. Samples contain a final concentration of 50 µM 5

peptide (AS1p-, AS2p- or a 1:1 mix) in 50 mM sodium phosphate buffer (pH 7.2) (left 6

panel), buffer with an additional 1 mM POPC (center panel) or buffer with an additional 7

1 mM POPC/POPG 4:1 (right panel). 8

9

Figure 2. Estimation of the extent of secondary structure in the presence of LUVs. The 10

far-UV CD spectra were further analyzed with Dicroweb [31] using the CONTIN method 11

[32]. The α-helical content of all 8 peptides in 50 mM sodium phosphate buffer (pH 7.2) 12

(white), buffer with an additional 1 mM POPC (light grey) or buffer with an additional 1 13

mM POPC/POPG 4:1 (dark grey) are shown. 14

15

Figure 3. Effect of increasing ionic strength on peptide-membrane interactions. 150 mM 16

KF was added to samples containing AS1p-NarXEc (left panel) and AS1p-TarEc (right 17

panel) in the presence of 50 mM potassium phosphate buffer (pH 7.2) and either 1 mM 18

POPC or 1 mM POPC/POPG 4:1. 19

20

Figure 4. Far-UV CD spectra of the AS1- and AS2-containing peptides in the presence of 21

neutral and acidic phospholipid bicelles. Samples contain a final concentration of 500 µM 22

    29

peptide, 50 mM potassium phosphate (pH 7.2) and 150 mM DMPC/DHPC (q=0.5) (left 1

panel) or 150 mM [DMPC/DMPG 4:1]/DHPC (q=0.5) (right panel). 2

3

Figure 5. Estimation of the extent of secondary structure from the far-UV CD spectra in 4

the presence of phospholipid bicelles. The α-helical content of all 8 peptides in 50 mM 5

sodium phosphate buffer pH 7.2 (white), buffer with an additional 150 mM 6

DMPC/DHPC (q=0.5) (light grey) or buffer with an additional 150 mM [DMPC/DMPG 7

4:1]/DHPC (q=0.5) (dark grey). 8

9

Figure 6. Secondary structure of AS1p-TarEc as determined by NMR. (a) Secondary Hα 10

chemical shifts of AS1p-TarEc in the presence of acidic bicelles.. (b) A summary of the 11

sequential and medium-range NOE-derived distances from the NOESY spectra. 12

13

Figure 7. (a) Ensemble of the 25 structures of AS1p-TarEc in 10% negatively charged 14

phospholipid bicelles with the lowest CYANA target function. The side-chains of Pro6, 15

Ile11, His13, Arg15, Ile17 and Gly19 from the average structure in the ensemble are 16

shown. (b) AS1 of TarEc adopts an amphipathic α-helical structure in the presence of 17

acidic bicelles. The position of the hydrophobic (yellow), postively charged (blue), 18

negatively charged (red) and partially positively charged residues (grayish green) are 19

indicated. The side chains of Lys9 and Arg15 that flank this region are indicated as well. 20

21

Figure 8. Add model here. 22

23

    30

Table legends 1

2

Table 1. Properties of the AS1- and AS2-containing peptides used in the study. 3

4

Table 2. Diffusion coefficients of the AS1- and AS2-containing peptides employed in this 5

study. 6

7

8

    31

1

2

3

4

5

a The receptor that served as the source of AS1- and AS2-containing peptides. The 6

position of the residues within the primary structure is provided in parentheses. 7

b The “GenInfo Identifier” sequence identification number. 8

c The hydrophobic moment (µH) of the peptides according to Eisenberg [53]. 9

d The energy of interfacial partitioning (ΔGwif) calculated within MPEx [54]. 10

11

12

Table 1. 13

14 15

16

17

18

Receptora GIb Peptide Sequence Net Charge

Hydrophobic moment (µH)c

ΔGwif (kcal/mol)d

Af1503 (278-296) 1484730 AS1p-Af1503 STITRPIIELSNTADKIAE -1 6.28 -0.71 EnvZEc (180-198) 947272 AS1p-EnvZEc RIQNRPLVDLEHAALQVGK +1 3.84 -1.51 NarXEc (177-195) 945788 AS1p-NarXEc RLLQPWRQLLAMASAVSHR +3 6.19 -6.35 TarEc (214-232) 946399 AS1p-TarEc RMLLTPLAKIIAHIREIAG +2 5.79 -4.32

Af1503 (310-328) 1484730 AS2p-Af1503 DEIGILAKSIERLRRSLKV +2 5.42 -0.29 EnvZEc (211-229) 947272 AS2p-EnvZEc SEVRSVTRAFNHMAAGVKQ +2 3.46 -1.51 NarXEc (208-226) 945788 AS2p-NarXEc EMAMLGTALNNMSAELAES -3 5.57 -2.25 TarEc (246-263) 946399 AS2p-TarEc EMGDLAQSVSHMQRSLTD -2 4.81 -0.29

    32

1 2 3 4 5 6

a Diffusion coefficients in D2O normalized according to the diffusion of HDO in order to 7

account for differences of viscosity. 8 b Estimation of the percentage of peptide bound to the phospholipid bicelle as calculated 9

by equation 1. 10

11

Table 2. 12

13 14

Diffusion coefficient (10-10 m2/s)a

Buffer DMPC/DHPC [DMPC/DMPG 4:1]/DHPC Peptide Dfree Dcomplex Dbicelle x (%)b Dcomplex Dbicelle x (%)b

AS1p-Af1503 1.6 ± 0.07 1.2 ± 0.07 0.38 ± 0.01 36 0.96 ± 0.1 0.36 ± 0.01 52 AS2p-Af1503 1.6 ± 0.02 0.39 ± 0.01 0.37 ± 0.01 98 0.40 ± 0.01 0.37 ± 0.01 98 AS1p-EnvZEc 1.6 ± 0.05 1.2 ± 0.04 0.38± 0.02 36 1.1 ± 0.04 0.39 ± 0.01 43 AS2p-EnvZEc 1.6 ± 0.04 1.0 ± 0.05 0.35 ± 0.01 48 1.1 ± 0.03 0.37 ± 0.01 40 AS1p-NarXEc 1.5 ± 0.05 0.40 ± 0.01 0.38 ± 0.01 98 0.35 ± 0.01 0.36 ± 0.01 100 AS2p-NarXEc 1.6 ± 0.04 1.0 ± 0.02 0.37 ± 0.01 49 0.92 ± 0.02 0.37 ± 0.01 58 AS1p-TarEc 1.4 ± 0.04 0.38 ± 0.01 0.37 ± 0.01 99 0.35 ± 0.01 0.36 ± 0.01 100 AS2p-TarEc 1.7 ± 0.06 1.2 ± 0.04 0.38 ± 0.01 42 1.2 ± 0.05 0.37 ± 0.01 40

    33

Supplemental information 1

2

Supplemental Figure 1. Helical wheel projections of the AS1- and AS2-containing 3

peptides used during this study. Various properties of the residue side chains are color-4

coded: hydrophobic (yellow), polar (purple), negatively charged (red) and positively 5

charged (blue). Partially positive (slightly blue), partially negative (slightly red) and 6

prolyl (green) residues are also indicated. An arrow representing the direction and 7

magnitude of the hydrophobic moment is also displayed. A distinct hydrophobic surface 8

is observed within AS1p-NarXEc and AS1p-TarEc and, in both cases, two positively 9

charged residues flank this hydrophobic surface. 10

11

12

    34

Supplemental Table 1. Structural statistics for the ensemble of 25 structures of AS1p-1

TarEc in 10% negatively charged phospholipid bicelles. 2

3

4

5

6

References 7

[1] R. Gautier, D. Douguet, B. Antonny, G. Drin, HELIQUEST: a web server to 8

screen sequences with specific alpha-helical properties, Bioinformatics 24 (2008) 9

2101-2102. 10

Number of constraints 133 (59, 41, 33)

CYANA target function 0.84 ± 0.07 Å2

Maximum distance violation 0.17 ± 0.02 Å

Backbone atom rmsd (Å)

All residues 1.04 ± 0.36

Residue 9-19 0.63 ± 0.17

Residue 12-18 0.32 ± 0.09

Ramachandran plot regions (%)

Most favored 83.8

Allowed region 13.2

Generously allowed 3

Disallowed 0