Soluble Regions of GlpG Influence Protein-Lipid Interactions and Lipid

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Soluble Regions of GlpG Influence ProteinLipid Interactions and Lipid Distribution Yasser Almeida-Hernandez, and Henning Tidow J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b06943 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 3, 2019

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The Journal of Physical Chemistry

Soluble Regions of GlpG Influence Protein-Lipid Interactions and Lipid Distribution Yasser Almeida-Hernandez1,2* and Henning Tidow1,2*

1

Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of

Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany 2

The Hamburg Centre for Ultrafast Imaging, University of Hamburg, Luruper Chaussee 149,

D-22761 Hamburg, Germany

* Corresponding authors Henning Tidow Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany Tel: +49 40428388984 e-mail: [email protected] Yasser Almeida-Hernandez Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany e-mail: [email protected]

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract The GlpG rhomboid protease from E. coli is a well-characterized intramembrane protease that cleaves transmembrane substrates inside the lipid bilayer. Most studies have focused on the GlpG transmembrane domain containing the catalytic site, while the full-length protein, also containing a soluble cytoplasmic domain, a linker region, and a small positively charged Cterminal fragment remains poorly understood. In this work, we used coarse-grained molecular dynamics (CGMD) simulations to investigate full-length GlpG embedded in a native-like model of the E. coli membrane. We identified differences in the distribution and clustering of phosphoglycerol(PG)-based lipids around GlpG in both leaflets depending on whether the soluble regions are present or absent. These data suggest a possible role of the cytoplasmic extensions of GlpG in the regulation of the lipid environment around GlpG, which may influence the activity of GlpG in vivo.


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Introduction Intramembrane proteases are present across all species and are involved in key physiologic functions such as cell signaling, pathogen invasion, cell growth, and epithelial homeostasis 1– 6.

These proteins perform their proteolytic function by cleaving peptide bonds inside the

hydrophobic environment of the lipid bilayer 7,8. How these enzymes perform their hydrolytic reaction in a lipidic environment has been investigated for 20 years but still remains poorly understood at a molecular level. The GlpG rhomboid protease from E. coli is one of the most extensively studied intramembrane proteases. GlpG is a serine protease with Ser201 and His254 forming the catalytic site. The protein contains a transmembrane domain (TMD) consisting of six αhelices, a linker region (Ln) and a soluble cytosolic N-terminal domain (CytoD) 9. Several crystal structures of wild-type and mutants of the TMD have been determined, either in apo form and in complex with inhibitors

10–17,

providing detailed structural information about the

catalytic site and its arrangement within the TMD. As the TMD shows activity in the absence of the rest of the protein 18, the influence of the Ln and the CytoD domain have been widely ignored. The atomic structure of the CytoD has been determined by NMR and X-ray crystallography, showing a compact α-β fold that can also form domain-swapped dimers in the presence of detergents

18,19.

The function of CytoD is still unknown. It has been shown

that cytosolic extensions of rhomboid protease Rho4 from Drosophila melanogaster regulate the activity by modulating the substrate gating by calcium binding

20,

while in yeast, the

cytosolic domain of Rbd2 rhomboid protease controls the lipids organization and actin assembly during clathrin-mediated endocytosis 21. In the course of the standard purification of TMD or CytoD by protease cleavage of full-length GlpG 10 the Ln region is eliminated; hence there is little structural information about this region, although it is expected to be flexible and



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unstructured. In addition, GlpG contains a small positively charged, C-terminal fragment, that it is eliminated during the purification of TMD. Using atomistic MD simulations of GlpG TMD inserted in POPE or POPC model membranes, differences in the hydrogen bonding between lipid head groups and the protein surface were found, which influence the orientation of GlpG in the bilayer 22. More recently, full-length GlpG was reconstituted in SMALP nanodiscs to study the influence of the native membrane lipids composition in the dynamics and accessibility of the protein using hydrogendeuterium exchange mass spectrometry (HDX-MS) 23. In this study, we used the previously determined lipid ratios in E. coli membranes

23

to build

systems of full-length GlpG (GlpG) and GlpG-TMD embedded in a model membrane composed of PE-, PG-derived lipids, and cardiolipin. Using coarse-grained molecular dynamics (CGMD) simulations we identified GlpG/lipid interaction sites and distinct lipid clustering in both leaflets, with differences between GlpG and GlpG-TMD. Our simulations revealed specific accumulation features of various lipids (differing with respect to saturation level and head group) to GlpG. While some lipids accumulate around the protein (annular lipids) and show lower presence at higher distances from the protein (e.g. DOPG), other lipids are less likely to interact with the protein and are more homogeneously distributed in the bilayer (bulk, DPPG).

Methods Systems setup A structural model for full-length GlpG was generated using the Phyre server 24. This model and a GlpG-TMD structure (PDB: 2XOV) were converted to CG models using the program martinize

25.

The proteins were inserted with the program insane

26

in 12 x 12 nm solvated

model membranes, containing PE- (DPPE: di-C16:0 PE, DOPE: di-C18:1(9c) PE, POPE: 4 ACS Paragon Plus Environment

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C16:0/18:1 PE), PG- (DPPG: di-C16:0 PE, di-C18:1(9c) PG, POPG: C16:0/18:1 PG) based lipids, and cardiolipin, at a ratio of 70.5 % / 27 % / 2.5 %, respectively (Table 1). Lipids topologies and coordinates were obtained from the MARTINI force field web site 27. Na+ and Cl- were added at concentration of 0.15 M for neutralization. 5 independent systems were built for each protein, where the bilayers were constructed placing the lipids randomly.

Simulations The simulations were performed with the GROMACS suite 5.1.2 28, using the MARTINI 2.2 force field

29.

Elastic network RubberBand, similar to ElNeDyn

30,

and implemented in

martinize, was used to restrain the entire protein during the simulations. We used all the default parameters in Martini force field defined in the martinize script. We built the ELNEDYN elastic network CG system with the default cut-off distance RC = 0.9 nm and the elastic bond constant KSPRING = 500 kJ mol-1 nm-2 30. We used the default reaction-field with the parametrized short-range shifted electrostatic interaction, with Coulomb cut-off distance rCoulomb = 1.2 nm. The systems were minimized with the steepest descent method up to a maximum of 10000 steps. Next, equilibration runs were performed using a Berendsen barostat 31

with a coupling time of 10 ps. Independent production runs for each system were executed

during 10 s using semi-isotropic pressure coupling to a reference pressure of 1.0 bar with the Parrinello-Rahman barostat

32

and compressibility of 3.4 x 10-4 bar-1. The temperature was

controlled at 300 K using the velocity rescaling thermostat

33

with a time step of 10 fs. Five

independently built trajectories were simulated for each system. Analysis In order to allow sufficient equilibration of the systems, all analyses were performed on the last 1 s of the simulations. Before the analysis, all the trajectories were structurally aligned



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to the last frame of the GlpG in the first run. XY radial distribution functions (RDF) were computed with the xy_rdf tool implemented in the LOOS suite (v2.3.2)

34

using the center of

mass of the transmembrane domain in both systems. 2D density maps were obtained with the densmap tool from GROMACS, with a grid size of 1 Å. Proteins were rotationally aligned and used as reference to obtain the maps in the same coordinate system. Protein/PO4 bead contacts were calculated with the tool select (implemented in GROMACS) and mapped onto the CG structure with in house scripts. Contacts were defined as protein backbone BB beads and lipid PO4 beads at a cutoff distance of a smaller than 6 Å. Structures and trajectories were visualized and analyzed with UCSF Chimera 35 and VMD 36.

Results and Discussion As the transmembrane domain (TMD) of GlpG retains proteolytic activity most previous studies on GlpG focused on the TMD, and only a few studies of the full-length protein are available. We performed coarse-grained molecular dynamics simulations of full-length GlpG as well as GlpG-TMD embedded in a model membrane composed of PE-, PG-derived lipids, and cardiolipin (Figure 1, Figure S1). The investigated lipids differ with respect to their head group and to the number of unsaturations (double-bonds) in their acyl chains. We computed XY radial distribution functions (RDF) for the phosphate group (coarse-grained PO4 bead) for each phospholipid, in the plane of both leaflets, for GlpG and GlpG-TMD (Figure 2). RDFs show clear differences between lipids with respect to the interaction with GlpG, depending on the presence of CytoD. DOPG dominates the interaction with the protein in both leaflets and systems, although this effect is more prominent in the bottom leaflet. POPG also shows a strong interaction with GlpG in the bottom leaflet, which is lost when CytoD is eliminated. The RDF plot for GlpG-TMD in the bottom leaflet shows that almost all


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lipids (in particular DOPG) are located closer to the center of mass of the transmembrane domain, filling the space that was occupied by the CytoD in GlpG. In order to study the spatial distribution of the lipids in the XY plane of the membrane surrounding GlpG and GlpG-TMD, we computed 2D density maps, averaged along the last 1 s of the simulations for PE- and PG-based lipids in both leaflets (Figure 3 and S2). Each pixel of the maps corresponds to a spatial bin of 1 Å2 and contains the number of events of the presence of a certain PO4 bead in a XY coordinate bin, averaged among all simulations for each system. The inspection of the maps revealed interesting features about the interaction of the lipids with the protein in each leaflet, in particular for POPG and DOPG (Figure 2). In the bottom leaflet, the absence of CytoD affects the protein/lipid interactions, in particular for POPG. We observed a clustering of POPG to two regions of full-length GlpG (Figure 3). The first region corresponds to the residues G170, S171, G172 and A272 (TMD), and the second to residues R81, R82 (Ln) and R227 (TMD). These two clusters were affected when the soluble region was eliminated. In particular, the second cluster is formed by the interaction of Arg residues from the TMD and the Ln region, which provides a positively charged, responsible for the clustering of the lipid in this area. However, when the soluble region is removed, the clustering and number of contacts between POPG and R227 were significantly reduced (Figure 4). For DOPG, there was also a strong clustering in the bottom leaflet in GlpG, in a region corresponding to the Ln segment, as well as an accumulation around the protein forming an annulus. This clustering was diminished in the area corresponding to the Ln segment. This indicated that, although residues located in the TMD seem to be sufficient for the lipid-interaction, the presence of certain residues in the cytoplasmic domain and the linker region provides further stabilization to retain the protein/lipid interaction. In this respect, the



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amount of positively charges residues located in Ln and the region of TMD facing the bottom leaflet may play a role. The differences between POPG and DOPG in the bottom leaflet of GlpG-TMD can be attributed to unsaturations and the hydrophobic mismatch effect. It has been reported that unsaturated lipids show stronger interactions with several membrane proteins through this effect, which can subsequently affect their activity

37–41.

Compared to DPPG, which is

completely saturated, POPG and DOPG both show increased interactions with the protein (Figures 3 and S2). In addition, the two double bonds of DOPG make the length of the acyl chains shorter compared to POPG (contains one double bond), leading to membrane thinning. This behaviour of DOPG could make it more prone to interact with GlpG by matching the hydrophobic surface area of GlpG forming the before mentioned annulus, which is thinner than the average thickness of the membrane (hydrophobic mismatch)

42.

POPG, however,

with only one unsaturation can be more extended and interacts with the protein in defined residues. The hydrophobic mismatch effect on GlpG and its impact on the protein diffusion in the membrane were recently also experimentally demonstrated by Kreutzberger and coworkers 43. Interestingly, we also detected some changes in POPG and DOPG clustering in the top leaflet (Figure 3), in a region corresponding to an interaction with the L5 loop, which is responsible for the protein gating to allow substrates to enter in the catalytic site. This effect was also evident in the number of protein/PO4 bead contacts (Figures 4,5 and S3). Previous studies have shown the importance of loop L5 in vitro and in vivo for the gating during substrate binding, and mutations in this region completely abolished the activity 44–46. These variations in the lipid clustering and contacts around the L5 loop may suggest that these could regulate the access of substrates to the active site and hence the activity of the protein. Similar behavior has recently been described in atomistic simulations, where lipid head groups could


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compete with the substrate in the membrane by transient contacts in the active site of the protein

47.

Furthermore, crystal structures of GlpG-TMD show a detergent molecule in the

active site as well as lipid molecules interacting with residues in the cytoplasmatic side of the molecule 48. We also found that the positively charged C-terminal fragment interacts with all lipids (Figure 5). This fragment together with CytoD and Ln is usually lost during the standard proteolytic purification of TMD

10

and thus not present in the available crystal

structures. In summary, our simulations reveal that GlpG preferentially interacts with unsaturated and PG-based lipids. These lipids preferentially accumulate in the annular belt of GlpG, which may lead to membrane thinning due to hydrophobic mismatch 42. In addition, residues in the linker and bottom region of GlpG are involved in the regulation of the lipid distribution in the bottom leaflet of the membrane. Although our simulations did not show large conformational changes for the linker region, it may still be flexible and adopt extended conformations, in which the cytoplasmic domain and the linker region are more distant from the membrane, resulting in an arrangement resembling the GlpG-TMD simulated in this study. In that case, the interactions with the lipids in the bottom leaflet would change, affecting their distribution in both leaflets. We propose this as a putative a mechanism to regulate the activity of GlpG, where the soluble regions of GlpG act as a relay by regulating the lipid distribution around the protein. As stated before, this mechanism has been demonstrated before in yeast, where the cytosolic domain of Rbd2 rhomboid protease influences the lipids organization, which in turn regulates actin assembly during clathrin-mediated endocytosis 21. Our results indicate that the cytoplasmatic extensions of GlpG affect the lipidic environment around its transmembrane domain.



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Conclusion We performed coarse-grained molecular dynamics simulations to study the interaction of GlpG with lipids of the cell membrane. Although most structural and functional studies of GlpG to date have been performed using the transmembrane domain only, our data indicate that cytoplasmic regions of GlpG (N-terminal cytoplasmic domain, linker region and Cterminal extension) play a role in the regulation of the activity of the protein by modifying the surrounding lipid environment. In addition, we show that unsaturated PG-based lipids interact preferentially with GlpG compared to PE-based and saturated lipids. Although further experimental work is required to prove this hypothesis, our simulations contribute to a better understanding of the possible regulatory role of the soluble fragments of GlpG, widely ignored to date.

Supporting Information Available 

Figure S1: Top and bottom views of GlpG and GlpG-TMD



Figure S2: Density maps of PE-based lipids and DPPG for GlpG and GlpG-TMD



Figure S3: GlpG/GlpG-TMD contacts with PO4 beads along the simulation

Author contributions Conceptualization, YAH and HT; Investigation, YAH; Analysis and Interpretation, YAH and HT; Manuscript Writing, YAH and HT; Funding Acquisition, HT; Supervision, HT.

Acknowledgments We thank Regionales Rechenzentrum (RRZ) at the University of Hamburg for access to the Hummel computer cluster. This research was funded by the excellence cluster 'The Hamburg


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Centre for Ultrafast Imaging - Structure, Dynamics and Control of Matter at the Atomic Scale' of the Deutsche Forschungsgemeinschaft (DFG EXC 1074).

Competing Financial Interest statement We declare no competing financial interests.



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Page 16 of 21

Figure legends Figure 1

Full-length model of GlpG embedded in a phospholipid bilayer. A) Coarse-grained (CG) representation of GlpG embedded in the bilayer. Regions of the protein are shown: CytoD (magenta), Ln (cyan), TMD (green), L5 loop (blue) and C-terminal fragment (red). The model was generated with the Phyre server using the crystal structure of the transmembrane domain (pdb:2XOV) and the NMR-structure of the cytoplasmic domain (pdb:2LEP), and transformed with martinize

25.

B) CG models of lipids used with the beads for every atoms group:

ethanolamine (blue), PO4 (orange), glycerol (magenta), acyl chain (gray). The white bead represents the unsaturation of the acyl chain.


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Figure 2

Lateral radial distribution function (XY-RDF) analysis. Displayed is the XY-RDF of the lipid phosphate beads in the top and bottom leaflets of the model bilayer, with embedded GlpG and GlpG-TMD. Curves for each lipid represent the average between five independent runs.



Page 18 of 21

Figure 3

2D density maps of the phosphate beads of POPG and DOPG, with embedded GlpG and GlpG-TMD. Each pixel of the maps represents a bin of 1 Å2 and contains the count of PO4 beads in each grid point along the last s of the simulations averaged between five independent runs. The red arrowheads (top leaflet) indicate the lipid accumulation close to the L5 loop and the substrate-gating site. Dark grey arrowhead (bottom leaflet, POPG) indicates an interaction cluster with G170, S171, G172 and A272 (TMD). Light arrow indicates an interaction cluster with R81, R82 (Ln) and R227 (TMD).


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Figure 4

Residue-based protein-lipid contacts. Number of contacts between backbone BB beads of the protein and PO4 beads of each lipid. Contacts were defined as the PO4 at a cutoff distance of smaller than 6 Å. The colored bars represent the regions in the protein. Magenta: CytoD, Cyan: Ln region, Green: TMD, Blue: L5 loop.



Page 20 of 21

Figure 5

3D mapping of the protein-lipid (backbone BB bead/lipid PO4 bead) contacts on the structure. The color scale and the size of the BB beads represent the contact frequency of each lipid PO4 bead with the protein residues, normalized against the total number of contacts of each lipid. The color of the bonds represents each region of the protein. Magenta: CytoD, Cyan: Ln region, Green: TMD, Blue: L5 loop.


Page 21 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1: Simulations setups GlpG full length

GlpG-TMD

Lipid

TOP

BOTTOM

TOP

BOTTOM

DPPE

47

48

49

48

POPE

47

48

49

48

DOPE

47

48

49

48

DPPG

18

18

18

18

DOPG

18

18

18

18

POPG

18

18

18

18

CDL

3

3

3

3

Solvent Water

8966-9036

9224-9239

Na+

133

138

Cl-

66

68

TOC Graphic

ACS Paragon Plus Environment

21

Soluble Regions of GlpG Influence Protein-Lipid Interactions and Lipid

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