Glycine Perturbs Local and Global Conformational Flexibility of a

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Glycine Perturbs Local and Global Conformational Flexibility of a Transmembrane Helix Philipp Högel, Alexander Götz, Felix Kuhne, Maximilian Ebert, Walter Stelzer, Kasper D. Rand, Christina Scharnagl, and Dieter Langosch Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01197 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 2018

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Biochemistry

Glycine Perturbs Local and Global Conformational Flexibility of a Transmembrane Helix

Philipp Högel1§, Alexander Götz2§, Felix Kuhne1, Maximilian Ebert1, Walter Stelzer1, Kasper D. Rand3, Christina Scharnagl2 and Dieter Langosch1*

1

Center for Integrated Protein Science Munich (CIPSM) at the Lehrstuhl Chemie der

Biopolymere, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany 2

Chair of Physics of Synthetic Biological Systems (E14), Technical University of Munich,

Maximus-von-Imhof Forum 4, 85354 Freising, Germany 3

Department of Pharmacy, University of Copenhagen

Universitetsparken 2, 2100 Copenhagen (Denmark)

*

Corresponding author

D. Langosch, Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany. Tel.: +49-8161-71-3500; Fax: +49-816171-4404; E-mail: [email protected]

ACS Paragon Plus Environment

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ABBREVIATIONS H-bond, hydrogen bond; DHX, deuterium/hydrogen exchange; ETD, electron transfer dissociation; MD, molecular dynamics; TMD, transmembrane domain; CD, circular dichroism


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Biochemistry

Abstract

Flexible transmembrane helices frequently support the conformational transitions between different functional states of membrane proteins. While proline is well known to distort and destabilize transmembrane helices, the role of glycine is still debated. Here, we systematically investigated the effect of glycine on transmembrane helix flexibility by placing it at different sites within the otherwise uniform leucine/valine repeat sequence of the LV16 model helix. We show that amide deuterium/hydrogen exchange kinetics are increased near glycine. Molecular dynamics simulations reproduce the measured exchange kinetics and reveal, at atomic resolution, a severe packing defect at glycine that enhances local hydration. Furthermore, glycine alters H-bond occupancies and triggers a redistribution of α-helical and 310-helical H-bonds. These effects facilitate local helix bending at the glycine site and change the collective dynamics of the helix.


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Introduction

It is increasingly appreciated that the function of integral membrane proteins is related to their conformational dynamics. This includes rigid-body motions of transmembrane helices relative to each other, their bending and twisting coordinated by flexible hinges, and local structural fluctuations.1,2 For example, analyses of high-resolution structures of multi-span membrane proteins revealed that their transmembrane helices exhibit different curvatures and that about half of them contain non-canonical elements, such as kinks and π- or 310-helical turns.3–5 Kinks and flexible hinges are often located in functionally important regions of membrane proteins like ion channels6,7 or G-protein-coupled receptors.8,9 Kink angles and coil regions tend to be conserved in evolution, implicating them in the functional role of transmembrane domains (TMD).1,2,10–13 In early studies, glycine and proline had the strongest destabilizing effects of all amino acids on model transmembrane helices, as measured by their helicity in detergent micelles.14,15 Subsequent analyses of high-resolution membrane protein structures refined this picture. In a helix, proline at position i can contribute to a kink, as it cannot donate an amide H-bond to residues i-4 and i-3, while its side chain clashes with the i-1 carbonyl oxygen.16 Proline can thus introduce permanent helix kinks and enhance helix flexibility in terms of bending and swivel motions.1,17 The role of proline in distorting transmembrane helices is reflected by a several-fold enrichment of proline at positions C-terminal of kinks.18–22 Compared to proline, the role of glycine in transmembrane helix flexibility is less clear. Depending on the used database and kink definition, glycine was reported to be slightly overrepresented in kinks19,20,23 or not.18,21 Around glycine, transmembrane helix bending angles generally do not exceed the moderate bending angles seen around other amino acids.24 In aqueous solution, glycine disfavors helix folding since the entropic cost of tethering glycine in a helix exceeds the gain of enthalpy upon helix formation.25–27 Within the apolar ACS Paragon Plus Environment

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Biochemistry

environment of transmembrane helices, this entropic penalty is compensated for by stronger amide hydrogen bonding.28,29 Not surprisingly, therefore, glycine is twice as abundant in transmembrane helices than in water-soluble helices.30 Due to its missing side-chain, glycine induces a packing defect which predisposes this residue to destabilize a transmembrane helix locally.31 In a prior study, short molecular dynamics (MD) simulations of poly-alanine host helices containing naturally occurring motifs of proline and/or glycine underscored the different impacts of these amino acids in distorting a transmembrane helix. The simulations confirmed that proline can induce pronounced anisotropic kink/swivel motions. In contrast, multiple glycine residues had only very slight effects on helix bending.32 However, the study noted that a poly-alanine host helix might be unstable by itself which may lead to an underestimation of the glycine effect. We recently compared the conformational dynamics of several natural transmembrane helices by amide deuterium/hydrogen exchange (DHX). Mutating their glycine residues slowed the DHX kinetics, thus suggesting a role of glycine in the conformational flexibility of natural transmembrane helices.33 In this work, we systematically investigated the effect of glycine on transmembrane helix flexibility, using novel variants of our low-complexity model helices, collectively termed LVpeptides.34 LV-peptides readily form transmembrane helices35 whose flexibility is connected to their ability to induce liposome fusion and lipid flip/flop.34,36,37 Previously, the conformational flexibility of LV helices has been found to increase with the ratio of helixdestabilizing valine to helix-promoting leucine residues and after introducing a glycine/proline pair.36,38 The uniform leucine/valine repeat sequence of the LV16 variant forms a helix with intermediate flexibility; it was used here as a template for leucine-toglycine substitutions. Our results reveal complex and distributed effects of glycine, which result from a local ACS Paragon Plus Environment

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packing defect and conformational destabilization. Glycine substitution is associated with increased local helix bending and alters concerted movements of the helix.


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Biochemistry

Materials and Methods

Peptide Synthesis: Peptides were synthesized by Fmoc chemistry by PSL, Heidelberg, or by the Core Unit Peptid-Technologien of Universitaet Leipzig, Germany. All other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, Missouri, USA).

Mass spectrometric experiments: All mass spectrometric experiments were performed on a Synapt G2 HDMS (Waters Co., Milford, MA). A 100 µl Hamilton Gastight Syringe was used with a Harvard Apparatus 11plus, the flow rate was set to 5 µl/min. Spectra were acquired in a positive-ion mode with one scan for each second and 0.1 s interscan time.

DHX experiments, ESI-MS, and data evaluation Solutions of deuterated peptide (100 µM in 80 % (v/v) d1-trifluoroethanol (d1-TFE) in 2 mM ND4-acetate) were diluted 1:20 with protonated solvent (80 % (v/v) TFE in 2 mM NH4acetate, pH 5.0) to a final peptide concentration of 5 µM and incubated at a temperature of 20.0°C in a thermal cycler (Eppendorf, Germany). Incubation times were 0, 1, 2, 5, 10, 20, 30, 40, 50 min, and 1, 2, 3, 4, 6, 8, 12, 24, 48, 72 h. Exchange reactions were quenched by placing the samples on ice and adding 0.5 % (v/v) formic acid, resulting in a pH ≈ 2.5. Mass/charge ratios were recorded and evaluated as previously described.36,39 The distribution of DHX rate constants, as well as population sizes, were analyzed by using the maximum entropy method (MEM) implemented in the program MemExp 4.1.40,41 Since we are interested in the exchange kinetics of potentially hydrogen-bonded amide deuterons, their theoretical number (19 D = D 22 N-D – 3 non-bonded N-D of an idealized α-helix of 23 residues) was taken as the initial data point (t = 0) for MEM analysis. The individual classes were delimited by the following rate constants: A (logkDX[h-1] > 1.6), B (0.1 < logkDX[h-1] < 1.6), C (-1.1 < logkDX[h-1] < 0.1), D (logkDX[h-1] < -1.1). ACS Paragon Plus Environment

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For electron transfer dissociation (ETD) we preselected the 5+ charged peptides via MS/MS and used 1,4-dicyanobenzene as reagent. Fragmentation of peptides and data evaluation was performed as described.42 Briefly, ETD MS/MS scans were accumulated over 10 min scan time, smoothed (Savitzky-Golay, 2 x 4 channels), and centered (80 % centroid top, heights, 3 channels). The amounts of remaining deuterons on the c- and z-fragment ions were calculated by subtracting the intensity-weighted centroid masses of the individual isotopic distributions of the non-deuterated reference spectra from the deuterated samples43 and corrected with the dilution factor.39 Several fragment ions could not be evaluated due to spectral overlap with other ions. The hydrogen scrambling rate was calculated with the ammonia loss method44 and was found to be negligible .

MD Simulation and Analysis MD simulations of ~200 ns length were conducted as described.33 In brief, the peptides were built initially as ideal α-helices and placed in a rectangular solvent box, containing 80 % TFE and 20 % TIP3 (v/v). Equilibration was carried out in multiple steps by reducing harmonic restraints over a total of 1.2 ns. Production runs were performed in a NPT ensemble (T = 293 K, p = 0.1 MPa) using NAMD2.945 and the CHARMM22 force field with CMAP corrections.46 The last 150 ns of each trajectory were subjected to analysis. Mean values θ and their standard errors of the mean (SEM) σ were estimated by block averaging using a block size of 30 ns, which is >> 2τ with τ being the first zero passage time of the autocorrelation. Distributions of τ values for investigated parameters are shown in Figure S1. If not mentioned otherwise, all analyses used custom-built Python scripts based upon the MDtraj library.47 Visualizations of structures were made using VMD1.9.3.48 Root-mean-squared deviations (RMSD) and fluctuations (RMSF) were calculated with respect to the average structure which was determined iteratively.49


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Biochemistry

DHX kinetics were calculated from the MD simulations as described.50 To account for sufficient access of the exchange catalyst to the amide, a distance cutoff of 0.35 nm was used for determining the fraction of exchange-competent amide hydrogens.51,52 The empirical factor correcting chemical exchange for TFE effects was computed for each peptide separately, thus accounting for differences in the local environment. The quality of the MDderived prediction of exchange kinetics was assessed by the normalized mean-squared deviation (χ2) with respect to the experiment.

Computation of structural features: Rise per residue (RPR) values were computed by a differential geometric approach which uses a cubic spline through three consecutive Cα atoms.53 To calculate H-bond occupancies a H-bond was considered as closed if the distance ⋯ was < 0.26 nm and the ⋯ − angle was in the range 180° ± 60°. We counted the H-bond for any amide i to be closed if either the H-Bond to Oi-4 (α H-Bond) or the H-Bond to Oi-3 (310 H-bond) is formed. The same distance and angle cutoffs have been used to access H-bond occupancies between the peptide backbone and surrounding water molecules. A water molecule can act as an acceptor of a H-bonds from the backbone amide as well as a donor of two H-bonds to backbone carbonyls. To access steric shielding of the amide proton we computed the normalized packing score Snorm. The packing score Si is computed as the sum of the inverse of the pairwise distance rij between the backbone amide proton and all other atoms raised to the sixth power.54 Backbone atoms belonging to the same residue i as the carbonyl are excluded from the computation. Normalization was done by dividing Si by the sum of Si over all residues. To access local helix deformations, bending Θ and swivel Φ angles as defined in Figure S2 were computed along the TMD helix. The axes of two neighboring helical domains, A (i-1 → i-4) and B (i+1 → i+4), were determined, using the differential geometric approach also applied for computing RPR. The angle between A and B represents the local bending angle Θι (Figure S2, side view). The local swivel angle Φi


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defines the rotation of B relative to the Cα atom of residue i in the plane orthogonal to A (Figure S2, top view).17 The program Dyndom55 was used to identify regions involved in hinge bending and twisting, to determine the location of the intersegmental screw axis and to calculate the rotation angles. Quasi-rigid body domains of at least 4 residues were identified using a sliding window of 5 residues. The motion around a hinge was classified by the orientation of the screw axis relative to the helix axis. Screw axes which are mainly perpendicular to the helix axis (%closure > 50 %) are classified as bending motions, while twisting motions are classified by a screw axis which is mainly parallel to the helix axis (%closure ≤ 50 %). We subjected snapshots every 50 ps to analysis. The conformation with the lowest RMSD from the iteratively determined average structure49 was used as reference.


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Biochemistry

Results To systematically examine the impact of glycine on helix backbone flexibility, we compared the previously36,38 investigated L16, LV16, and LV16-G8P9 reference peptides to a series of novel LV16 derivatives that hold single glycine residues at different positions (termed LXG peptides, Table 1). Since the different ‘X’ positions within LV16 have identical amino acid neighbors, we asked whether an impact of glycine depends on its location within terminal or central parts of the helix. In the LV16-L3,9,15G variant, three substitutions were combined.

Table 1: Peptides used in this work

Peptide L16 LV16 LV16-G8P9 LV16-L3G LV16-L5G LV16-L7G LV16-L9G LV16-L11G LV16-L13G LV16-L15G LV16-L3,9,15G

Sequence KKKW LLLLLLLLLLLLLLLL KKKW LVLVLVLVLVLVLVLV KKKW LVLVLVLGPVLVLVLV KKKW LVGVLVLVLVLVLVLV KKKW LVLVGVLVLVLVLVLV KKKW LVLVLVGVLVLVLVLV KKKW LVLVLVLVGVLVLVLV KKKW LVLVLVLVLVGVLVLV KKKW LVLVLVLVLVLVGVLV KKKW LVLVLVLVLVLVLVGV KKKW LVGVLVLVGVLVLVGV

KKK KKK KKK KKK KKK KKK KKK KKK KKK KKK KKK

Glycine accelerates the DHX kinetics of a helix Intrahelical amide H-bond stabilities were probed by DHX experiments in a buffered TFE/water (80/20) mixture where our peptides form helices as previously shown by circular dichroism (CD) spectroscopy for L16, LV16, and LV16-G8P9 (~83 %, ~78 %, and ~65 % helix content, respectively).35,36 This solvent therefore mimics helix stabilization by a lipid membrane while allowing for water access. DHX experiments of a membrane-embedded helix are not practical since a bilayer completely protects large parts of a transmembrane helix


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from exchange.36,39 For amide DHX, exhaustively (> 98 %) deuterated peptides were diluted into proteous solvent and exchange of amide deuterons to protons was followed over time at pH 5.0 and 20°C by electrospray ionization mass spectrometry. Under these conditions, DHX of LV-peptides results in a gradual shift of isotopic envelopes towards lower mass/charge values (data not shown).36 A gradual shift in addition to the lack of a bimodal shape of the isotopic distributions is diagnostic of EX2 kinetics where individual deuterons exchange in an uncorrelated fashion upon local unfolding.56,57 Compared to our previous experiments36, the incubation period was extended to 72 h where L16 exchanges ~75% of amides while nearly complete exchange is seen for all other peptides (Figure 1). A qualitative comparison of the global DHX kinetics shows the same rank order for our basic set of peptides (L16 < LV16 < LV16-G8P9) within the first 10 min (Figure 1 A) and at later time points (Figure 1 B). Curve fitting with a maximum-entropy method (MEM)41 subdivides the backbone amide deuterons into four kinetically distinct classes A, B, C, and D that show decreasing average rate constants (Table 2). We find that the rate constants tend to increase in the rank order L16 < LV16 < LV16-G8P9 within all populations. At the same time, the sizes of the populations tend to increase in populations A, B, and C at the expense of the slowest population D. Within the series L16, LV16, and LV16-G8P9, therefore, the acceleration of amide exchange is paralleled by a redistribution of deuterons from slower to faster populations; this matches the qualitative rank order of the kinetics (Figure 1). The situation is somewhat more complex with LXG peptides. Qualitatively, the rank order of LXG relative to LV16 DHX kinetics, as seen within the first 10 min of exchange (LV16 ≈ L7G, L9G, L11G, L13G < L15G < L3G ≈ L5G < L3,9,15G, Figure 1 A), differs from the order seen after 72 h (L15G < L13G ≈ LV16 < L5G ≈ L3G < L7G < L11G < L9G < L3,9,15G, Figure 1 B). Back-calculations showed that differences in the intrinsic chemical exchange rate constants kch of the peptides are negligible for DHX (Figure S3).


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Biochemistry

Due to the fact that all LXG peptides share the same composition with a glycine residue that is shifted over the core region and always flanked by two valines, the sum of kch is identical for all LXG peptides. Hence, differences can only be caused by the secondary structure. To illustrate the discrepancies between these early and late time points, we plot the differences between the numbers of amide deuterons on LV16 and its LXG variants detected after the various incubation periods. As exemplified by three cases in Figure 1 C and shown for all LXG in Figure S4, glycine close to a terminus (L3G, L5G, L15G) accelerates DHX early on while glycine closer to the core of the helix (L7G, L9G, L11G, L13G) accelerates DHX at a later stage. It is well known that terminal regions of a helix exhibit fraying and thus exchange faster than core regions.58,59 Therefore, the observed acceleration of early DHX by glycines close to both termini and the acceleration of late DHX by glycines within the helix core fits to expectation. It remains unclear why some variants (L13G, L15G) approach LV16 at later stages of DHX (Figure 1 C, Figure S4). With the triple mutant LV16-L3,9,15G, DHX is very fast documenting the cumulative effect of multiple glycines. Attempts to obtain rate constants and populations of kinetically distinct LXG amides by curve fitting using MEM or multiple exponentials was not practical due to the limited deviations from LV16, thus preventing a quantitative analysis (data not shown).


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Figure 1. Global amide DHX kinetics. (A and B) The number of remaining deuterons plotted as a function of time for (A) the initial 10 min and (B) the complete 72 h of incubation (n = 3, SEM are smaller than the size of the symbols). For a better visualization of differences in the DHX kinetics shown in (B), the y-axis is truncated at 10 deuterons. Due to the cyclic side chain of proline, LV16-G8P9 lacks one amide deuteron/proton leading to a lower (≤ 1 D) ACS Paragon Plus Environment

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Biochemistry

initial deuterium content. (C) Differences between the numbers of deuterons on LXG-peptides and LV16 as a function of incubation time. For better visualization, only L5G, L9G and L13G are shown. Note the accelerated exchange within fast, intermediate, or slow amides of L5G, L13G, and L9G, respectively.

Table 2: DHX rate constants and sizes of kinetically distinct populations of amide deuterons

Peptide

A1: log k/h-1 (DA)

B: log k/h-1 (DB)

C: log k/h-1 (DC) D: log k/h-1 (DD)

L16

1.812 (6.643)

0.37 (1.24)

-0.70 (2.55)

-2.19 (8.38)

LV16

1.81 (6.29)

0.58 (2,52)

-0,52 (5.02)

-1.62 (4.79)

LV16-G8P9

2.20 (7.12)

0.77 (3.22)

-0.17 (8.39)

-1.64 (0.26)

1 A to D correspond to the four kinetic classes of amide deuterons 2 logarithm of DHX rate constants 3 population sizes

DHX experiments were performed three times ($2 of MEM fits ranged between 0.08 and 0.229)

Next, we identified sites of preferential exchange along the helices by employing electron transfer dissociation (ETD) in the gas phase after different periods of DHX in solution. ETD fragmentation produces series of c- and z-fragment ions that extend from a given residue position towards the N- or C-terminus, respectively. Analyzing the deuterium content of the fragments reveals the deuteron distribution across the helix at different incubation times (Figure S5). Figure 2 shows the deuteron contents of z-fragments that are aligned with the respective amino acid sequences (see Figure S6 for the distributions of the corresponding cfragments). In general, a flat region of this curve reflects rapid DHX, while a steep slope indicates a rigid domain characterized by slow exchange. With all peptides investigated here, exchange is faster at both termini (N-terminus faster than C-terminus) than at the helix cores. As exemplified by LV16, exchange gradually progresses towards the cores with ongoing incubation time. After 6 h, the region between residues 8 and 14 still is largely protected and ACS Paragon Plus Environment

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thus represents the most stable part of the helix (Figure 2 A). Compared to LV16, progression of exchange is slower with L16 and faster with LV16-G8P9 (Figure S6). Figure 2 B directly compares the site-specific DHX of L16, LV16, and LV16-G8P9 after 30 min. Clearly, exchange within the hydrophobic core of the helix follows the rank order L16 < LV16 < LV16-G8P9 which is reminiscent of the global DHX kinetics (Figure 1 B). Relative to LV16, the flattening of the LV16-G8P9 curve N-terminal of position 12 reveals site-specific acceleration of DHX (missing fragments prevented us from an unequivocal assignment of enhanced DHX to position 10 or 11). In order to probe site-specific effects of glycine, we examined the three LXG peptides with the most central glycines where terminal helix fraying is minimal. Comparing site-specific DHX of LV16, L7G, L9G, and L11G by ETD after 240 min of incubation reveals that exchange is enhanced from about two residues C-terminal of the respective glycine site (X+2), as indicated by a tendency of the respective curves to flatten locally (Figure 2 C). Attempts to derive exchange rate constants for each individual amide were unsuccessful due to significant overlap of many fragment ions.

Taken together, the rate of amide DHX at the N-terminus of a helix generally exceeds that at the C-terminus and the helix core. Individual glycines cause local accelerations of exchange. The apparent dependence of these glycine-induced changes on location is likely determined by the extent of helix fraying at the respective site.


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A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

KW L V L V L V L V L V L V L V L V K K K LV16 1 min

12 remaining deuterons

LV16 10 min LV16 30 min 9

LV16 120 min LV16 240 min LV16 360 min

6

3

z2 z22 z21 z10 z19 z18 z17 z16 z15 z14 z13 z12 z11 0 z9 z8 z7 z6 z5 z4 z3 z2 z1

0

z-fragments

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

12

L16 30 min

remaining deuterons

LV16 30 min 9

LV16-G8P9 30 min

6

3

z2 z2 2 z2 1 z1 0 z1 9 z1 8 z1 7 z1 6 z1 5 z1 4 z1 3 z1 2 z1 1 0 z9 z8 z7 z6 z5 z4 z3 z2 z1

0

z-fragments

C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

LV16

remaining deuterons

LV16 L7G 6

LV16 L9G LV16 L11G

4

2

0 z2 z22 z21 z10 z19 z18 z17 z16 z15 z14 z13 z12 z11 0 z9 z8 z7 z6 z5 z4 z3 z2 z1

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Biochemistry

z-fragments


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Figure 2. Impact of helix-destabilizing residues on local DHX. (A to C) Peptide sequence aligned against deuterium contents of z-fragments as determined after ETD (n = 3, SEM ≈ 0.1 deuterons). A given amino acid aligns with the z-ion type containing the respective amide. Missing z-ions are due to overlap with other fragments in the mass spectra that prevent their identification. The corresponding c-fragments are shown in Figure S6. (A) Progression of DHX along the LV16 sequence over time (data for L16 and LV16-G8P9 are shown in Figure S6 A to D) (B) Comparison of L16, LV16, and LV16-G8P9 after 30 min of exchange (data are extracted from Figure 2 A and Figure S6 A, C). Arrows indicate the positions of glycine and proline. (C) Comparison of LV16, L7G, L9G, and L11G after 240 min of exchange. Arrows indicate positions of glycine.

Glycine affects DHX kinetics by changing local helix flexibility and hydration In order to interpret the observed impacts of glycine on experimental DHX kinetics at the atomistic scale, we performed MD simulations in the same solvent environment. For their validation, we back-calculated global DHX kinetics from the local fractions of open H-bonds (fopen), chemical exchange rates, and the levels of hydration.50 Comparing calculated and experimental kinetics shows excellent agreement up to 4 h of exchange (χ2 = 0.196 to 1.759). A somewhat poorer agreement over the total 72 h (χ2 = 0.307 to 7.635) (Figure S7, Table S1) is probably the consequence of insufficient sampling of very slowly exchanging amides. Calculated site-specific exchange rates (kDHX) reveal a W-shaped pattern for the parental LV16 helix. Minima localize around V6 and V13, while two maxima within the helix core are found at V8 and V10 (Figure 3 A). The impact of glycine is exemplified for L5G, L9G, and L13G in Figure 3 A while complete data are given in Figure S8. To compare the role of glycine at different positions along the helix, we calculated the ratios of exchange rates


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kDHX,LXG/kDHX,LV16 at different positions (X–4 to X+4)

near the various glycines and

computed averages (Figure 3 B). Depending on the individual peptide variant, glycine enhances this ratio from ~10- to ~1000-fold at its own site and at downstream positions while decreasing DHX to various extents upstream. A systematic dependence of how glycine affects amide exchange on its location within the helix is not apparent although smaller changes were seen with L15G. In general, kDHX depends on local amide H-bonding (fopen). Our results reveal significant glycine-induced alterations in fopen (Figure 3 C) since the ratios fopen,LXG/fopen,LV16 change by up to ~100-fold (Figure 3 D). Although the changes in fopen largely agree with the changes in computed DHX rates (Figure 3 B), they do not fully mirror them, especially around the glycines. To resolve these discrepancies, we determined the local levels of hydration which determine the concentration of hydroxide ions, the catalyst in DHX, and may affect the intrinsic exchange rates (kint). Indeed, local water coordination is consistently enhanced at each glycine, its immediate neighbours, and X+3, as indicated by H-bond formation between water molecules and the given amide protons and/or the carbonyl oxygens at the respective X-3 and X-4 sites (Figure 3 E, F).

We conclude that glycine shapes the DHX kinetics by altering amide H-bonding as well as by increasing the accessibility of the amide to water.


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Figure 3. Local exchange rates kDHX and underlying parameters computed from MD simulations. (A) Local kDHX for parental LV16, L5G, L9G, and L13G. (B) Ratios of kDHX,LXG / kDHX,LV16 at positions X–4 to X+4 that were averaged for the different glycines. (C) Local fopen of exchange-competent amides. (D) Ratios fopen,LXG/fopen,LV16 from X-4 to X+4. (E) Water coordination, describing the probability of H-bonds from water molecules to the amide proton at position i and/or the carbonyl oxygen at i-4. (F) Change in water coordination between LXGs and LV16 (∆ = LXG - LV16) from X-4 to X+4. Note that changes in exchange rates originate from changes in fopen and are modulated by altered hydration. All data points represent mean values (n = 5) with SEM determined by block averaging. Error propagation has been applied for ratios and differences. Leucine residues which have been substituted to glycine are highlighted by arrows.

The glycine-mediated packing defect distorts helix backbone geometry To assess the extent of packing defects as introduced by glycine, we calculated normalized local packing scores Si,norm around amide hydrogen atoms.54 As expected, leucine to glycine ACS Paragon Plus Environment

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substitutions cause a strong packing defect at glycine; they also cause slightly increased local packing two residues upstream of glycine (Figure 4 A, B and Figure S9). What are the consequences of this packing defect? First, we gauged the impact of glycine on the geometry of the helix backbone. For an ideal α-helix, the rise per residue (RPR) equals 1.5 Å (Figure S8). This is true for L16 while RPR values of LV16 alternate between 1.9 Å at leucine residues and 1.2 Å at valine residues (Figure 4 C). Similar deviations from the geometry of an idealized helix have also been observed for other natural helices4,11,22 and may result from steric hindrance between β-branched side chains and the peptide backbone.60 Introducing glycine enhances the distortion, as we detect differences (∆RPR = RPRLXG – RPRLV16) of up to ~0.3 Å (Figure 4 C, D). Second, we compared the occupancies for α-helical (i, i-4), 310-helical (i, i-3) and total (α or 310) intrahelical H-bonds (Figure 4 E, G, I) and calculated the differences between the respective H-bond occupancies of LV16 and LXG (Figure 4 F, H, J). The total intrahelical H-bond occupancy is > 80 % with all helices, reduced occupancies at positions 1 and 2 are attributed to N-terminal helix fraying. Leucine-to-glycine substitutions tend to decrease the total H-bond occupancy at glycine and positions downstream (Figures 4 E, F and S10). (Note that H-bond occupancies are based on more stringent geometrical criteria than the fopen values shown in Figure 3 C.) Interestingly, glycine changes the relative contributions of α- and 310-helical occupancies to the total occupancies. Specifically, α-helical H-bonding is lower at glycine and at various downstream residues. In some cases, a slight increase is detected upstream (Figures 4 G, H and S10). Notably, the changes in α-helical H-bonding are largely compensated for by opposite changes in 310 Hbonding (Figures 4 I, J and S9). In Figure 4 K we visualize the packing defect by computing the space that is explored by some representative helices (LV16-L5G, L9G, L11G) in the course of the respective trajectories.


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Figure 4. Local backbone dynamics determined by MD simulation. (A) Normalized packing scores Si,norm along the TMD. (B) Change in local packing scores (∆Si,norm= Si,norm,LXG Si,norm,LV16) from positions X-4 to X+4. (C) Rise per residue (RPR) between Cα atoms. (D) Difference in RPR (∆RPR) between the parental LV16 and LXGs (∆RPR = RPRLXG RPRLV16) from X-4 to X+4. (E) Occupancy of total intrahelical H-bonds (α or 310). (F) ACS Paragon Plus Environment

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Difference of occupancies between LV16 and LXG mutants. (G) Occupancy of α-helical backbone H-bonds (amide proton at residue i extending to carbonyl oxygen at i-4). (H) Differences in α-helical backbone H-bond occupancies between LV16 and LXG mutants. (I) Occupancy of 310-helical H-bonds (amide proton at residue i extending to the carbonyl oxygen at i-3). (J) Differences in 310-helical H-bond occupancies between LV16 and LXG mutants. All data points represent mean values (n = 5) with SEM obtained by block averaging. Error propagation has been applied when calculating the differences. Leucine residues which have been substituted to glycine are highlighted by arrows. (K) A visualization of the space that is explored in the course of the trajectories by the atoms of some representative helices (LV16-L5G, L9G, L11G); note the voids at the sites of glycine.

Third, we assessed the impact of glycine on helix bending. To this end, we placed the Cα atom of glycine or its neighbors at the pivot point between both helical turns bordering the respective pivot points and calculated local bending and swivel angles (see: Figure S2). Indeed, LXG substitutions shift the distribution of bending angles of LV16 to slightly higher values at glycine and the X±1 residues (Figure 5 A). Figure 5 B shows a different representation of the data that emphasizes the more extreme deviations of LXG variants relative to LV16. While helix bending of LV16 rarely exceeds ~10°, introducing glycine permits bending up to ~30°, although these glycine-induced bends are rare (see: Table S2). Swivel angles characterize the direction of bending and remain around 0° thus showing that glycine localizes at the concave face of the bends. The shifted swivel angle population of L13G is likely related to helix fraying near the C-terminus.


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In sum, glycine-induced packing defects appear to induce a partial switch from α to 310 helical H-bonds downstream of glycine. This is connected to variations in helix backbone geometry and an increased population of bent helices.

Figure 5. Local bending Θ and swivel Φ angles. (A) Local bending angles at and around glycine. A bin width of 1° has been used for discretization. (B) Normalized 2D probability density distributions of bending and swivel angles at different sites of LV16 (left panels) and at the respective sites within LXG variants (right panels). To reduce noise in the data, a bin width of 2° has been used for both angles.

Glycine modulates collective helix dynamics Finally, we assessed the collective dynamics of the helix which depends on the correlation of local fluctuations. We analyzed large-scale global helix bending and twisting within the conformations sampled in the trajectories using the program DynDom.55 A mechanical hinge ACS Paragon Plus Environment

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is defined as a region of high flexibility, constraining and/or correlating the movement of flanking quasi-rigid domains.61,62 In about 15 % to 25 % of the investigated conformations, no hinge motions could be detected (Figure 6 A). Among the annotated conformations, ~70 % exhibit single hinges (BEND1/TWIST1 ratio ~ 60/40 %). In ~30 % of the annotated conformations, a pair of hinges connects three rigid segments (BEND2/TWIST2). A slight reduction of single bending motions (BEND1) is a consistent difference observed between LV16 and LXG mutants. Figure 6 B, C exemplarily visualizes single hinge regions of BEND1 or TWIST1 type motions, respectively, of LV16. The dominating single hinge bending and twisting motions are mainly located in the center of the helix (residues 8-9) or the flanking regions (residues 6-7 and 10-12). These distinct populations overlap in the distributions shown in Figure 6 D that depict the probability for each residue to be involved in a BEND1/TWIST1 motion. With LV16, residues 7 – 8 exhibit the highest probability to act as single hinge residues, with a shoulder at residue 10. The preference of hinge formation around these positions explains the W-shape of the predicted local amide exchange rates that peak around residues 8 to 10 (Fig. 3 A). Interestingly, glycine shifts the most probable hinge location towards its own position (Figure 6 D, E). However, none of the glycine positions are identical with the points of highest hinge probability.

In sum, introducing glycine has only a minor impact on the type of collective helix motion but shifts hinge regions along the helix backbone.


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Figure 6. Analysis of collective helix motions. (A) Probability of single bending (BEND1) and twisting (TWIST1) as well as double bending (BEND2) and twisting (TWIST2) motions. (B) Visualization of exemplary bending (BEND1) motions. (C) Visualization of exemplary twisting (TWIST1) motions. Hinge sites are colored in green, the regions moving as quasirigid bodies with respect to the flexible hinges are shown in blue and red color. Screw axes are shown as grey arrows. The rotation angles for all motions shown are ~35°. (D, E) Residual probability to be detected as hinge residue joining two segments (type BEND1 or TWIST1). Values are normalized to the total number of detected conformations exhibiting motions of type BEND1 or TWIST1.


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Discussion Our data indicate that the local packing defect that results from substituting the bulky leucine for a glycine can locally distort the backbone of a transmembrane helix and change its collective dynamics. To which extent does the role of glycine depend on its position within a helix? DHX experiments reveal that glycine close to a helix terminus (L3G, L5G, L15G) accelerates the early phase of the overall amide exchange kinetics, while glycine closer to the helix center (L7G, L9G, L11G) accelerates later phases (Figure 1). Site-specific DHX data uncover gradual progression of amide exchange from the helix termini towards its center which indicates helix fraying near the termini (Figure 2 A), as previously found for water-soluble helices.58,59 The early phase of exchange thus reflects DHX near helix termini while later phases result from DHX closer to its center. It follows that the position-dependent impact of glycine on early and late phases likely reflects its differential impact on more flexible terminal parts or more rigid central parts, respectively. Apart from this apparent position-dependent effect of glycine on the overall DHX kinetics, carefully validated MD simulations do suggest minor differences in computed DHX rates at and around glycine located at different sites (Figure 3 B). What determines the observed changes in DHX? Comparing the effects of glycine in the different LXG peptides on local exchange rates, fopen, and local hydration (Figure 3) reveals that glycine affects fopen as well as local hydration. Notably, the changes in DHX rates at different amides around a glycine correlate much more with the changes in fopen than with changes in hydration. In other words, it is the effect of glycine on the stability of H-bonds that primarily specifies the change in amide exchange.63


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What is the mechanism by which glycine mediates these effects? The packing defect introduced by glycine results in local distortions of helix geometry, as reflected by an altered RPR (Figure 4). In addition, the water molecules attracted by glycine can H-bond to the backbone and thus compete with intrahelical H-bonding. Moreover, glycine causes a redistribution from α-helical to 310-helical H-bonding. The packing defect, enhanced local hydration, and changes in the backbone H-bond network are connected to rarely occurring bending motions at and near glycine (Figure 5) as well as to altered collective motions of the helix backbone as indicated by shifts of the single-hinge-center (Figure 6 D, E). One important aspect of this scenario is that glycine at the pivot point of local helix bending motions localizes to their concave side (Figure 5 B). This suggests that the direction of helix bending is mainly determined by the lack of steric hindrance between glycine and side chains at upstream and downstream sites. Thus, glycine appears to differ from other amino acids in the way by which it promotes helix flexibility. For example, helix distortion by proline14,15 results from its cyclic side chain that prohibits amide H-bond formation and introduces steric hindrance.16 The differential flexibility of transmembrane helices with varying leucine/valine ratios, such as L16 and LV16, had previously been connected to the extent of side-chain/sidechain interactions, where the low volume of the valine side-chain and its slow rotamer switching is destabilizing.38 Transmembrane helix flexibility can also be modulated by Hbonding between polar side chains and the main chain.42,64–66 Our results help to resolve the controversies on the role of glycine in transmembrane helix flexibility. While some database analyses did not detect a role of glycine in TMD helix kinking18,21,24, other studies found such a connection.19,20,23Also, relating TMD kink formation to sequence conservation did not unequivocally ascribe a role of glycine in transmembrane helix flexibility.18–21, However, these studies are based on multi-span proteins where an impact of glycine might be obscured by stabilizing helix-helix packing. Further, our current analysis suggests that glycine does not so much introduce permanent kinks but enable ACS Paragon Plus Environment

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bending dynamics that are not detected in a static structure. Our results also contrast a previous MD study which detected a clear impact of proline, but not of glycine, on the bending of model helices.32 The inability of that study to detect a clear effect of glycine might stem from the use of an oligo-alanine host helix whose intrinsic flexibility might have minimized further destabilization by glycine. Further, modelling within a membranemimicking octane slab is likely to have stabilized intrahelical H-bonding thus rendering them insensitive to the effects of a glycine-induced packing defect.

The very same argument provokes the question to which extent a glycine-mediated packing defect can destabilize a transmembrane helix in a natural lipid membrane, where intrahelical H-bonds are more stable than in water. In other words, how relevant are our results obtained in water-containing solvent to the situation in a membrane? Even if the modest increases in total H-bond occupancy observed here do not occur in a membrane, we find that glycine leads to a pronounced redistribution of α-helical to 310-helical H-bonds. A similar H-bond shifting has previously been observed near the di-glycine hinge of the amyloid precursor protein (APP) TMD50, upon mutational straightening of helix B of bacteriorhodopsin, and within TMDs of different conformers of the sarcoplasmic calcium ATPase.67 Moreover, systematic structural analysis have uncovered hundreds of short 310-helical fragments within transmembrane helices.22 It has been argued that redistributing H-bond patterns in a membrane may confer conformational flexibility to TMD helices without the considerable energetic cost that is associated with H-bond opening.67 Thus, the ability to shift H-bond patterns may render glycine an effective helix-destabilizing amino acid in a membraneembedded helix. Moreover, the role of glycine is likely to be enhanced within transmembrane helices within water-containing multi-span membrane proteins, such as intramembrane proteases, channels, and transporters. For example, one of the catalytic aspartate residues of presenilin, the enzymatic component of γ-secretase, is part of a conserved and functionally ACS Paragon Plus Environment

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required GxGD motif of TMD7. Located within the aqueous environment of the catalytic cleft68, glycine may increase the flexibility of TMD7 and thus facilitate catalysis. A GxGD motif is also highly conserved in presenilin-like proteins69, signal peptide peptidases70, and the type-4 prepilin peptidases,71 thus underscoring its importance for intramembrane proteolysis. Simulations have suggested that the di-glycine hinge of the APP TMD, a substrate of γ-secretase,50,72–74 attracts solvent50; this may enhance the flexibility required for the correct positioning of scissile bonds within the catalytic cleft.75 Other examples, where glycine residues are associated with functional TMD kinks or bendings include the ammonia channel AmtB31,76 and fluoride ion channels of the Fluc family.77 Moreover, glycine residues are abundant within transmembrane helices proposed to transiently unfold during conformational transitions of the leucine transporter LeuT78 and the multihydrophobic amino acid transporter MhsT.79 Consistent with this, a statistical analysis of high-resolution membrane protein structures found that glycine and proline in partially unfolded TMDs of channels and transporters are significantly more conserved than other residues.10 The demonstrated ability of glycine residues to induce local flexibility and H-bond destabilization in a TMD model helix in the present study could provide a molecular rationale for how functionally-important helical dynamics is achieved in some natural integral membrane proteins.


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ASSOCIATED CONTENT Supporting Information Supporting information contains Figures S1 – S10, and Tables S1 and S2. AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected]

Author Contributions PH and FK performed all experimental work. AG did MD simulations and most evaluations of the trajectories. ME did initial analysis of MD simulations. WS did initial DHX experiments and gave valuable advice to PH. KDR contributed his expertise with HDX-ETD measurements and data analysis, CS contributed her expertise in MD simulations and did the DynDom analysis. DL designed and supervised the project and wrote the manuscript with PH and AG. All authors have commented and given approval to the final version of the manuscript. § These authors contributed equally. Funding Sources This work was supported by grant LA699/16-1, LA699/20-1 and SCHA630/3-1 of the Deutsche Forschungsgemeinschaft and the Center of Integrative Protein Science Munich (CIPSM). Computing resources were provided by the Leibniz Supercomputing Centre (LRZ) through grants ta511 (Linux Cluster) and pr42ri (SuperMUC). Notes The authors declare no competing financial interests.


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Faster Exchange / Dynamics

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G5

G9

G13 Slower


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Glycine Perturbs Local and Global Conformational Flexibility of a

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