Mechanisms by Which Lipids Influence Conformational Dynamics of

Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

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Mechanisms by Which Lipids Influence Conformational Dynamics of the GlpG Intramembrane Protease Ana-Nicoleta Bondar* Freie Universität Berlin, Department of Physics, Theoretical Molecular Biophysics Group, Arnimallee 14, D-14195 Berlin, Germany

J. Phys. Chem. B Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 05/11/19. For personal use only.

S Supporting Information *

ABSTRACT: Rhomboid intramembrane proteases are bound to lipid membranes, where they dock and cleave other transmembrane substrates. How the lipid membrane surrounding the protease impacts the conformational dynamics of the protease is essential to understand because it informs on the reaction coordinate of substrate binding. Atomistic molecular dynamics simulations allow us to probe protein motions and characterize the coupling between protein and lipids. Simulations performed here on GlpG, the rhomboid protease from Escherichia coli, indicate that the thickness of the lipid membrane close to GlpG depends on both the composition of the lipid membrane and the conformation of GlpG. Transient binding of a lipid headgroup at the active site of the protease, as observed in some of the simulations reported here, suggests that a lipid headgroup might compete with the substrate for access to the GlpG active site. Interactions identified between lipid headgroups and the protein influence the dynamics of lipid interactions close to the substrate-binding site. These observations suggest that the lipid membrane environment shapes the energy profile of the substrate-docking region of the enzyme reaction coordinate.

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INTRODUCTION Intramembrane proteases catalyze cleavage of peptide bonds of transmembrane (TM) substrates. Such chemical reactions are implicated in, for example, cleavage of amyloid precursor protein by presenilin1 or by rhomboid RHBDL4,2 formation of adhesive peptides required by the malaria parasite Plasmodium falciparum,3 and mitochondrial dynamics.4 An intriguing, yet poorly understood aspect of intramembrane proteases is that their catalytic activities depend on the composition of the surrounding lipid membrane.5−8 An excellent model system to dissect the mechanism by which lipids influence catalysis is GlpG, the intramembrane rhomboid protease from Escherichia coli. Knowledge of the three-dimensional structure of GlpG in different conformations9,10 and of its catalytic activity in various lipid membranes5,11,12 makes it possible to use computer simulation techniques to explore the mechanism by which lipids participate in GlpG’s reaction mechanism. GlpG has a small size and a relatively simple architecture (Figure 1). It contains six TM helices of different orientations relative to the membrane normal. The catalytic S201 and H254 are located close to the membrane center (Figure 1A). At the extracellular side, the loop connecting TM5 and TM6, denoted as the cap loop L5, was proposed to control accessibility to the active site;12 substrate docking from the membrane side involves lateral motions of TM5, denoted as the “lateral substrate gate”.13 At the cytoplasmic side, the loop connecting TM4 and TM5, L4, plays an important, yet poorly understood role: mutations of hydrophobic groups from this region can largely inhibit catalysis.14 Networks of hydrogen (H) bonds © XXXX American Chemical Society

interconnect helices 1-2-3 and 3-4-6, and the region of the active site, to the lipid bilayer via groups from loop L1.15−17 These H-bond networks are likely important structural elements of the mechanism of long-distance protein conformational coupling, and for coupling GlpG to the surrounding lipid membrane.15−17 Crystal structures of GlpG suggest that L5 and TM5 are mobile, and indeed a lateral displacement of TM5 distinguishes two crystal structures thought to represent the open and closed states of the enzyme.9 These structural differences may, however, be influenced by the crystallization conditions,12 and TM5 appears less mobile in proteoliposomes than in detergent.8,18 Time scales associated with lateral motion of TM5 during the transition from the closed to open state, when substrates are present, remain unclear; they must, however, be longer than ∼100 ns because during molecular dynamics (MD) simulations on this time scale, no conformational transitions of TM5 could be observed.19 Motions of TM5 or other structural elements that interact with lipids might depend on lipids. Although TM5 appears highly dynamic in simulations with an implicit membrane,20 its motions when GlpG is reconstituted in proteoliposomes are restricted relative to detergent.8 For TM5 to move laterally, lipids from the immediate vicinity would need to be displaced; these lipid displacements could involve breaking and reforming Received: November 21, 2018 Revised: April 10, 2019

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DOI: 10.1021/acs.jpcb.8b11291 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 1. Architecture and lipid interactions of GlpG. (A) GlpG embedded in a hydrated 1-palmytoyl-2-oleoyl-sn-glycero-3-phsphatidylethanolamine (POPE) lipid bilayer. In addition to bulk waters, waters whose oxygen atom is within 9 Å of the catalytic groups S201 and H254 are shown explicitly as van der Waals spheres. The cap loop L5 and helix TM5 are thought important for substrate access to the active site,12−14 loop L4 carries hydrophobic groups whose mutation inhibits function,14 and loop L1 appears to have a structural role.13 (B) View from the periplasmic side of GlpG. Water molecules from the cytoplasmic side of the protein are colored pink. All molecular graphics were prepared using visual molecular dynamics (VMD).50

lipid membranes (see, e.g., ref 16). GlpG can be embedded in lipid membranes of various compositions, and GlpG/lipid interactions can be sampled at room temperature. Here, I probe the conformational dynamics of wild-type GlpG starting from three different crystal structures, and using five different lipid membrane environments, for a total of 11 independent simulations (Sims). Four additional Sims on two GlpG mutants probe perturbations at selected sites at the protein/ lipid interface. The protein structures were chosen so as to probe the dynamics and lipid interactions of GlpG starting from crystal structures thought to represent the open vs closed states.9,10 Two main conformations of GlpG, open- and closed-gate, have also been observed with electron paramagnetic resonance spectroscopy.8 For the closed state, I used chain A from PDB ID: 2IRV.10 For the open state, I used two structures, chain B from PDB ID: 2IRV10 and chain A from PDB ID: 2NRF.9 The two structures suggested to represent open conformations9,10 are distinguished largely by the orientation of TM5, whose lateral displacement is more pronounced in 2NRF than in 2IRV.28 For simplicity, in what follows, the 2IRV open-state structure10 is denoted as “open”, whereas structure 2NRF chain A9 is denoted as “open-TM5”. I chose the lipid bilayers so as to evaluate the dynamics of GlpG in one-component bilayers distinguished by their Hbonding properties and/or their lipid alkyl chains, and in a two-component lipid bilayer (3:1 mixture of POPE and POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylgycerol) proposed to model the E. coli membrane.29 To probe the effect of the lipid headgroup H bonding, I consider POPE vs 1palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine, POPC; the ethanolamine group of POPE can easily donate H bonds, whereas in POPC, the methyl groups hinder H bonding at the choline region.15 The lipid alkyl chains considered include monounsaturated (POPC), biunsaturated (1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol, DOPC), and shorter, saturated alkyl chains (1,2-dymyristoyl-sn-glycero-3phosphatidylcholine, DMPC). The simulations indicate that thinning of the lipid membrane close to GlpG depends on the composition of the lipid bilayer and on the conformation of GlpG. This suggests a complex dynamics of the lipid/protein interactions, whereby the thickness of thinner lipid membranes close to GlpG can

of H bonds of lipid headgroups and rearrangements of hydrophobic contacts between lipid alkyl chains and the protein. Presence in the crystal structure of open-state GlpG of a phosphatidylglycerol (PG) lipid headgroup H-bonded to the catalytic groups10 suggests that, in lipid membranes, unbinding of a lipid from the active site might be a distinct event along GlpG’s reaction coordinate, required before the substrate could dock. The propensity of the lipid headgroup to visit the catalytic site likely depends on whether there are favorable electrostatic and/or hydrophobic interactions with protein groups and on the local thickness of the lipid membrane. Close to the protease, membrane thickness is determined by the lipid composition, which gives the intrinsic thickness of the proteinfree membrane, and by interactions between lipids and the protein surface, which may cause the membrane to adjust to the hydrophobic thickness of the protein itself. Estimations of the thickness of the lipid membrane close to GlpG came from inspection of a crystal structure21 and from computer simulations of GlpG in hydrated one-component lipid membranes.15,19,22 All-atom simulations revealed that a hydrated lipid membrane composed of 1-palmytoyl-2-oleoylsn-glycero-3-phsphatidylethanolamine (POPE) lipids adjusts to the presence of GlpG such that, on the average, there is nonuniform thinning of the membrane by ∼3 Å relative to the bulk membrane.15 Since PE lipids are the major phospholipid component of the E. coli membrane23−25 and GlpG is catalytically active when reconstituted in PE,5 POPE lipid membranes appear suitable as model system to study the motions of GlpG. But the 42−45 Å phosphate-to-phosphate thickness of a model POPE bilayer26 is larger than that of the E. coli lipid membrane: The thickness of the E. coli cytoplasmic membrane estimated from experiments is 37.5 ± 0.5 Å and that of liposomes composed of E. coli lipids is 33.5 ± 0.4 Å.27 This difference between the thickness of E. coli and model POPE membranes raises the possibility that GlpG sits differently in E. coli vs POPE membranes. Different lipid interactions and motions of GlpG could provide a molecular interpretation of why GlpG cleaves poorly a model Spitz substrate when embedded in E. coli lipids extract as compared to PE.5 All-atom MD simulations are valuable tools to derive knowledge on how membrane proteins interact with fluid B


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The Journal of Physical Chemistry B Table 1. Summary of Simulations Performeda Sim Sim1 Sim2 Sim3 Sim4 Sim5 Sim6 Sim7 Sim8 Sim9 Sim10 Sim11 Sim1a Sim1b Sim6b Sim7b

starting structure

protein conformation

2IRV, chain B

wild type, open

2IRV, chain A 2NRF, chain A

wild type, closed wild type, open-TM5

2IRV-B, K191A 2IRV-B, D243A 2IRV-A, D243A 2NRF-A, D243A

K191A, open D243A, open D243A, closed D243A, open-TM5

lipids

temp. (K)

length (ns)

POPE POPC DOPC POPE/POPG DMPC POPE POPE POPE/POPG DMPC POPC DOPC POPE POPE POPE POPE

310.15 303.15 303.15 310.15 303.15 310.15 310.0 310.0 303.0 303.15 303.15 310.15 310.15 310.15 310.15

204.0 197.0 183.0 190.0 204.0 204.0 190.0 204.0 186.0 204.0 204.0 204.0 204.0 190.0 195.9

rmsd (Å) 0.9 0.9 1.5 1.2 1.1 0.8 1.6 1.1 2.1 2.1 1.9 1.1 0.9 1.0 2.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.2 0.5 0.1 0.1 0.1 0.1 0.2

a

Sim1−Sim11 are simulations on wild-type GlpG. Sim1a and Sim1b are simulations on mutant GlpG started from the protein structure PDB ID: 2IRV-B, chain B. Sim6b and Sim7b are simulations of the D243A mutant started from, respectively, PDB ID: 2IRV (chain A) and 2NRF (chain A). The length of the simulation, in nanoseconds, is the length of the unconstrained simulation. The root-mean-square distance (rmsd) indicates the average Cα rmsd of the TM helical segments computed from the last 100 ns of each Sim.

Lipid Membranes. Independent simulations were prepared and performed for GlpG embedded in five different lipid membrane environments (Table 1). POPE and POPC have the same length of their alkyl chains (16:0, 18:1) and one double bond at position C9 = C10 of the sn-2 chain. DOPC and DMPC lipids have alkyl chains 18:1, 18:1, and, respectively, 14:0, 14:0; together with POPC, the DOPC and DMPC simulations serve to assess the response of GlpG to changes in lipid alkyl chain dynamics and membrane thickness. With simulations on GlpG embedded in a 3:1 POPE/POPG lipid mixture, I probe the dynamics in an environment proposed to approximate that of E. coli.29 Force-Field Parameters and MD Protocol. MD simulations were performed using CHARMM force-field parameters35−39 with TIP3P water40 and NAMD41,42 to generate the trajectories. Initial equilibration was performed using velocity reassignment and the scheme for restraint forces as suggested by CHARMM-GUI.32 Briefly, in this equilibration scheme, during the first five steps of equilibration, 50−100 ps each, force constants are placed on protein heavy atoms and gradually reduced from 10 to 0.5 kcal/(mol Å2) for the backbone, and from 5.0 to 0.1 kcal/(mol Å2) on side chains. Force constants of 10 kcal/(mol Å2) are placed on the ions only during the first equilibration step. Additionally, during this equilibration scheme, restraint forces used for waters and lipids are gradually released. In the sixth step of the equilibration scheme, 100 ps long, a force constant of 0.1 kcal/(mol Å2) is placed on the backbone heavy atoms. All constraints are then switched off for the production run. The first two equilibration steps, 25 ps each, were performed in the NVT ensemble (constant number of particles, constant volume, and constant temperature); remaining equilibration steps and the production run were performed at constant pressure (1 bar) and constant temperature (NPT ensemble). The temperature used in each Sim is reported in Table 1. The length of the covalent bonds to H atoms was fixed.43 For NPT simulations, I used a Langevin dynamics scheme44,45 with a damping coefficient of 5 ps−1, the Langevin piston period set to 200 fs, and piston decay set to 100 fs. A switch function was used between 10 and 12 Å for short-range real space interactions, and smooth particle

change during the reaction coordinate of GlpG, and could not be easily predicted based only on the composition of the lipid bilayer. Whether or not a lipid headgroup samples the region of the active site depends on the properties of the lipid’s headgroup and alkyl chain and on the conformation of GlpG. Specific protein groups that interact with lipid headgroups help control the orientation of the protein in the hydrated lipid membrane environment, and how lipids interact with the region of the substrate-binding site.

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METHODS Protein Structures. Independent MD simulations were performed using three different protein structures deposited in the Protein Data Bank (PDB): chain A from PDB ID: 2IRV,10 chain B from the same PDB structure, and chain A from PDB ID: 2NRF.9 H254 was protonated on Nδ;15 all other titratable groups were standard protonated, with Arg and Lys positively charged, Asp and Glu negatively charged, and histidine side chains protonated on Nε. Chain A from PDB ID: 2IRV10 lacks coordinates for the cap loop L5, residues 244−249. Coordinates for the missing loop were constructed and refined using Modeller30,31 version 9.16. Chain B from PDB ID: 2IRV contains a phosphatidylglycerol lipid bound at the active site of the protease;10 this lipid molecule was removed from the structure. Removing the lipid headgroup from the starting crystal structure alleviates uncertainties regarding the coordinates of the alkyl chains of this lipid molecule and makes it possible to probe with the simulations whether a lipid molecule could spontaneously bind to the active site. The K191A mutant protein was prepared with CHARMMGUI32,33 starting from the crystal structure PDB ID: 2IRV, chain B.10 Three independent D243A mutant proteins were generated with CHARMM-GUI32,33 starting from the crystal structure PDB ID: 2IRV10 chain A and chain B and from PDB ID: 2NRF chain A.9 I used the web-based servers PPM34 and CHARMMGUI32,33 to orient and place GlpG in hydrated lipid membrane patches, as summarized in Table 1. In all Sims, ions were added for charge neutrality as necessary. C


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Figure 2. Lipid bilayer thinning depends on the composition of the lipid membrane and on the conformation of GlpG. (A) Lipid bilayer thinning as estimated from the difference between peaks of the distribution of phosphate atoms in the two lipid leaflets, far from GlpG as compared to far close to GlpG, computed from the last 35 ns of each Sim. Each dot indicates the estimated thinning computed from a Sim. “PC lipids” indicates POPC, DOPC, or DMPC lipid membranes. As lipids close to GlpG I considered lipids whose phosphate atoms are within 15 Å of protein heavy atoms at the end of each simulation,15 all other lipid molecules are considered as far from GlpG. (B) Estimated thickness of the lipid membrane. The squares and the dots indicate the thickness of the lipid membrane far away from GlpG and close to GlpG, respectively. (C) Box plots of the number of water molecules whose oxygen atom is within 6 Å of the catalytic S201-Oγ atom during the last 100 ns of 12 of the 15 Sims performed. For Sim4, Sim5, and Sim9, in which the number of waters close to S201 changes during the last ∼30 ns of the corresponding Sim, time series of the number of water molecules are presented in Figure S5 together with time series of the other Sims. (D) Histograms of the number of water molecules whose oxygen atom is within 6 Å of the catalytic S201-Oγ atom during the last 50 ns of simulations on wild-type GlpG in POPE: Sim1 (green), Sim6 (purple), and Sim7 (blue). (E) Box plots of the distance between S201 and H254, monitored as the shortest distance between S201Oγ and H254-Nδ1 or H254-Nε2 during the last 100 ns of all Sims except for Sim8, in which the distance increases during the last ∼20 ns segment of the trajectory segment. Time series of this distance along the entire length of each Sim are presented in Figure S6. (F) Box plots of the shortest distance between S201-Oγ and lipid phosphate atoms sampled during the last 35 ns of each Sim. Time series of this distance throughout the entire length of the Sims are presented in Figures 5E, S12G,J and L,M, S15G−I and L,M, and S16A−C and G−L.

mesh Ewald summation46,47 for Coulomb interactions. Equilibration and the first 1 ns of production were performed with an integration timestep of 1 fs; for the remaining of the MD, a reversible multiple timestep algorithm48,49 was used with 1 fs for the bonded forces, 2 fs for short-range nonbonded forces, and 4 fs for long-range electrostatic forces. Coordinates were saved each 10 ps. Trajectory analyses were performed using visual molecular dynamics (VMD),50 ProFit (Quantum Soft), and our own scripts. Distances between selected atoms and the number of specific atoms within a certain distance from a selection of atoms were extracted from the trajectories using tcl scripting in VMD. The thickness of the lipid membrane was estimated

from the peaks of the normalized number density of the lipid phosphate atoms in the two leaflets of the bilayer. Time series for various parameters are reported for the entire length of the unconstrained Sims. Unless specified otherwise, average values and histograms were computed using the last 100 ns of each Sim.

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RESULTS AND DISCUSSION All Sims show overall good structural convergence with relatively small values of the Cα root-mean-square distances (rmsd) of the TM segments relative to the starting crystal structures. Sims started from the closed- and open-state structures10 (Sim1−Sim6, Sim1a, Sim1b, Sim6b) give small D


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The Journal of Physical Chemistry B values of the rmsd, typically ≤1.5 Å, and relatively small values of the loop and termini, up to ∼2.5 Å, when starting from the open GlpG structure (Table 1, Figures S1A−I and S2A−I). Overlaps of structural snapshots from these Sims further support good structural stability of the protein (Figure S3A− I). In Sims initiated from the open-TM5 structure from ref 9 (Sim7−Sim11), the average TM rmsd values for the wild-type protein are ∼1.1 Å in POPE/POPG, ∼1.6 Å in POPE, and ca. 1.9−2.1 Å in PC lipids (Table 1 and Figure S1J−P). Loop L5 has little motion in the open-state Sim1 (Figure S2A), but it is rather dynamic in the closed-state Sim6 (Figure S2H) and the open-TM5 Sim7 (Figure S2J). Loops L1 and L4 have overall small rmsd values in all Sims performed, which indicates structural stability (Figure S2). Taken together, the rmsd profiles (Figures S1 and S2) and coordinate snapshots (Figure S3) indicate that, for all membranes and starting GlpG structures used here, the TM helical core of GlpG has overall good structural stability (Table 1). Thickness of the Lipid Membrane Close to GlpG Depends on the Membrane Composition and on the Conformation of GlpG. The thickness of the lipid membrane close to GlpG could impact the binding and catalytic cleavage of the substrate.15 To estimate the thickness of the lipid membrane in each of the 15 independent Sims performed (Table 1), I use the distance between the peaks of the lipid phosphate-atoms distribution in the two lipid leaflets. To estimate the thinning of the lipid membrane close to GlpG, I report the difference between the thickness of the lipid membrane far away from GlpG and close (within 15 Å) to the surface of GlpG.15 The values estimated for the bilayer thickness and bilayer thinning in all Sims performed here are summarized in Figure 2A,B. Far from GlpG, the thickness of the POPE bilayer in Sims on wild-type GlpG (Sim1, Sim6, and Sim7, Table 1) is 42.2− 42.6 Å (Figure 2B), which is in excellent agreement with recent simulations on a pure POPE bilayer.51 Close to GlpG, the POPE membrane thins by ∼4 Å in Sim1 and Sim6 (open and closed GlpG), which is consistent with previous simulations on open-state GlpG15,19 and on another closed-state GlpG structure.19 A different result is observed in Sim7 (openTM5), where the thinning close to GlpG is ∼1 Å (Figure 2A,B). The average thickness of the POPE/POPG bilayer far away from GlpG is 40−41 Å in both Sim4 and Sim8 (Figure 2B). This value of the POPE/POPG bilayer thickness appears compatible with the ∼42 Å value reported in ref 51 for a 3:1 POPE/POPG membrane, and with the ∼40 Å value estimated elsewhere52 for a 5:1 POPE/POPG membrane. Close to GlpG, there is little thinning in the open-state Sim4, whereas in the open-TM5 Sim8, there is an estimated thinning of ∼3.8 Å (Figure 2A,B). The average thickness of the POPC membrane far from GlpG, ∼39 Å in Sim2 and 39.4 Å in Sim10 (Figure 2B), is close to the ∼40 Å value reported in ref 51, 38.9 ± 3.1 Å in ref 53, and 39.1 ± 0.6 Å reported in ref 54 for the phosphate groups. Likewise, the thickness of the DOPC membrane estimated here, ∼38 Å in Sim3 and ∼38.4 Å in Sim11 (Figure 2B), is compatible with the 38.6 ± 2.8 Å bilayer thickness from ref 53 and 39.6 ± 0.6 Å from ref 51. For DMPC, the estimated average thickness far away from GlpG is 37 Å in both Sim5 and Sim9 (Figure 2B), which is close to 36.2 ± 3.4 Å in ref 53.53 In the open-state GlpG Sim2 (POPC), Sim3 (DOPC), and Sim5 (DMPC), there is barely any thinning of the membrane

close to GlpG (Figure 2A,B). By contrast, close to open-TM5 GlpG the DMPC (Sim9) and POPC membranes (Sim10) thin by ca. 3−4 Å, and the DOPC membrane thins by about 2 Å (Figure 2A,B). Taken together, analyses of the lipid bilayer thinning close to wild-type GlpG indicate that thinning of the bilayer close to GlpG is up to ca. 3−4 Å, that is, up to about one helical turn. The POPE membrane thins by ca. 3−4 Å close to open- and closed-state GlpG, but it thins only slightly in the vicinity of wild-type open-TM5 GlpG. The POPE/POPG membrane undergoes only minor thinning close to open-state GlpG (Sim4), but it thins by about 3.8 Å near open-TM5 GlpG (Sim8). Close to open-TM5 GlpG, the DOPC (Sim11), DMPC (Sim9), and POPC membranes (Sim10) thin by ca. 1.8−3.8 Å; by contrast, the thickness of PC membranes close to open GlpG remains largely the same as far away (Figure 2A). It thus appears that interactions between wild-type GlpG and lipids depend on both the lipids and the protein conformation. Lipid Composition and Protein Conformation Influence Water Interactions at the Active Site. Proteolysis involves the direct participation of a water molecule in the chemical reaction.55 Waters are present at the active site in all three crystal structures used for the simulations (Figure S4). To find out whether solvation at the active site could depend on the lipid environment and on the protein conformation, I extracted from the simulation trajectories the number of water molecules whose oxygen atom is within 6 Å of S201-Oγ (Figures 2C,D and S5). In all Sims, the number of active-site waters fluctuates with time (Figure S5), and it depends on protein conformation and lipid composition. The three Sims performed on wild-type GlpG in POPE lipids indicate that there are, on the average, slightly fewer active-site waters in open- and closed-state GlpG (ca. 6.5−6.9 on the average) than in open-TM5 GlpG (∼8.6 ± 1.8), reported as the mean value ± mean absolute deviation (see Figures 2D and S5A,H,J). Sims on open-state GlpG in different lipid environments suggest that, overall, there are on the average about three more active-site waters in POPC (Sim2) and DOPC membranes (Sim3) than in POPE (Sim1, Figure 2C); in POPE/POPG and in DMPC. there are on the average about seven to eight active-site waters for most of Sim4 and Sim5, with an increase to ca. 11−12, or even more waters, during the last ∼20 ns (Figure S5D,E). In the case of the open-TM5 structure, there are similar numbers of active-site watersabout eight to nine on the averagewhen GlpG is embedded in POPE (Sim7) or POPE/POPG (Sim8); in DMPC, there are on the average about six to seven active-site waters during the last ca. 20−30 ns of Sim9, compared to ca. 11−12 at time point ∼100 ns of this Sim (Figure S5L). To find out whether the number of active-site waters might associate with the dynamics of interactions between S201 and H254, I monitored the shortest distance between S201-Oγ and H254-Nδ1 or H254-Nε2 (Figures 2E and S6). Overall, in wildtype GlpG, S201 and H254 sample H-bond distances only infrequently (Figure 2E). In Sim2 and Sim3, where there are more waters close to S201 than in Sim1, S201 tends to be further away from H254 (Figure 2E). In Sim10, where there are ∼6 waters in the vicinity of S201 (Figures 2C and S5M), S201 and H254 are predominantly within H-bonding distance (Figures 2E and S6M). In Sim9, where the number of waters fluctuates significantly with time (Figure S5L), S201 and H254 E


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Figure 3. Lipid binding to charged groups of GlpG. (A) Molecular graphics of open-state GlpG based on a coordinate snapshot from Sim1. Charged groups of GlpG are shown as van der Waals spheres; side chains whose H bonding to lipids is discussed explicitly in the text are shown in atom colors, other side chains are in white. The side chains of S201 and H254 are shown as green surfaces. H atoms are not shown. (B) Time series of the number of lipid phosphate oxygen atoms within H-bonding distance from K191-Nζ in Sim4. (C) Summary of the number of lipid atoms within H-bond distance from selected protein groups. For Arg and Lys, the numbers indicate the median number of phosphate oxygen atoms within H-bond distance from nitrogen atoms of these side chains; for D116 and D243, numbers show the median number of POPE nitrogen atoms within H-bond distance of the carboxylate oxygen atoms. For simplicity, Sims performed in the same type of membrane are grouped together, and the range of median values is given; numbers in bold indicate the median value in most of the POPE Sims. For example, the median number for K132 in POPE is 1 in all Sims except for Sim1 and Sim6, where the median number is 2 and, respectively, 0. Further analyses of the number of lipid atoms close to selected protein groups are collected in Figures S6 and S7. (D) Molecular graphics of open GlpG in POPE/POPG from Sim4. The protein is viewed from the periplasmic side. K132, K191, and S243 are shown as yellow van der Waals spheres; lipid molecules within 3.5 Å of these three protein groups are shown in atom color. In the coordinate snapshot used for illustration, K132, K191, and D243 are part of a cluster that includes six POPE molecules and one POPG lipid molecule.

in POPE or POPE/POPG membranes (Sim1, Sim4, Sim6; Figures 3A,C, S7C,P, and S8C); there is only sporadic H bonding of D243 to POPE lipids when open-TM5 GlpG is embedded in POPE/POPG (Sim8, Figure S8F,I). In the vicinity of D243, S248 of the cap loop L5 (Figures 4C and S12A) comes frequently to within H-bond distance from POPE phosphate groups when Sims are performed with openTM5 GlpG (Sim7, Figures S11J and S12C), but not with closed (Sim6, Figures S11H and S12B) or open GlpG (Sim1, Figures S11A and S12A). Likewise, S248 H-bonds transiently to lipid phosphate groups during the last ∼40 ns of Sim8 (open-TM5, Figure S11K), whereas in Sim4 (open GlpG), it does not (Figure S11F). Such differences in how POPE and POPE/POPG lipids interact with the periplasmic tip of TM5 and with the cap loop in open-TM5 vs open GlpG could contribute to the different values of the estimated thinning of these membranes close to the protein (Figure 2A). An extended protein/lipid cluster that extends from the vicinity of K132 to D243 via K191 can be sampled (Figures 3D and 4A). To find out whether lipid/protein interactions at this site could impact the conformational dynamics of GlpG, I probed the dynamics of the K191A mutant in POPE (Sim1a, Figure 4B). Since K191A was prepared starting from the open GlpG structure, I use as a reference Sim1.

are mostly within distances too long for H bonding (Figures 2E and S6L). Dynamics at Lipid-Anchoring Sites on the Surface of GlpG Depend on Lipids and on the Protein Conformation. Charged and polar groups on the surface of GlpG anchor the protein into the membrane via H bonding.15 The long side chains of the positively charged groups Arg and Lys can H-bond to lipid phosphate groupsthese H bonds would then be independent of the lipid headgroup, but could depend on the length of the alkyl chain. A negatively charged carboxylate side chain could make direct H-bonds with a lipid headgroup that can easily donate H bonds, such as the ethanolamine group of POPE. K191, R217, and R227 (Figure 3A,D) H-bond to lipid phosphate groups in all Sims performed here (Figure 3C). Although lipid−protein H bonds are dynamic (Figures 3B, S7−S11), overall, these three protein groups remain within Hbond distance from lipid phosphate groups (Figure 3C). The frequent H bonding of K191 to lipids observed here is consistent with previous simulations on GlpG in POPE and POPC.15 The R227 lipid-anchoring site (Figure 3A,C) is known from experiments as a functionally important group whose substitution to Ala reduces proteolytic activity.56 At the periplasmic tip of TM5, D243 samples H-bond to POPE lipids when wild-type closed or open GlpG is embedded F


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Figure 4. Dynamics of the K191A mutant. (A, B) Molecular graphics illustrating lipid and protein interactions at the K/A191 site in wild-type Sim1 (A) vs K191A Sim1a (B), viewed from the periplasmic side. Heavy atoms of selected protein side chains are shown as van der Waals spheres using the following color coding: K132, K/A191. and D243yellow, S201 and H254green, other side chainswhite. For clarity, H atoms are now shown. POPE lipids shown have at least one atom within the 3.5 Å distance of K132-Nζ, K191-Nζ, or the carboxyl oxygen atoms of D243. (C) Molecular graphics showing the cap loop L5 in K191A (Sim1a, magenta structure) vs wild-type GlpG (Sim1, green structure). The lipid membrane is shown as a cutaway view based on coordinates from Sim1, with carbon atoms shown as van der Waals spheres colored white, oxygenred, nitrogenblue, and phosphorousbrown; Cα atoms of selected protein groups are shown as small spheres, and the catalytic groups are shown as bonds. For simplicity, H atoms are not shown. The arrows indicate the location of G246 and S248 at the end of the two Sims. Histograms of selected interatomic distances in Sim1a vs Sim1 are shown in Figure S13. (D) Histogram of the distance between I121-Cα and F127-Cα computed from the last 100 ns of Sim1 and Sim1a.

POPE headgroup near the periplasmic interface between helices 2 and 5 samples a relatively wide region (Figures 5E and S15A−C), and most of the time, the distance between S201 and the closest POPE lipid headgroup remains at ∼12 Å (Figures 2F and 5E). Close interactions between a lipid headgroup and the active site are not observed for K191A (Figure S15G). Interactions between PC lipids and GlpG depend on both the lipid alkyl chains and the protein conformation. In the case of open GlpG, POPC (Sim2) and DMPC (Sim5) lipid headgroups tend to remain away from the active site (Figures 2F, 6B, and S16A,C,D,F,G,I), and a DOPC lipid headgroup (Sim3) visits the active site only infrequently (Figures 2F, 6A,B, and S16B,E,H). Although POPC lipid headgroups remain away from the active site of open-TM5 GlpG in Sim10 (Figures 2F, 6D, and S14K,N,Q), a DOPC lipid headgroup visits the vicinity of the active site of open-TM5 toward the end of Sim11 (Figures 2F and S16L,O,R). Short distances between the active site of open-TM5 GlpG and a DMPC lipid headgroup are sampled frequently in Sim9, where a DMPC lipid can fit between TM2 and TM5 (Figures 2F, 6C,D and S16J,M,P). Transient visits of DOPC at the region of the GlpG active site in Sim3 and Sim11 (Figures 2F, 6A, and S16B,E,H,L,O,R) are likely facilitated by the DOPC lipids being kinked at the double bond of both alkyl chains, which could make it easier for a lipid to dock transiently at the active site (Figures 6A and

In K191A, the side chain of F127, which in Sim1 locates between the hydrophobic regions of the K132 and K191 side chains (Figure 4A), reorients to interact closely with L121 on loop L1 and with W196 on the loop connecting TM3 and TM4 (Figure 4A,B,D). Relative to wild-type open GlpG (Sim1), in K191A (Sim1b), the cap loop L5 has closer interactions with the inner region of the TM domain (Figures 4C and S13); differences in the conformational dynamics of the cap loop L5 (Figure S13A−C), and the altered lipid interactions close to the K/A191 site, may contribute to the slightly smaller thinning of the POPE bilayer observed in Sim1b compared to Sim1 (Figure 2A). Lipid Binding at the Active Site of GlpG. The observation from crystallography that a lipid headgroup can bind at the active site of open GlpG10 (Figure S14A) raises the important question as to whether, in a fluid membrane at room temperature, lipids could indeed visit the active site of the enzyme, thereby competing with substrate docking. On the time scales of the Sims performed here, transient visits to the vicinity of the active site largely depend on the composition of the lipid membrane and on the conformation of GlpG. When GlpG sits in POPE, transient visits of a lipid at the active site are observed for open-TM5 during the initial stages of Sim7 (Figure S15I); for the remaining of Sim7, and during Sims on closed (Sim6, Figure S15H) and open GlpG (Sim1, Figures 2F and 5E), POPE lipid headgroups tend to remain away from the active site (Figure 2F). In Sim1, the G


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Figure 5. Lipid visits at the lateral gate region of the D243A mutant in open GlpG, Sim1b. (A, B) Molecular graphics illustrating interactions between a POPE lipid and D243A open GlpG in Sim1b. In (A), carbon atoms of the POPE lipid that binds between TM2 and TM5 are colored green. In (B), phosphate atoms of POPE lipids close to the gate region are shown as van der Waals spheres colored yellow, orange, and white. (C) Close view of D243A illustrating interactions between a POPE lipid and G246 at the end of Sim1b. (D) Time series of the number of phosphate oxygen atoms within H-bond distance of the backbone amide group of G246. (E) Time series of the distance between S201-Oγ and the nearest POPE lipid phosphate atom in wild-type open GlpG (Sim1, green) vs D243A (Sim1b, magenta).

Figure 6. Interactions between GlpG and PC-type lipids. (A) Molecular graphics illustrating a coordinate snapshot in which two DOPC lipids visit the vicinity of the active site of open-state GlpG (Sim3). (B) Histograms of the shortest distance between S201-Oγ and the phosphate atom of POPC (Sim2) vs DOPC (Sim3) and DMPC (Sim5) lipids in Sims started from open GlpG. (C) Molecular graphics illustrating interactions between a DMPC lipid and the active-site region of open-TM5 GlpG from Sim9. (D) Histograms of the distance between S201-Oγ and the phosphate atom of the closest DMPC lipid in Sim9 vs POPC (Sim10) and DOPC (Sim11). Time series corresponding to the histograms in (B) and (D), and for open-TM5 GlpG in DOPC, are presented in Figure S16A−C,J,K. Additional molecular graphics illustrating interactions between protein groups, and the lipids closest to S201 are presented in Figure S13D−I,M−R.

S16E,H,O,R). The frequent visits of a DMPC headgroup at the active site of GlpG in Sim9 could be enabled by the lipid alkyl

chains being short, and by the protein having an open-TM5 conformation such that a DMPC lipid can fit between TM2 H


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Figure 7. Lipid interactions at the active-site region of open-TM5 GlpG in POPE/POPG. (A) View from the periplasmic side of GlpG interacting with selected POPE and POPG lipids. For clarity, alkyl chains of the lipids and H atoms are not shown. The small ice blue spheres indicate water molecules within 3.5 Å of the phosphate oxygen atoms O13 and O14, and within 3.5 Å of the nitrogen atom of POPE and headgroup glycerol oxygens of POPG. (B) Surface representation of open-TM5 GlpG; selected protein groups are shown as surface colored yellow (F153, W236) or green (S201), or as van der Waals spheres colored blue (K132, K191, and D243). Two POPE lipids that lean over the active site are shown as van der Waals spheres colored magenta. H atoms are not shown. (C) Close view of interactions at the D243 and S248 sites. (D) Local environment of the D243. The histograms show the number of backbone amide nitrogen atoms within 3.5 Å of the two D243 carboxylate oxygen atoms in Sim1 (green curve), Sim7 (magenta), Sim8 (yellow), and Sim9 (brown curve).

and TM5 (Figures 6C,D and S16F,I). Since a DMPC lipid may visit frequently the vicinity of the active site of open-TM5 GlpG (Sim9, Figure 6C,D), but not that of open GlpG (Figures 6B and S16C), sufficient space between TM2 and TM5 might be required for a DMPC lipid to dock here. In a POPE/POPG membrane, lipids remain outside of the active site of both the open and open-TM5 GlpG (Sim4, Sim8, Figures 2F, 7A−C, and S12F−J). Intriguing interactions between lipids and the cap loop L5 are observed for openTM5 GlpG (Sim8, Figures 7A−C and S12F): The carboxylate group of D243 engages in H bonds with backbone nitrogen atoms within the loop (Figure 7C,D), and two POPE lipid headgroups can lean above the loop (Figure 7B,C). Lipid interactions at the active site of GlpG are thus governed by a complex interplay between protein conformation, dynamics of the lipid alkyl chains, and details of how specific protein groups such as D243 interact with lipids and nearby protein groups. To further evaluate how interactions between D243 and lipid headgroups could influence dynamics of lipid−protein interactions at the active site of GlpG, I studied the dynamics of the D243A mutant in POPE. I performed three independent Sims on D243A starting from the closed state (Sim6b), open GlpG (Sim1b), and open-TM5 GlpG (Sim7b, Table 1). Similarly to the case of the wild-type GlpG (Sim6, Figure S15H), POPE lipid headgroups remain away from the active site of closed-state D243A (Sim6b, Figures 2F and S15J,L). Loop L5 samples a conformation whereby it caps the active site and it H-bonds with other protein groups (Figure S17D− F); the closer interactions of loop L5 with the inner region of the TM domain could help explain why the number of waters close to the catalytic S201 is smaller in Sim6b than in Sim6 (Figures 2F and S5I,H). In open-GlpG D243A, a POPE lipid headgroup visits transiently the vicinity of the active site during the trajectory segment ranging from ∼95 to ∼150 ns of Sim1b (Figures 5

and S15D−F). Likewise, a POPE lipid headgroup visits transiently the vicinity of the active site of open-TM5 D243A toward the end of Sim7b (Figure S15K,M). The analyses here indicate that, although the carboxylate side chain of D243 samples transient H bonds to the ethanolamine group of POPE (Figures 3C, S7C,D, and S8C,F), it can also repel lipid phosphate groups, hindering visits by a POPE lipid at the active site of wild-type openGlpG. This suggests that D243 might help guard access of lipids to the active site. Extended H-Bond Network Extends Across GlpG and Connects to Lipids. The catalytic S201 and H254 are hosted by TM4 and TM6, both of which contribute to interhelical Hbond clusters with potential role in controlling the conformational dynamics of GlpG.15,16 To assess whether lipids and the conformation of GlpG impact internal H bonds of GlpG, I monitored the H bonds of selected protein groups throughout the 15 Sims performed here (Figure 8 and Table S1). GlpG is spanned by a network of H bonds that reaches from the periplasmic to the cytoplasmic side of GlpG (Figure 8A− C); this network has as its end points groups that neighbor membrane-anchoring groups, and as intermediate points groups located near the catalytic site (Figure 8C). Overall, the dynamics at internal interhelical H bonds is largely similar in the Sims performed. In all Sims, the hydroxyl group of TM3−T178 H-bonds to L174, a group whose bulky hydrophobic side chain is close to TM2−W158 (Figure 8A,B), and adjacent to K173a site where GlpG may anchor to the membrane (Figures 3C and 8A−D). In most Sims, the hydroxyl group of S181 H-bonds to the backbone carbonyl group of T178 (Table S1). Whether interhelical H bonding of T178 and S181 has a functional role is unclear. T181 is often replaced by Tyr, and S181 is present as Ser/Thr in some of the rhomboid sequences.57 Ala substitution of T181 has a modest effect on catalytic activity, whereas S281A has the same activity as wild-type GlpG.58 I


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Figure 8. H bonds mediate intraprotein and protein/lipid coupling. (A) Molecular graphics of the H-bond network. Selected protein groups are shown as bonds, and S201 and H254 as green surface. The H-bond network shown here relies in part on previous analyses of GlpG’s H bonds from crystal structures17 and simulations.15 (B) Schematic representation of the H-bond network. (C) Cutaway view of GlpG in POPE (Sim1) illustrating the location of the H-bond network relative to lipid-anchoring sites. Protein groups are shown as black bonds; the pink spheres indicate Cα atoms of protein groups that can H-bond to lipids (see Figure 3A). (D, E) Time series of the distance between selected protein groups. Data analyses for the H-bond network are summarized in Table S1.

this loop further hosts D243 and S248, which sample H bonding to lipid headgroups (Figures 3C, 8A,B,E, S7, and S11). H bonding of Q189 to M249 and A253 is sampled in Sims on wild-type closed and open GlpG in POPE, whereas for open-TM5 GlpG in POPE, these distances remain too long for H bonding (Table S1). At the cytoplasmic side, the H-bond network extends to TM2 via H bonding between TM1-T97 and TM2-E166 (Figure 8A,B,D and Table S1). H bonding between T97 and E166 was observed in previous computations on GlpG,15 and is sampled in all wild-type Sims performed here (Figure 8D and Table S1). Interhelical H bonding of E166 could explain why its substitution by Ala reduces catalytic activity and the unfolding temperature of GlpG.58 An important functional role of interhelical H bonding at the cytoplasmic side of GlpG is further supported by the homology model of the C-terminal rhomboid domain of Parl, the mitochondrial rhomboid protease,59 in which T168 (corresponding to GlpG T97) is within H-bond distance from TM3−Q242.59

S185 is located close to the catalytic S201 (Figure 8A), and its mutation to Val is known to inhibit catalysis.19 Simulations on open GlpG suggest that dynamics at the S185 site depends on lipids: The H bond between S185 and the backbone carbonyl of S181 is largely stable at 2.7 ± 0.1 Å when GlpG is embedded in POPE (Sim1) or POPE/POPG (Sim4), but not when in POPC (Sim2), DOPC (Sim3), or DMPC (Sim5) here, the average distance between S185-Oγ and the S181 backbone carbonyl is ca. 4.3−4.4 Å (Table S1), i.e., too long for direct H bonding. In addition to its H-bonding with S181, S185 can sample H-bonding distances to the backbone carbonyl of A182, which further H-bonds to H141 of loop L1 (Figure 8A,B and Table S1). In all Sims, H141 samples Hbond distances to the backbone carbonyl of Y138; this H-bond pair is located relatively close to two lipid-anchoring sites, S147 and K132, respectively (Figures 3C, 8A−C, and S10), and is likely important for the structural stability and functionality of GlpG. Relative to wild-type GlpG, Y138F and H141F have reduced activity;13 H141A has reduced activity and unfolding temperature.58 One helical turn to the periplasmic side of S185, Q189 is a key functional group whose mutation inhibits proteolysis.19 Depending on the conformation of GlpG, Q189 can sample dynamic H bonds with M249 and A253 on the cap loop L5;

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CONCLUSIONS I studied the conformational dynamics of the E. coli rhomboid protease GlpG starting from three different protein crystal J


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The Journal of Physical Chemistry B structures and using five different lipid membrane environments. The ensemble of the 15 simulations performed, with a total of ∼2.9 μs sampling time, inform on mechanisms by which lipid interactions influence conformational dynamics of GlpG. How the lipid membrane adjusts itself to the presence of GlpG is important to understand because thinning of the lipid membrane might impact the dynamics of an incoming TM substrate.15 Depending on the composition of the lipid membrane and on the conformation of GlpG, the estimated membrane thinning ranges from 0 to ∼4 Å (Figure 2A). In POPE, such thinning of the membrane close to GlpG is observed for wild-type closed and open GlpG, and close to the D243A mutant of open-TM5 GlpG; close to wild-type openTM5 GlpG, the estimated membrane thinning is reduced to ∼1 Å (Figure 2A). The mixed POPE/POPG membrane thins by ∼4 Å in the vicinity of open-TM GlpG and by ∼1 Å near open GlpG (Figure 2A); likewise, thinning by ca. 1.8−3.8 Å is observed for PC membranes in the vicinity of open-TM5 GlpG, whereas the thickness of PC membranes close to open GlpG remains largely the same as far away from the protein (Figure 2A). Differences in how lipid bilayers interact with GlpG in, e.g., the closed- vs open-TM5 conformations likely originate from details of how specific protein groups interact with lipids. Particularly important appear to be interactions at the cap loop L5 and at the active site of GlpG (Figures 2F, 4C, and 5−7). The result here that lipid membrane thinning close to GlpG depends on the conformation of GlpG is compatible with previous observations on the SERCA calcium pump.60 Water molecules are present close to S201 in all Sims reported here (Figures 2C and S5). The number of waters at the active site can, however, depend on the protein conformation and on the lipid membrane. The smallest number of active-site waters, ∼5, is observed in the D243A mutant of closed GlpG (Sim6b, Figure 2C), in which loop L5 caps the active site and H-bonds with other protein groups (Figure S17D−F); by contrast, open-TM5 D243A has ∼11 waters close to S201 (Sim7b, Figure 2C). Open GlpG has about three more active-site waters in POPC (Sim2) than in POPE (Sim1, Figure 2C), whereas open-TM5 GlpG has about two fewer waters in POPC (Sim10, Figure 2C) than in POPE (Sim7, Figure 2C). This suggests that the structural dynamics of GlpG along its reaction coordinate are likely accompanied by changes in the number of active-site waters. Direct interactions between protein amino acid residues and lipid headgroups can anchor GlpG into the membrane, thus helping control how GlpG orients relative to the lipid membrane.15 Encounters between lipids and protein surface groups are dynamic, being characterized by fluctuations of the number of lipid atoms within H-bond distance of a protein group (Figures 3B and S7−S11). At the periplasmic side of the protein, a cluster of protein side chains and lipid headgroups extending from K132 of loop L1 to D243 at the periplasmic tip of TM5 and via TM3−K191 can be sampled (Figures 3D and 4A). Sampling of such a lipid−protein cluster during dynamics of GlpG in a hydrated lipid membrane is compatible with the crystal structure of GlpG solved in a lipid/detergent mixture, in which fragments of DMPC lipids are located in the vicinity of K132 and K19161 (Figure S14B). K132 is close in sequence to the conserved WR motif57W136 and R137, whose individual alanine substitution reduces the catalytic activity of GlpG in cell

experiments.21 Since the WR motif appears essential for catalytic activity only when GlpG is in the membrane,62 lipid−protein interactions close to the WR motif appear important for the functioning of GlpG. Intraprotein and lipid−protein H bonds in the vicinity of the catalytic site are dynamic. The backbone of A253, an amino acid residue adjacent in the sequence to H254, samples dynamic H-bonding with Q189 (Figure 8A,B and Table S1). Close to S201, H bonds between S185 and A182, and between H141 and A182, are relatively weak, with average distances of ca. 3−3.5 Å (Table S1). Dynamic H bonding at loop L5 is compatible with the notion that this is a flexible structural element of GlpG;12,28 such H bonding might facilitate local structural plasticity when the substrate binds to GlpG. H bonds with distinct substates (e.g., the H bonds of Q189, Figure 8E and Table S1), might experience a shift in their preferred state upon substrate binding.16 S185, Q189, and A253 were suggested to contribute to a substrate-binding site,63 and M249, whose backbone carbonyl can to Q189, makes van der Waals contacts with the substrate in a crystal structure of inhibitor-bound GlpG.64 The A253T mutant, in which H bonding of the dynamic Q289 cluster is likely altered (Figure 8B), has reduced catalytic activity,56 and the backbone carbonyl of S248 H-bonds with the substrate in the crystal structure of GlpG from ref 11. The observation here of dynamic H bonds in the region of substrate docking and cleavage is compatible with the notion that weak H bonds might be important for protein function.65 The crystal structure of open GlpG10 captured the protein in a conformation in which a lipid headgroup H-bonds at the catalytic site (Figure S14A). On the time scale of the Sims reported here, transient visits of a lipid at the region of the catalytic site of GlpG depend on the composition of the lipid membrane and on the conformation of GlpG. Transient lipid visits at the vicinity of the active site were sampled for wildtype closed- and open-TM5 GlpG in DOPC (Figures 2F, 6A,B, and S16L,O,R), and for D243A closed- and open-TM5 GlpG in POPE (Figures 2F, 5, and S15K,M); more frequent visits of a lipid headgroup at the active site were observed for openTM5 GlpG in DMPC (Figures 2F, 6A,C, and S16J). In openTM5 GlpG embedded in POPE/POPG, two lipids interact closely with the cap loop L5 (Figure 7). Such binding of a lipid headgroup at the region of the access to the active site could interfere with substrate binding. In vitro, the D243A mutation has mild effects on the catalytic activity of GlpG,13 and D243 is not conserved among rhomboid proteases.57 The lipid-dependent dynamics at the D243 site (Figure 3C) suggest that the lipid environment could influence how substrates bind to GlpG. Interactions between a particular rhomboid protease and the membrane might rely on specific amino acid residues whose lipid binding is optimal for the physiological lipid membrane environment of that rhomboid. An example here is offered by the crystal structure of Haemophilus influenzae GlpG.66 Instead of an Asp side chain on the cap loop L5 (D243), this rhomboid has a Glu, E163, and K132 and K191 of loop L1 are missing. Nevertheless, the crystal structure suggests that, similar to E. coli GlpG, in H. influenzae GlpG,66 loop L1 could still bridge to TM3 via a lipid molecule (Figure S14C). Another rhomboid, the mammalian RHBDL4, is thought to bind cholesterol via two Tyr groups.67 In the future, new crystal structures of rhomboids from different organisms, and complementary MD simulations, might shed light on the relationship between K



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ACKNOWLEDGMENTS This work was supported in part by funding from the Excellence Initiative of the German Federal and State Governments provided via the Freie Universität Berlin, by an allocation of computing time from the HLRN, the NorthGerman Supercomputing Alliance, and by local computing resources of the Freie Universität Berlin, Department of Physics. The author thanks Dr Sin Urban for valuable discussions, and Ms. Konstantina Karathanou for technical support.

organism-specific lipid interactions and catalytic activities of the proteases. In spite of the systematic approach taken here, whereby the dynamics of GlpG were studied with different protein structures and different membrane environments, information on the conformational dynamics of GlpG could be limited by the time scale of the simulations. A full description of the molecular movie of structural rearrangements of GlpG along its reaction coordinate will need additional, longer computations, and validation by experimental data. The work presented here suggests that it will be important to use the same lipid membrane composition in experiments and simulations. The reaction coordinate of GlpG can be thought of as three main events: docking of the substrate to the enzyme active site, cleavage of the substrate, and release of substrate fragments from the active site. A priori, lipids could influence each of these regions of the reaction coordinate. The computations performed here highlight the complex interplay between lipid and protein motions that impact how GlpG anchors in the membrane, and between the protein and lipid dynamics near the substrate-binding region. Such sensitivity of the conformational dynamics of GlpG to its surrounding lipid membrane could influence the early part of the reaction coordinate of GlpG.

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REFERENCES

(1) Kopan, R.; Ilagan, M. X. γ-Secretase: Proteasome of the Membrane? Nat. Rev. Mol. Cell Biol. 2004, 5, 499−504. (2) Paschkowsky, S.; Hamzé, M.; Oestereich, F.; Munter, L. M. Alternative Processing of the Amyloid Precursor Protein Family by Rhomboid Protease RHBDL4. J. Biol. Chem. 2016, 291, 21903− 21912. (3) Baker, R. P.; Wijetilaka, R.; Urban, S. Two Plasmodium Rhomboid Proteases Preferentially Cleave Different Adhesins Implicated in All Invasive Stages of Malaria. PLoS Pathog. 2006, 2, e113. (4) McQuibban, G. A.; Lee, J. R.; Zheng, L.; Juusola, M.; Freeman, M. Normal Mitochondrial Dynamics Requires Rhomboid-7 and Affects Drosphila Lifespan and Neuronal Function. Curr. Biol. 2006, 16, 982−989. (5) Urban, S.; Wolfe, M. S. Reconstitution of Intramembrane Proteolysis in Vitro Reveals That Pure Rhomboid Is Sufficient for Catalysis and Specificity. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1883−1888. (6) Narayanan, S.; Sato, T.; Wolfe, M. S. A C-Terminal Region of Signal Peptide Peptidase Defines a Functional Domain for Intramembrane Aspartic Protease Catalysis. J. Biol. Chem. 2007, 282, 20172−20179. (7) Osenkowski, P.; Ye, W.; Wang, R.; Wolfe, M. S.; Selkoe, D. J. Direct and Potent Regulation of Γ-Secretase by Its Lipid Microenvironment. J. Biol. Chem. 2008, 283, 22529−22540. (8) Moin, S. M.; Urban, S. Membrane Immersion Allows Rhomboid Proteases to Achieve Specificity by Reading Transmembrane Segment Dynamics. eLife 2012, 1, No. e00173. (9) Wu, Z.; Yan, N.; Feng, L.; Oberstein, A.; Yan, H.; Baker, R. P.; Gu, L.; Jeffrey, P. D.; Urban, S.; Shi, Y. Structural Analysis of a Rhomboid Family Intramembrane Protease Reveals a Gating Mechanism for Substrate Entry. Nat. Struct. Mol. Biol. 2006, 13, 1084−1091. (10) Ben-Shem, A.; Fass, D.; Bibi, E. Structural Basis for Intramembrane Proteolysis by Rhomboid Serine Proteases. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 462−466. (11) Cho, S.; Dickey, S. W.; Urban, S. Crystal Structures and Inhibition Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug Design Implications for Rhomboid Proteolysis. Mol. Cell 2016, 61, 329−340. (12) Wang, Y.; Ha, Y. Open-Cap Conformation of Intramembrane Protease GlpG. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2098−2102. (13) Baker, R. P.; Young, K.; Feng, L.; Shi, Y.; Urban, S. Enzymatic Analysis of a Rhomboid Intramembrane Protease Implicates Transmembrane Helix 5 as the Lateral Substrate Gate. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8257−8262. (14) Brooks, C. L.; Lazareno-Saez, C.; Lamoreux, J. S.; Mak, M. W.; Lemieux, M. J. Insights into Substrate Gating in H. Influenzae Rhomboid. J. Mol. Biol. 2011, 407, 687−697. (15) Bondar, A.-N.; del Val, C.; White, S. H. Rhomboid Protease Dynamics and Lipid Interactions. Structure 2009, 17, 395−405. (16) Bondar, A.-N.; White, S. H. Hydrogen Bond Dynamics in Membrane Protein Function. Biochim. Biophys. Acta, Biomembr. 2012, 1818, 942−950.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b11291. Average values for selected H-bond distances; Cα rmsd profiles decomposed for the transmembrane segments vs loops and termini; Cα rmsd profiles for loops L1, L4, and L5; molecular graphics of overlaps between structure snapshots from each Sim; molecular graphics illustrating crystal structure waters; time series of the number of water molecules close to S201; time series of the distance between S201 and H254; time series of the number of lipid atoms within H-bond distance of selected protein groups in Sim1−Sim5, Sim1a, Sim1b, and in Sim6−Sim11, Sim6b, Sim7b; number of lipid N atoms within H-bond distance of D116; number of lipid phosphate oxygen atoms within H-bond distance of S147 and S248; illustration of lipid interactions in POPE and POPE/POPG membranes; dynamics of the cap loop L5 in K191A; molecular graphics of lipid/protein interactions in crystal structures; analyses of lipid interactions in Sims on wild type and D243A GlpG embedded in POPE; analyses of lipid interactions in Sims with GlpG embedded in POPC, DOPC, or DMPC; analyses of the cap loop in D243A (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49 30 838 53583. ORCID

Ana-Nicoleta Bondar: 0000-0003-2636-9773 Notes

The author declares no competing financial interest. L


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