Reactions at Biomembrane Interfaces - Chemical Reviews (ACS

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Reactions at Biomembrane Interfaces Ana-Nicoleta Bondar*,† and M. Joanne Lemieux*,‡ †

Freie Universität Berlin, Department of Physics, Theoretical Molecular Biophysics Group, Arnimallee 14, D-14195 Berlin, Germany University of Alberta, Department of Biochemistry, Membrane Protein Disease Research Group, Edmonton, Alberta T6G 2H7, Canada

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‡

ABSTRACT: Membranes surrounding the biological cell and its internal compartments host proteins that catalyze chemical reactions essential for the functioning of the cell. Rather than being a passive structural matrix that holds membrane-embedded proteins in place, the membrane can largely shape the conformational energy landscape of membrane proteins and impact the energetics of their chemical reaction. Here, we highlight the challenges in understanding how lipids impact the conformational energy landscape of macromolecular membrane complexes whose functioning involves chemical reactions including proton transfer. We review here advances in our understanding of how chemical reactions occur at membrane interfaces gleaned with both theoretical and experimental advances using simple protein systems as guides. Our perspective is that of bridging experiments with theory to understand general physicochemical principles of membrane reactions, with a long term goal of furthering our understanding of the role of the lipids on the functioning of complex macromolecular assemblies at the membrane interface.

CONTENTS 1. Introduction 2. Membrane Reactions: From Basic Science to Medical Applications 2.1. Intramembrane Proteolysis: The γ-Secretase Complex and the GlpG Rhomboid Protease 2.2. Bacterial Sec Protein Secretion: Role of Lipids and Proton Binding 2.3. The Programmed Death-1 Pathway and Protein−Protein Interactions at Complex Membrane Interfaces 3. Reducing Complexity to Understand Chemical Reactions at Biomembrane Interfaces 3.1. Structural Biology 3.2. Kinetics 3.3. Activation Energy, Kinetic Isotope Effects, and Transition-State Inhibitors 4. Role of Lipids in the Reaction Coordinates of Membrane Proteins 4.1. Lipid Interactions of Bacteriorhodopsin 4.2. Lipid Interactions of Rhomboid Proteases 5. Using Computer Simulations to Understand Reaction Mechanisms of Membrane Proteins 5.1. A Molecular Picture of the Lipid Interactions of Intramembrane Proteases 5.2. Combined Quantum Mechanical−Molecular Mechanical Computations of Reaction Mechanisms: GlpG and the Bacteriorhodopsin Proton Pump 6. Structural Motifs of H Bonding in Membrane Proteins 6.1. Interhelical Hydroxyl-Carboxylate H-Bond Motifs in Membrane Proteins © XXXX American Chemical Society

6.2. H-Bond Dynamics at Negatively Charged Protein Interfaces 7. Future Directions Author Information Corresponding Authors ORCID Author Contributions Notes Biographies Acknowledgments References

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M M O O O O O O O O

1. INTRODUCTION Biomembranes are complex entities whose main constituents are lipid molecules that assemble in a bilayer and proteins embedded in the membrane (Figure 1) or bound at the membrane interface. There are numerous types of lipid molecules, with specific lipids being found in a membrane depending on the organism, cell type, and cellular compartment.1,2 The composition and distribution of lipid molecules in the two lipid leaflets are tightly regulated by the cell machinery, which gives a membrane its physicochemical properties.3 At a macroscopic level, altered composition or distribution of lipids has been associated with human disease states and exploited as biomarker in medical imaging. Knowledge of how specific features of lipid membranes impact

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Figure 1. Lipid membrane interface. (A) Side view of a hydrated lipid membrane with atoms shown as van der Waals spheres. Lipid headgroups are colored green; glycerol, pale blue; carboxyl groups, pink; alkyl chains, purple; for clarity, only heavy atoms are shown. Two lipid molecules are shown in atom-color representation using the following color code: carbon, cyan; hydrogen (H), white; nitrogen, blue, phosphate, brown; oxygen, red. Note the different conformations sampled by the lipid alkyl chains, which highlight the fluidity of the lipid bilayer. (B) Close view of the lipid headgroup interface. Heavy atoms of selected lipid headgroups, carbonyl, and carboxyl groups are shown in atom colors; other lipid heavy atoms are shown in transparent ice-blue spheres. Water molecules shown are within 4.5 Å of the oxygen atoms of the selected lipids. The images are based on a MD simulation of a lipid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-snglycero-3-phosphatidylglycerol (POPG) in a ratio of 5:1 from ref 6 Unless specified otherwise, molecular graphics were prepared using Visual Molecular Dynamics, VMD.7

the membrane interface is complicated by the need to account for the chemical complexity of the lipid bilayer (Figure 1), as lipid interactions can influence membrane reactions (Table 2). In what follows, we discuss three prominent examples of large protein complexes with important biomedical applications, whose functioning involves chemical reactions, protein conformational dynamics, and lipid interactions. Our focus is on systems whose understanding requires consideration of both proton binding and lipid interactions to gain full insight into the role of the membrane interface on the reactions occurring with these protein complexes. Deciphering how large protein complexes work in complex lipid environments is facilitated by existing knowledge on model systems and structural motifs, and we discuss examples of observations from experiments and computations that provide a framework for assessing reaction coordinates of membrane proteins.

the functioning of membrane-associated enzymes is essential because it has the potential to assist with drug design. Understanding the reaction mechanism of an enzyme requires access to the conformations sampled by the enzyme along its pathway from the reactant to the product state and to the free energy profile of this pathway. The influence of lipids on the reaction mechanism of a membrane-embedded enzyme requires an understanding of how the enzyme couples to lipids. While this concept was known for some time,4,5 we posit that such molecular descriptions of membrane reactions can only be accomplished by bridging experiments with molecular modeling. We consider here membrane reactions from a broad perspective that includes enzymatic catalysis mediated by membrane-embedded proteins, proton binding at protein− membrane interfaces, and protein−protein interactions within the complex environment of the lipid membrane. A unifying question is how lipids, given their remarkable diversity,2 shape reactions at the biomembrane. We build upon knowledge about membrane reactions to discuss a general framework of the role of lipids in membrane protein mechanisms and implications for biomedical applications. We focus on the usefulness of studying relatively simple proteins and bilayers systems to understand general physicochemical principles of membrane reactions and using this knowledge to approach ever more complex biosystems.

2.1. Intramembrane Proteolysis: The γ-Secretase Complex and the GlpG Rhomboid Protease

Membrane-embedded proteases cleave other transmembrane (TM) substrates, liberating molecules that participate in cell signaling or other essential cellular processes.8 Classes of intramembrane proteases that have been identified include serine, aspartyl, metallo, and glytamyl proteases.8 A few representative structures that have been solved with protein crystallography or cryoelectron microscopy (cryo-EM) for serine,9−12 metallo-,13−15 aspartyl-,16 and glutamyl11 forms reveal that, despite the distinct environments in which they work, the active sites of intramembrane proteases have strong similarities with those of soluble proteases. Well characterized forms include γ-secretase, which is an aspartyl protease, and rhomboid proteases, which are serine proteases. The γ-secretase complex includes four membrane-bound proteins: the protease subunit presenilin, nicastrin, the anterior pharynx-defective 1 protein APH-1, and the presenilin enhancer 2, PEN2 (Figure 2A).17 Presenilin cleaves TM substrates such as amyloid precursor protein,17 whose amyloid-

2. MEMBRANE REACTIONS: FROM BASIC SCIENCE TO MEDICAL APPLICATIONS Protein functions at membrane interfaces comprise a wide range of reactions that occur with breaking and forming of chemical bonds and interactions in which at least one reaction partner is associated with the cell membrane. Protein complexes, including complexes between proteins and substrates, can play essential roles in the cell and perturbed functionality can associate with human disease (Table 1). Understanding how proteins and protein complexes work at B

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C

biological function of the substrate

ATP is used to power chemical reactions in the cell adds an acyl group to diacylglycerol to generate triacylglycerol (TAG) TAG is a lipid important in metabolism, and seed oil production mediate cell signaling in response to a variety of external stimuli; example, opioid receptors regulation of pain sensation (in the case of the opioid receptor ligands) TM proteins that provide passage for ions transferred down their electrochemical gradient, as important in action potential propagation a result of gating (ligand, electrical, etc.) TM proteins that translocate ions across cell membranes, using ATP hydrolysis to transport ion homeostasis (proton, Na, K, Ca2+)223 ions against a gradient Na+−Ca2+ exchangers couple movement of Na+ down the electrochemical gradient with the Ion homeostasis uphill translocation of Ca2+ DGAT2 building blocks of the lipid membrane ATP-powered, directional movement of lipids from the outer to the inner membrane leaflet building blocks of the lipid membrane (flippase) or in the opposite direction (floppase). nonspecific, nondirectional movement of lipids between the bilayer leaflets228 building blocks of the lipid membrane presenilin, the catalytic subunit in γ-secretase, cleaves amyloid precursor protein (APP), notch is involved in cell signaling17 Notch, and numerous other substrates17 the receptor, found on the surface of an immune system cell, binds the ligand found on the down-regulates the action of the immune tumor cell system cell231 233 mitochondrial rhomboid protease; cleaves PINK1 kinase whose action has been implicated in Parkinson’s disease234 Cleave TM adhesin proteins in Plasmodium falciparium236 establishing infection by the parasite236

synthesizes ATP from ADP and phosphate

biological function of the enzyme−receptor

a

Disease associated with mutations in the enzyme or receptor.

mediates incorporation of TM helical segments into the membrane and secretion of soluble newly synthesized proteins with various roles in proteins the cell ZMPSTE24 intramembrane zinc modifies carboxy termini of residues nuclear scaffold protein lamin A metalloprotease

programmed-death-1 receptor (PD-1) presenilin associate rhomboid like (PARL) Plasmodium rhomboid proteases 1, 4 SecY-Sec61 protein translocon

lipid scramblases γ-secretase (presenilin)

lipid transfer proteins lipid flippases, floppases.

ion exchangers

ion pumps

ion channels

diacylglycerol acyltransferase (DGAT) GPCRs

ATP synthase

enzyme−receptor

Table 1. Examples of Membrane-Bound Enzymes and Receptors and Their Biological Function

lipodystrophies premature aging239

kidney disease238

parasitic infection237

Parkinson’s disease235

cancer232

blood disorders229 familial Alzheimer’s disease230

metabolic syndroms, lipodystrophies226 Tangier’s disease (flopase) ABC1227

neuronal and cardiac disorders225

neuronal and cardiac disorders224

immune dysfunction, hypothyroidism, hypogonadism, infertility221 neuronal and cardiac disorders222

obesity, congenital bowel disorders220

neuromuscular disorders219

diseasea

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Table 2. Examples of Membrane Proteins Whose Functioning Is Influenced by the Composition of the Surrounding Lipid Membrane protein ATP synthase bacteriorhodopsin β-secretase cytochrome-c oxidase DGAT1 γ-secretase GlpG rhomboid protease mechanosensitive ion channel TRAAK lipid phosphatase PgP SecA-SecYEG protein translocase signal peptide peptidase visual rhodopsin, GPCR

physiologic lipid environment mitochondrial membrane (eukaryotes) or plasma membrane (bacteria) purple membrane of Halobacterium salinarium endosomes, trans-Golgi of brain cells241 mitochondrial membrane(eukaryotes) or plasma membrane (bacteria) endoplasmic reticulum, predominant in intestinal cells (eukaryotes) and plant seeds presenilins: the endoplasmic reticulum of neuronal cells245 E. coli inner membrane

impact of altered membrane composition activity increases when DOPG is added to DOPC liposomes240 removal of squalene alters photocycle kinetics132,133 PS, PA, and cerebroside lipids stimulate catalytic activity242 activity is sensitive to the alkyl chain length of the lipids243 PC with different chain lengths and saturation modified enzymatic activity244

bacterial membrane

cleavage of APP depends on the composition of the lipid membrane; relative to PC-only membranes, activity is higher when PE, PS, or PA are added, but not when PI is added27 GlpG cleaves a model Spitz substrate when in PE lipids, but not in PG, PC, or E. coli lipids extract246 arachidonic acid promotes activity249; reconstitution in PC lipids with branched alkyl chains promotes ion channel activity250 PE lipids inhibits activity251

bacterial plasma membrane

stable assembly of the translocase requires negatively charged lipids252

endoplasmic reticulum253

catalytic activity is observed in PC, PC/PE, or E. coli lipid extract, but not in heart lipid extract254 changes in lipid composition alter conformational dynamics of the receptor’s activation (e.g., refs 256−258)

specific regions of the brain247,248

disk membranes of rod cells255

Figure 2. Architecture and lipid interactions of the γ-secretase complex. Both molecular graphics are based on the cryo-EM structure PDB 6IYC solved to a resolution of 2.6 Å.25 (A) The γ-secretase complex bound a fragment of the amyloid precursor protein.25 Side chains of the catalytic carboxylates are shown as van der Waals spheres colored red, phosphatidylcholine lipids, navy blue, and cholesterol molecules, cyan. (B) Close view of protein groups that interact with lipids or cholesterol in the cryo-EM structure. Protein groups shown have at least one atom within 3.5 Å from the lipid or cholesterol molecules.

β protein fragments can form plaques that are a hallmark of Alzheimer’s disease, and the Notch1−4 receptor,17−19 a singlespanning TM protein involved in cell signaling and cancer.20 Notably, γ-secretase is reported to have over 80 substrates,21 and the reaction coordinates for the cleavage of these substrates are largely unclear. Cryo-EM structures of the human γ-secretase (Figure 2A) suggest that the catalytic D257 and D385 of the presenilin subunit are exposed to the membrane22 and, indeed, during computer simulations of presenilin, a phosphatidylethanolamine (PE) lipid was observed to visit the active site and H bond to the catalytic carboxylate groups.23 The two recent high-resolution cryo-EM structures of γ-secretase in complex with Notch24 or amyloid peptide substrate models25 indicate coordinates for PC lipids and cholesterol molecules (Figure 2B). That PC lipids bind to γ-secretase (Figure 2B) agrees with earlier biochemistry experiments indicating that PC is compatible with substrate cleavage by γ-secretase.26,27 The impact of cholesterol on catalytic cleavage by γ-secretase

depends on the relative concentration and details of the lipid composition of the membrane.27 When bound to γ-secretase, the substrate remains partially exposed to the lipid membrane environment,25 where it could interact with both lipid alkyl chains and headgroups. We suggest that such lipid interactions could help position the substrate at the active site of γ-secretase. Lipid exposure of the substrate during catalytic cleavage could further assist release of cleaved substrate fragments without major conformational rearrangements of γ-secretase or of the surrounding lipid membrane, but whether and how surrounding lipids regulate substrate accessibility to the active site of γ-secretase are essential questions yet to be addressed. Another important feature of γ-secretase that is poorly understood is the sensitivity of its cleavage activity to pH. Earlier biochemistry experiments indicated that, when γsecretase cleaves amyloid precursor protein, the ratio between two cleavage products of different length depends on pH, the shorter cleavage product Aβ40 being more sensitive to pH D

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Figure 3. Proteins of the bacterial Sec protein secretion pathway. (A) Molecular graphics of the SecYE-SecA complex bound to a translocating peptide substrate. The image is based in part on ref 57 and uses the coordinates of the crystal structure of Geobacilus thermodenitrificans SecYE, bound to B. subtilis SecA, which was fused with a secretory protein segment (PDB 5EUL, from ref 58). In the crystal structure, SecY-E124, is within H-bond distance from T764 of the translocating substrate.57 (B) Molecular graphics of T. thermophilus SecDF based on the crystal structure PDB 3AQP from ref 59. D340 and D637, thought to be important for function,59 are shown as van der Waals spheres with carbon atoms colored cyan and oxygen atoms, red. The Cα atoms of other Asp and Glu groups and of Arg and Lys groups are shown as small spheres colored red and blue, respectively. For clarity, the segment of the protein ranging from P656 to L689 is shown as a transparent ribbon.

than Aβ4226 (the number in the cleavage product name indicates its number of amino acid residues). More recent data indicated that activity is optimal at pH 6.5, and it decreases at lower or higher pH values.28 The molecular origin of the pH sensitivity of the γ-secretase activity, and whether the pH sensitivity is also substrate specific, are unclear. In the case of the amyloid precursor protein, the conformation of its ectodomain E1 was found to be more open at neutral than at acidic pH, being thought that protein conformation couples to the protonation state at a histidine site and a hydrogen-bonded carboxylate pair.29 pH-dependent changes in the conformational dynamics and/or intermolecular interactions of the enzyme and substrate could impact the conformational dynamics of the γ-secretase−substrate complex and the reaction coordinate of substrate cleavage. A much simpler model system for studying intramembrane proteolysis is GlpG, the intramembrane rhomboid protease of Escherichia coli. GlpG, belongs to the rhomboid protease class, which includes proteases ubiquitously expressed in all kingdoms of life, the E. coli GlpG and Haemophilus influenzae hiGlpG being the best studied bacterial isoforms.30 HiGlpG, like GlpG, has a 6-TM catalytic core but is missing a 100 amino-acid residues cytoplasmic domain, while other topological forms include 7-TM.31 Several crystal structures have been solved for the 6-TM catalytic core of GlpG,32−34 which include structures in bicelles,35 with inhibitors,36−38 and substrate-based peptide inhibitors.39,40 These structures reveal a 6-TM helical bundle with a buried serine-histidine in the active site. Mobile elements include a loop on the periplasmic face that exposes the buried active site and slight tilt of helix 5.41 For hiGlpG, two structures have been solved where large variations on helix 5 and the periplasmic loop are observed.10,42 Because bacterial rhomboid proteases such as GlpG act alone without cofactors,43 they are excellent models to study the structure−function relationship of intramembrane proteolysis. In subsequent sections of this work, we will use

GlpG as a model system to discuss specific issues pertaining to chemical reactions in membrane proteins. 2.2. Bacterial Sec Protein Secretion: Role of Lipids and Proton Binding

The bacterial Sec protein secretion pathway is a major secretion pathway of interest for the development of antibiotics.44−46 In post-translational protein secretion, soluble proteins newly synthesized by the ribosome are pushed across the plasma membrane by the soluble SecA protein motor, an ATPase that binds to the membrane-embedded SecYEG protein translocation channel (Figure 3A) and couples the binding and hydrolysis of ATP with stepwise movement of the secretory protein through the translocon.47−50 At late stages of the process, when ATP and the secretory protein are not bound to SecA, the proton motive force (pmf) can drive protein translocation.51 Both the pH gradient across the membrane and the TM potential components of the pmf are required for optimal protein secretion.52 The reaction coordinate of SecA involves interactions between SecA and several binding partners: soluble chaperones such as SecB,48,53 the secretory protein,47,54 the membraneembedded SecYEG protein translocon,47,48 SecD and SecF,50 the lipid membrane,47 and the nucleotide.50,51,55 In addition to these interaction partners, SecA can bind to RNA and might be involved in transient coupling between protein secretion and protein translation.56 A fascinating open question regarding the functioning of SecA is how lipid interactions and the pmf impact protein secretion. The importance of lipids for bacterial protein secretion has been underlined by early experiments indicating that an E. coli mutant defective in phosphatidylglycerol (PG) has reduced protein translocation60 that can be restored by adding PG61 and that liposomes can stimulate the ATPase activity of SecA in the absence of SecY.47 In experiments with SecYEG reconstituted in nanodiscs, the fraction of SecA bound to SecYEG was significantly higher when the nanodisc E

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contained a mixture of PC and PG, as compared to PC only,62 being thought that binding of SecA to negatively charged PG lipids surrounding the translocon is essential for initiating protein secretion.63 A particularly important lipid is the negatively charged cardiolipin (CL), which promotes dimerization of SecY, binding of SecA to SecY, and stimulates ATP hydrolysis.64 Interactions between SecA and the membrane are thought to involve direct contacts between protein groups and lipids. Initial observations were interpreted to suggest that SecA inserts into model membranes65,66 and lipid monolayers,66 and that binding of SecA perturbs lipid packing and restricts lipid motion.67 Because SecA is a large protein of ∼840−860 amino acid residues (for Bacillus subtilis and E. coli) and its surface is decorated with numerous charged and polar protein groups,68 the molecular picture of a SecA protein inserted into the lipid bilayer remained largely unclear. Recent measurements with site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy indicated that when SecA binds to SecYEG, its N-terminus lies parallel to the membrane surface and inserts ∼5 Å.69 That would mean that when SecA binds to the translocon, its interactions with the lipid membrane might be largely restricted to the headgroup region of the membrane, and to the proximity of the translocon. Membrane interactions close to the translocon appear important for the influence that the pmf can exert on protein translocation. Two Lys groups of the translocon bind CL, and these lipid interactions appear to be required for the pmf to stimulate protein translocation.64 The pmf favors deinsertion of SecA from the membrane, which is thought to promote protein secretion by making soluble SecA available when the concentration of SecA is low.70 Because the pmf had a lesser impact on protein secretion and deinsertion of SecA from the membrane when a SecY mutant was used, the hypothesis was put forward that the pmf might act primarily on SecY.70 Moreover, early experiments on pmf-dependent protein secretion in D2O as compared to H2O were interpreted to suggest that protein secretion involves proton transfer.52 Bacterial Sec protein secretion may be assisted by SecDF (Figure 3B), a proton channel that interacts with the secretory protein exiting the translocon59,71 and couples the flow of protons down their electrochemical gradient with protein conformational changes,59 resulting in movement of the secretory protein whose backsliding is thus prevented.59,71 SecDF is a member of the resistance-nodulation-cell division (RND) superfamily that includes bacterial drug and heavy metal transporters.72 Deletion of SecDF increases the sensitivity of Staphylococcus aureus to antibiotics73 and reduces the secretion or virulence factors in S. aureus74 and Listeria monocytogenes.75 In T. thermophilus, SecDF a conserved carboxylate located in the TM region of the protein, D340 (Figure 3B), is essential for the proton channel activity of SecDF, as D340N is inactive.59 Computer simulations of SecDF from Deinococcus radiodurans showed that the protonation state of the corresponding carboxylic group, D365, impacts internal water dynamics and couples to structural rearrangements of SecDF.76

2.3. The Programmed Death-1 Pathway and Protein−Protein Interactions at Complex Membrane Interfaces

The programmed death (PD)-1 pathway involves interactions between two type I membrane proteins, the PD-1 receptor and the PD-L1 ligand, bound to the membrane of the immune system T-cell and, respectively, the antigen presenting cell (cancer cell).77 Because this binding can lead to downregulation of the immune cell response and loss of tumor elimination,78 the PD-1−PD-L1 pathway is a prominent target for modern immunotherapy cancer treatments.78−82 PD-1, which is a member of the immunoglobulin superfamily,83,84 binds PD-L1 via its ectodomain85 (Figure 4). This

Figure 4. Charged protein groups at the binding interface of PD-1 and PD-L1. (A) Illustration of selected H-bonding groups at the binding interface between PD-1 and PD-L1. The molecular graphics is based on the crystal structure of the Homo sapiens proteins, PDB 4ZQK, solved in ref 85 at a resolution of 2.45 Å. Ref 85 identified H bonds at the interface between PD-1 and PD-L1. (B) The surface of the ectodomains of PD-1 and PD-L1 comprises numerous charged groups. Cα atoms of Asp/Glu groups are shown as spheres colored red, Arg/Lys, blue, and Ser/Thr, ice-blue. The molecular graphics is based on crystal structure of the complex between H. sapiens PD-L1 and Mus musculus PD-1 (chain A and, respectively, chain B from crystal structure PDB 3BIK, ref 95).

protein-binding event leads to phosphorylation of a conserved Tyr group at the cytoplasmic, C-terminal side of PD-1, recruitment of cytoplasmic phosphatase SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2,86−88 colocalization of PD-1 with T cell receptors (TCRs),87 and altered TCRmediated signaling.89 The PD-1−PD-L1 pathway thus involves a complex set of protein binding and chemical reactions that effectively couple binding of two membrane proteins across the intercellular region with a chemical reaction inside the immune system cell and altered cell signaling. The molecular picture of how PD-1 and PD-L1 interact with each other is further complicated by the observations that the pH close to the surface of cancer cells can be acidic,90−93 with excess protons in the tumor tissue and a proton gradient at the interface between tumor and normal tissue,92 and that binding between PD-L1 and a high-affinity mutant PD-1 was stronger at acidic pH.94 Crystal structures of fragments of the ectodomains of PD-1 and PD-L1 bound to each other (Figure 4) provide valuable clues about titratable protein groups whose protonation state could change and impact protein interactions: At the protein binding interface, there is a H-bond network that includes F

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Figure 5. Open and closed conformations of GlpG as indicated by crystal structures of the E. coli GlpG rhomboid domain and the structure of the soluble cytoplasmic domain. (A, B) The open and closed conformations are colored green and purple, respectively. Water molecules within 6 Å of S201 in the open conformation are shown as small yellow spheres. (A) Overlap of the open and closed states of GlpG from PDB 2IRV, ref 34. The structure of the open conformation indicates coordinates for a PG lipid bound at the active site (phosphatidylglycerol,2-vaccenoyl-1-palmitoyl-snglycerol-3-phosphoglycerol). (B) Overlap of the open and closed states of GlpG from PDB 2NRF, ref 33. Structural alignments in A and B were performed using VMD.7 (C) The soluble NMR structure of the cytoplasmic domain of GlpG from ref 103. Cα atoms of the charged groups of the first N-terminal 61-amino acid residue segment of the cytoplasmic domain are shown as van der Waals spheres colored red and blue for Asp/Glu and Arg/Lys, respectively. Note that the orientation of the cytoplasmic domain relative to the rhomboid domain and its lipids interactions are not known.

TM α-helical segments to be seen.96 In recent years, structures as complex as that of a cyanobacterial photosystem II, containing some 19 protein subunits, cofactor molecules, special lipids, and numerous water molecules, could be solved with X-ray crystallography to a resolution of 1.9 Å.97 For bacteriorhodopsin, the most recent crystal structure was solved at a resolution of 1.3 Å, which allowed an accurate view of the conformation of the retinal chromophore.98 A proteolytic enzyme reaction involves breaking and forming of covalent bonds at the enzyme’s active site and at the substrate whose chemical structure is being altered by the enzyme. That is, the functioning of an enzyme associates with changes in the spatial location of atoms of the substrate and of at least some of the atoms of the enzyme. Structural snapshots of the enzyme caught at various moments of time along its reaction coordinate obtained from methods such as X-ray crystallography, NMR, or cryo-EM provide valuable glimpses into the structures that may be sampled by the enzyme along its reaction cycle, and are often used to propose models for how an enzyme works. As an example of a membrane-embedded enzyme crystallized in different conformations, we consider here GlpG. It has been proposed that docking of the TM substrate might involve lateral displacement of TM helix 5 of GlpG (Figure 5A,B)42,99 and/or loops that link this helix to the adjacent helices.41 Crystal structures solved for GlpG (for reviews see, refs 100, 101) differ mostly in the orientation of TM5 relative to the core of the protease, and in the conformation of loop L5.102 The two crystal structures solved by Ben-Shem and colleagues33,34 present GlpG in the closed vs the open states, which are distinguished mainly by the orientation of TM5 (Figure 5A). In the open conformation, GlpG binds a PG lipid at the active site (Figure 5A).34 A qualitatively similar picture of the structure of GlpG in the open vs closed states is indicated by two crystal structures (Figure 5B) solved by Wu and colleagues;33 in one of these structures, denoted as the open conformation, TM5 is displaced laterally toward the lipid membrane and away from the TM core of the protein (Figure

charged and polar groups of PD-L1 and PD-185 (Figure 4A), and both the ligand and the receptor have numerous charged and polar groups on their surface (Figure 4B). The relatively small ectodomains of PD-1 (115 amino acid residues) and PDL1 (211 amino acid residues) of the ectodomain complex95 have together 41 Asp/Glu groups and 34 Arg/Lys, that is, the complex is overall negatively charged when assuming all carboxylate groups to be negatively charged. Because carboxylate groups could bind protons, and the pH at the surface of the cancer cell can be acidic, an important question here is whether proton binding to carboxylate groups could impact interactions between PD-1 and PD-L1.

3. REDUCING COMPLEXITY TO UNDERSTAND CHEMICAL REACTIONS AT BIOMEMBRANE INTERFACES Deciphering how membrane-embedded proteins work often requires the use of simplified model systems that allow us to dissect how various components or interactions within the system impact the reaction mechanism. Information about the three-dimensional arrangement of the atoms that constitute the active site of the enzyme is invaluable for hypotheses regarding the way that enzyme might work. In the case of macromolecular protein complexes such as those discussed above as examples, knowledge about simpler model systems establishes the foundation for working models that can be tested with experiments and computation. Experimental strategies for this analysis are discussed below. 3.1. Structural Biology

To obtain a detailed view of the structure of a membrane protein experimentally, the protein is first extracted from the cell membrane, purified in detergent and, provided suitable samples can be obtained, its three-dimensional structure is solved using X-ray crystallography, Nuclear magnetic resonance (NMR) or cyro-EM. The first three-dimensional structure of a membrane protein was that of bacteriorhodopsin, solved in 1975 by Henderson and Unwin using cryo-EM;96 the resolution of this first structure was 7 Å, allowing the seven G

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5B).33 Such a lateral displacement of TM5 was suggested to allow binding of a substrate to the active site.33

biosynthesis and importantly are the drug target for NSAIDs.111 COX is a peripheral membrane protein that interacts with the lipid bilayer via a small hydrophobic region to gain access to lipid substrates.112 COX adds molecular oxygen to arachadonic acid, the precursor of prostaglandin, which requires abstraction of a bis-allylic H atom. Because arachidonic acid has three bis-allylic carbon atoms, KIE studies of COX have been challenging.113 Recently, KIE measurements were combined with lipidomics to characterize the physiological KIE, PKIE, defined as the ratio between the amount of nondeuterated (light) vs deuterated metabolite by accounting for the amount of light vs deuterated metabolite and substrate.113 The PKIE measured upon deuteration of a specific carbon atom of arachidonic acid (C10) were interpreted to suggest that this carbon atom contributes to the reaction coordinate of COX.113 Temperature is a critical variable that influences the rate of an enzymatic reaction and the ability of an enzyme to overcome this energy barrier.114,115 Enzymes, being proteinaceous macromolecules, have a large heat capacitance, and furthermore a difference in heat capacity between the enzyme−substrate and enzyme−transition state species for enzyme-catalyzed reactions. This has been measured both experimentally116 and in simulations.117 Rhomboid intramembrane protease were reported to have low intrinsic thermostability, with relatively small values of the free energy of stability in the range of 2.1−4.5 kcal/mol.118 Nevertheless, denaturant-induced unfolding of these proteases can also be largely reversible.119 The catalytic effect, or acceleration of reaction rates, by enzymes rely on charged groups that stabilize transition states or function as acid−base catalysts in the reaction. This stabilization reflects the underlying nature of the rate enhancement.120 Transition state analogues can be used with X-ray crystallography to visualize intermediates along the reaction cycle. For serine proteases, several covalent inhibitors exist that mimic either the tetrahedral or acyl intermediate.18,19 The crystal structure of GlpG from a membrane bicelle, solved in the presence of a peptide aldehyde derived from the native rhomboid substrate Gurken, is thought to mimic the tetrahedral intermediate of the protease.39 The substrate is covalently bound to the catalytic Ser, which H bonds to the catalytic histidine group. A second GlpG co-crystal structure with a peptidyl ketoamide inhibitor shows covalent binding to the catalytic Ser.40 The fact that the overall conformation of the protease bound to substrate models remains surprisingly close to a closed conformation solved in the absence of substrate indicates that, in the presence of a small model substrate and in a crystallographic environment, the conformation of GlpG at the tetrahedral intermediate transition state is very similar to that of the reactant state. The structure of GlpG bound to a full substrate in a fluid, hydrated lipid membrane environment, remains an important open issue for future studies.

3.2. Kinetics

The reaction coordinate of an enzyme consists of the sequence of structural changes and associated free energy profile of the enzyme and substrate from the initial reactant state to the final product state passing through the transition state(s) that give the mechanism of the reaction.104 Biochemistry experiments provide fundamental insight into the energetics of the chemical reaction. The rate of the reaction, k, measures the relative concentration of product vs reactant molecules as a function of time.105 These experiments can be conducted through measurement of reactant at various time points throughout an assay either in a discontinuous manner, which includes measurements at time intervals such as in chromatographic or radiometric assays, or a continuous based assay where the reaction is followed continuously over time, which includes fluorometric and spectrometric assays. On the basis of the Michaelis−Menten equation, the main factor that determines the enzymatic reaction rate is the substrate concentration.106 The Michaelis−Menten equation assumes that a thermodynamic equilibrium exists between the enzyme and substrate; this also assumes conformational equilibrium. In a nonequilibrium reaction, conformational heterogeneity of the enzyme, substrate, and their environment could influence this balance and thus the enzymatic turnover. Conformational heterogeneity might be particularly important for an enzyme such as GlpG, which performs its catalytic function in a lipid membrane. Kinetic measurements of GlpG were interpreted to propose that GlpG needs >2.5 min to cleave one substrate when in E. coli cells or when reconstituted in proteoliposomes;107 when GlpG was reconstituted in detergent, substrate cleavage occurred ∼6.5-fold faster.107 The reduced catalysis in a membrane environment was attributed to conformational changes required for substrate binding being slower in the membrane than in detergent.107 A distorted lipid bilayer close to the protease was suggested to facilitate diffusion at a high rate in lipid bilayers and enhance product formation.108 Slow cleavage of the substrate was also obtained for γsecretase: kcat for the cleavage of β-amyloid substrate by γsecretase is ∼4.3 h−1 in vitro and ∼6 h−1 in cells,109 which means that γ-secretase is slower than most soluble proteases, e.g., 2−3 orders of magnitude slower than caspase-3, which is a cysteine protease.109 3.3. Activation Energy, Kinetic Isotope Effects, and Transition-State Inhibitors

The activation energy barrier of a chemical reaction is the free energy difference between the reactant(s) and the transition state.104 Complex biochemical reactions might have more than one transition state, and the rate-limiting step of the reaction will be defined by the highest energy transition state along the path from the reactant to the product state.104 Kinetic isotope effects (KIE) can be used to study enzyme mechanisms by taking advantage of the rate change when one atom is substituted for its isotope to determine the relative contribution of individual components on steady state kinetics parameters.110 Advancements in these methods reveal new insights into enzymatic reactions, including those at the lipid− water interface. Cyclooxygenases (COX; prostaglandin G/H synthase) are enzymes that catalyze the first two steps of prostaglandin

4. ROLE OF LIPIDS IN THE REACTION COORDINATES OF MEMBRANE PROTEINS Mixtures of lipids, specifically in their headgroups, acyl chain length, and saturation level, provide a deeply complex environment that varies among different intracellular membrane compartments.121 Furthermore, each lipid has a unique overall shape, and packing defects such as small charged H

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Figure 6. H. influenzae rhomboid protease, hiGlpG, in membrane vs detergent environments. (A) Illustration of hiGlpG in a hydrated POPE lipid membrane environment. The catalytic Ser and His side chains are shown as yellow surfaces. The image is based on a coordinate snapshot from a molecular dynamics simulation at room temperature. (B) Illustration of hiGlpG in a detergent micelle composed of 130 n-decyl-N,Ndimethylglycine (DDMG) molecules. Protein heavy atoms are shown as yellow van der Waals spheres, DDMG heavy atoms are in atom colors, and water molecules (cut-away view) are transparent. The detergent micelle was generated using CHARMM-GUI161,166 starting from the crystal structure PDB 2NR9 from ref 10.

membrane in which it is found: the purple membrane of Helibacterium salinarium.96,128,129 Bacteriorhodopsin organizes as trimers that pack tightly in the membrane as the twodimensional hexagonal arrays such that there are only about 10 lipid molecules associated with each monomer.129 This hexagonal arrangement of bacteriorhodopsin trimers could not be observed when the protein was reconstituted in DMPC,130 but it was re-established when negatively charged native lipids of the purple membrane (either 2,3-di-Ophytanyl-sn-glycero-1-phosphoryl-3′-sn-glycerol 1′-phosphate, DPhPGP, or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3′-snglycerol 1′-sulfate, DPhPGS), and salt, were added to the system.131 In addition to playing such an important structural role, endogeneous lipids of the purple membrane directly impact the kinetics of the pump: squalene, a neutral lipid, and the negatively charged PGP-Me (phosphatidyl glycerophosphate methyl ester), are required for the normal kinetics of some of the steps in the proton-pumping reaction cycle.132,133 Crystal structures of bacteriorhodopsin provide clues about its lipid interactions. The crystal structure solved by Belrhali and colleagues using lipidic cubic phase crystallization at 1.9 Å resolution129 included coordinates for nine lipid molecules modeled as phytanyl moieties; because the precise nature of the lipid headgroups could not be readily identified,129 important details of how lipid headgroups might interact with the protein remained somewhat unclear. In a more recent crystal structure analysis of bacteriorhodopsin, a glucose− manose−galactose−sulfate headgroup was proposed to mediate interactions at the central region of the protein trimer.134 The precise mechanism by which lipids influence the kinetics of bacteriorhodopsin remains, to our knowledge, unclear at the atomic level of detail. A description of this mechanism is important because it could inform on how another microbial retinal protein, channelrhodopsin, which is used in optogenetics applications,135 could respond to changes in its lipid membrane environment from that of the native cyanobacteria vs that of a mammalian brain.

headgroups can assist with destabilization and curvature of membranes.122 The complexity of the cell membrane is drastically reduced when membrane proteins are studied in in vitro because such studies typically rely on detergent solubilization of the membrane protein, meaning that the environment of the membrane protein is significantly simpler than in a cell or even than in a lipid bilayer model (Figure 6). This issue is alleviated by the fact that lipids can be added to detergent-extracted membrane proteins to form mixed micelles123 or reconstituted with lipids to form bicellular124 (discoid shaped species) or liposomes107 (circular sealed lipid vesicles containing membrane proteins). Experimental methods used to probe various aspects pertaining to membrane protein function have limitations depending on the parameters to be tested. For example, protein diffusion might be largely different in detergent vs lipid membranes and in membranes of different lipid composition: Tightly bound lipids could slow down lateral diffusion of a membrane protein,125 and a membrane protein can undergo nonspecific, detergent-mediated aggregation.126 Liposomes and bicelles provide the lateral pressure needed to form a sturdy three-dimensional structure, however, not all lipid classes are suited for curved liposomes. Bicelle preparation typically require the usage of 1,2-dimyristoyl-sn-glycero-3phosphocholine lipids (DMPC)124 whose acyl chain length (14:0) is shorter than that found with some membrane compartments in vivo. The use of DMPC may thus introduce hydrophobic mismatch between the bicelle and the membrane protein, which in the case of a rhomboid protease can be inhibitory.127 To illustrate how specific lipids could influence the reaction coordinate of membrane proteins, in what follows we discuss the lipid interactions of bacteriorhodopsin, which is a model system for biological proton pumps, and of the GlpG rhomboid protease. 4.1. Lipid Interactions of Bacteriorhodopsin

Bacteriorhodopsin is a small seven-TM helical membrane protein that accounts for ∼75% of the total mass of the I

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close to the values of ∼31−33 Å estimated by Foo and colleagues145 for the thickness of the hydrocarbon phase of the 12-carbon detergent micelles. An important question that remains open is how full-length GlpG interacts with lipid bilayers. Crystal structures of the catalytic membrane domain9 and of the cytoplasmic domain of GlpG147 have been solved independently, but a full-length structure is lacking. Although the cytoplasmic domain is not required for catalytic activity of GlpG,32 it appears that this domain can regulate the functioning of the protease.103 Furthermore, a recent study with various bacterial intramembrane proteases indicated an enhanced catalytic turnover in the presence bicelle environment compared to detergent solubilized protein,148 but only with rhomboid proteases that have a soluble domain.148 In other words, in detergent, soluble domains may impede functioning of the membrane embedded GlpG protease. Because soluble substrates were used here, the effect of the lipids could be attributed to changes in the membrane-embedded enzyme.148 In vivo, the influence of the cytoplasmic domain on the catalytic activity of GlpG might be exerted indirectly, via interactions with lipids: The solution-NMR structure of the soluble cytoplasmic domain of GlpG149 displays a helical fragment that comprises six Asp/Glu groups and three Arg/Lys groups (Figure 5C). We speculate that this charged segment of GlpG might locate close to the lipid membrane interface of GlpG. Because the E. coli membrane contains negatively charged PG lipids,137,150 electrostatic interactions between carboxylate, Lys, and Arg groups of the cytoplasmic domain of GlpG and lipid headgroups could impact the lipid interactions of the TM rhomboid domain of GlpG, e.g., it could help anchor GlpG in the membrane and enforce a particular orientation of GlpG relative to the membrane surface. In this scenario, charged groups of the cytoplasmic domain of GlpG would play a role similar to that of the amphipathic helix of SecE located approximately at the lipid headgroup interface (Figure 3A); this helix contains charged protein amino acid residues that interact with lipids.151 The observation that the effect of the cytoplasmic domain on GlpG′s catalytic activity depends on lipids148 could also be interpreted to suggest that the rhomboid and, potentially, substrate domains extraneous to the protease domain may influence the surface electrostatics and thus the free energy profile along the reaction coordinate of proteolytic bond cleavage. Thus, the role of lipid in reactions at the membrane might need to be considered on an individual basis for each protein and substrate.

A first step toward assessing the impact of lipids on the reaction mechanism of channelrhodopsin was made in recent computations of the potential of mean force for proton transfer in a channelrhodopsin variant.136 These computations indicated that the energy barrier for proton transfer from the Schiff base of the retinal chromophore to a nearby carboxylate group was little affected by the presence of a hydrated lipid membrane environment.136 The absence of a significant impact of lipids on the reaction energetics for retinal deprotonation might be explained by the retinal Schiff base being located approximately at the center of the protein, i.e., far away from the lipid headgroups.136 4.2. Lipid Interactions of Rhomboid Proteases

Intramembrane proteases are particularly valuable model systems for studying the role of lipids in the reaction coordinates of membrane proteins. When bacterial rhomboid proteases were reconstituted in proteoliposomes of various lipid composition, catalytic cleavage of a model substrate depended drastically on the composition of the lipid membrane.43 The GlpG rhomboid from E. coli cleaved the substrate in, e.g., detergent and PE lipids, but not in PG lipids or in E. coli lipid extract.43 By contrast, the YqgP rhomboid from B. subtilis was inhibited in detergent and in PE lipids, but was active in PG and E. coli lipids.43 Because E. coli and B. subtilis membranes have different relative amounts of PE and PG lipids,137,138 the observation that GlpG and YqgP respond differently to these lipids could be interpreted to suggest that a rhomboid’s catalytic activity could be influenced by the relative concentration of lipids.139 The particular step along the reaction coordinate of rhomboid proteases that is influenced by the lipid composition is unclear. Because the substrate is a membrane protein,140 and the catalytic site of GlpG is exposed to the lipid membrane,34 the entire reaction coordinate of a rhomboid protease could be influenced by the lipids interacting with enzyme and substrate. Lipid composition is known to impact the α-helical content of a peptide,141 interactions between membrane proteins,142 and membrane protein orientation.143,144 How the enzyme and substrate interact with each other and orient relative to the membrane normal is likely essential for the efficiency with which the enzyme−substrate complex forms. To find out whether and how a hydrophobic mismatch between the lipid membrane environment and the rhomboid itself might impact catalytic activity, Foo and colleagues145 probed the cleavage activity of GlpG using a construct consisting only of the TM core protease domain, i.e., without the cytoplasmic domain of GlpG. Cleavage of a soluble substrate model (fluorogenic casein) was measured in detergent micelles of different chain length.145 It was found that phosphocholine detergents with relatively short alkyl chains with 10−12 carbon atoms allowed maximum substrate cleavage and that cleavage activity decreased when using detergents with longer alkyl chains.145 The lower activity of GlpG in lipid−detergent bicelles relative to detergent micelles was suggested to originate in a reduced conformational dynamics of helix 5 of GlpG.145 The observation that the TM catalytic core of GlpG has robust activity in detergents with short alkyl chains of up to 12 carbon atoms145 suggest a preference of GlpG for relatively thin lipid bilayers. This suggestion is compatible with the observation that liposomes of E. coli lipids are thin, with an average phosphate-to-phosphate thickness of ∼33 Å;146 this is

5. USING COMPUTER SIMULATIONS TO UNDERSTAND REACTION MECHANISMS OF MEMBRANE PROTEINS 5.1. A Molecular Picture of the Lipid Interactions of Intramembrane Proteases

Classical mechanical molecular dynamics simulations of membrane proteins provide a powerful tool to study membrane protein dynamics in fluid lipid membrane environments.152 Current issues pertaining to membrane protein simulations are addressed in detail by several other contributions to this topical issue.153−159 Briefly, the simulation of a membrane protein requires the threedimensional structure of the protein, i.e., the Cartesian coordinates of the atoms of the protein, which are typically J

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Figure 7. Interhelical carboxylate-hydroxyl H-bond motifs in membrane proteins. (A) The D96/T46 and D115/T90 sites in bacteriorhodopsin. The image is based in part on refs 104 and 186 and uses the crystal structure PDB 5B6V.180 Selected water molecules are shown as small pink spheres. (B) The multidrug transporter AcrB from the crystal structure PDB 1IWG.190 At ∼5−6 Å, the distance between D408 and S481 is too long for H bonding, but it might shorten during the reaction cycle of the transporter. (C) The Hv1 proton channel, PDB 3WKV.193 (D) The SecY protein translocon from the archaeon Methanocaldococcus jannaschii, PDB 1RHZ.201 (E) The proton channel SecDF from T. thermophilus, PDB 3AQP, chain A.59 (F) The human μ-opioid receptor bound to G protein, PDB 6DDE, chain R.194 (G) The human δ-opioid receptor, PDB 4N6H.196 The sodium ion is shown as a small yellow sphere.

atomistic simulations on simulation systems of ∼160k atoms reported trajectory lengths of up to ∼35 ns;143 atomistic simulations on GlpG were then prolonged to ∼110 ns.164 The most recent atomistic simulations with GlpG in a small membrane patch of 100 POPE lipids per leaflet, for ∼60k atoms in total, were prolonged to 450−750 ns.165 The trajectory, which gives the coordinates of the simulation system as a function of the simulation time, is then used to calculate various parameters that allow us to characterize the system of interest.152 For GlpG, particularly important was to first understand how the lipid bilayer adjusts to the presence of the protein: GlpG has a rather irregular shape, with largely different orientations of the TM helices and a long loop between the first two helices (Figures 5A,B, 6A). Simulations indicated that lipid membranes composed of POPE adjust to the presence of GlpG by nonuniform thinning, such that close to GlpG the membrane is about 4 Å thinner than far away from GlpG.143 Thinning of POPE membranes close to GlpG was reproduced by more recent simulations with all-atom164 and coarse-grain descriptions.167 When simulations were performed starting from a crystal structure that includes a PG lipid bound at the active site (Figure 5A),34 the lipid, modeled as POPG, remained close to the active site.143 That is, the active-site of GlpG can be directly accessed by lipids in both crystal and fluid bilayer environments. As discussed in section 2.1 above, a similar observation was made in atomistic simulations on a homology model of presenilin embedded in POPE.23 These simulations could then be interpreted to suggest that lipids could be directly involved in controlling access of the substrate to the active site of membrane-embedded proteases. A caveat of the simulations is that they rely on relatively simple one-

provided by experimental structural analyses. In the absence of a starting structure of the protein provided by experiments, coordinates for the protein could be derived by homology modeling but accurate homology models might require significant sequence identity between the target and template proteins. Where needed, coordinates are constructed for amino acid residues of flexible internal loops with insufficient electron density in the experimental structure and, in atomistic simulations, for missing H atoms. A suitable orientation of the protein in the membrane is set, for example, by using the Orientation of Proteins in Membrane server,160 and the protein is then embedded in a hydrated lipid membrane patch of the desired lipid composition, e.g., by using CHARMMGUI161,162 (Chemistry at Harvard Molecular Mechanics163 Graphical User Interface) or VMD.7 Interactions between atoms are computed according to a potential energy function whose mathematical expression depends on the particular force field used. This potential energy function will include terms to describe stretching of chemical bonds, bending of valence angles, dihedral angle torsions, and nonbonded interactions. Thus, in a simulation system for a membrane embedded in a hydrated lipid−membrane environment, a lipid molecule will interact with the protein via nonbonded, electrostatic, and van der Waals interactions. In the case of GlpG, the first atomistic simulation reported had the protein embedded in a hydrated lipid membrane patch of ∼250 lipids per leaflet and ∼30k waters, with a total of ∼160k atoms.143 The molecular dynamics simulation is performed by integrating numerically the classical equations of motions; the length of the simulation is given by the integration step, which is on the order of femtoseconds (fs) in atomistic simulations times the number of steps. For GlpG, the first K

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6. STRUCTURAL MOTIFS OF H BONDING IN MEMBRANE PROTEINS Analyses of structural motifs associated with relatively simple protein systems might inspire hypotheses regarding the mechanism of action of complex macromolecular systems. In what follows, we summarize structural motifs relevant to the membrane proteins and reactions we used as examples.

component lipid bilayers, such that the molecular picture they provide for the protein−lipid interactions might be rather simplistic. Moreover, a systematic comparison of simulations and experiments using the same lipid bilayer composition is still lacking. 5.2. Combined Quantum Mechanical−Molecular Mechanical Computations of Reaction Mechanisms: GlpG and the Bacteriorhodopsin Proton Pump

6.1. Interhelical Hydroxyl-Carboxylate H-Bond Motifs in Membrane Proteins

Understanding how the lipid membrane impacts the reaction mechanism of a membrane-embedded enzyme requires a description of the reaction coordinate of that protein, i.e., a description of the sequence of structural changes and free energy profile along the path of covalent bond breaking and forming. A valuable theoretical approach for studying enzyme reaction mechanisms is the combined quantum mechanical/ molecular mechanical approach (QM/MM).168−170 In this approach, groups directly involved in the chemical reaction are treated with QM, whereas the rest of the protein and solvent environment are treated with MM; when the frontier between the QM and MM regions of the system crosses covalent bonds, interactions between the QM and MM regions of the simulation system include electrostatic (Coulomb) and van der Waals interactions and bonded interactions. To illustrate the usefulness of the QM/MM approach for studying reaction mechanisms, we use as an example the first proton-transfer step in bacteriorhodopsin. In the resting retinal all-trans state of bacteriorhodopsin, the primary proton donor group, the retinal Schiff base, H bonds to a water molecule (w402) that further bridges to two carboxylate groups, D85 and D212, both of which are negatively charged (Figure 7A). The H-bond network at the active site of bacteriorhodopsin is perturbed when retinal photoisomerizes from all-trans to 13-cis, which starts a reaction cycle whose net effect is the transfer of one proton from the cytoplasmic to the extracellular side of the membrane. The issue of the retinal geometry prior to the first proton transfer step in bacteriorhodopsin has been controversial (see, e.g., refs 171−175). QM/MM computations have demonstrated that a 13-cis, 15-anti cytoplasmic-oriented retinal Schiff base is compatible with productive proton pumping.176−179 Predictions from QM/MM computations regarding the cytoplasmic orientation of the retinal Schiff base in the proton-transfer reactant state and the involvement of T89 in proton transfer176,179 are compatible with recent X-ray crystallography180 and NMR data,181 which highlights the usefulness of the theoretical approach for finding reaction coordinates for complex protein environments. QM/MM computations of the reaction mechanism of intramembrane proteases are still in their early days. Uritsky and colleagues reported computations in which the active site of GlpG was treated with QM at the PM6 level, and the protein, lipids, and waters interacted with QM via uniform dielectric continuum model.182 These computations indicated that distances between the catalytic Ser and His groups depend on the protonation states of these groups.182 When GlpG is placed in a lipid membrane, both catalytic groups (S201 and H254, Figures 5A,B) are found to be neutral at the Michaelis complex, suggesting that deprotonation of S201 by H254 contributes to the activation barrier.182

The GlpG rhomboid protease has a highly asymmetrical distribution of charged and polar side chains, which locate preferentially at loop L1, the cytoplasmic regions of the TM helices, and along TM3.143 In molecular dynamics simulations interhelical H bonds were observed to interconnect all TM helices of GlpG, except for the helix thought to move laterally, TM5.143 One of the interhelical H bonds identified from simulations is that between the highly conserved TM2-E166, which bridges to TM1-T97 and to TM3-S171143 (Figure 7A,B) Subsequent experimental work verified the importance of this interhelical carboxylate-hydroxyl connection: The E166A mutant has decreased thermostability and lower activity than the wild-type protein, and the double mutant T97A/ E166A has greatly reduced activity.118 The functional importance of the interhelical carboxylatehydroxyl H bond is further underlined by the observation of a similar arrangement in the crystal structure10 of H. influenzae GlpG, in which the carboxylate group of TM2-E81 is within Hbond distance from TM1-T12 and TM3-S86.139 More recently it was noted that a similar interhelical H bond might be present in the rhomboid domain of PROD, the PARL-rhomboid domain, in which TM3-Q242 could H bond with TM1T168.183 A carboxylate-hydroxyl interhelical H bond is observed at the heart of the archaeal SecY protein translocon, where a dynamic H-bonded cluster includes T80 and E122151,152 (Figure 7D). This cluster of H bonds includes highly conserved groups151 and groups whose mutation perturbs the functioning of the translocon,184 being thought that it plays an important role in controlling the conformational dynamics of SecY.57,152 It was noted recently that the TM2−TM3 interhelical H-bond motif of SecY could be directly involved in interactions with the translocating peptide.57 In SecDF, D340 and D637 are thought to be important for proton transfer185 (Figure 7E). The crystal structure of SecDF (PDB 3AQP, ref 59) indicates that both carboxylate oxygen atoms of D637 engage in interhelical H bonding with T672 and S676, respectively (distances of 2.9−3.2 Å); D340 is within ∼3.9 Å from S679 (Figure 7E). That is, D340 and D637 are located close to helical segment that contains three closely spaced Ser/Thr groups (Figure 7E). Similar interhelical carboxylate-hydroxyl motifs are observed in other membrane transporters186 and in receptor proteins (Figure 7). Examples of interhelical carboxylate-hydroxyl motifs in other membrane transporters include T46/D96 and T90/D115 of bacteriorhodopsin (Figure 7A), S767/D800 in the SERCA calcium pump,186 and D407/T978 in the multidrug transporter AcrB (Figure 7B). These interhelical H-bond motifs include groups thought to be important for the functioning of the transporter, including proton transfers:186 D96 of bacteriorhodopsin functions as proton donor for the retinal Schiff base,187−189 and D407/D408 of AcrB190,191 and D800 of SERCA.192 A similar arrangement of H-bonding L

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groups is observed for Hv1, a voltage-gated proton channel in which protons are transferred via the voltage-gating domain.193 E115 of Hv1 is located relatively close (within 4.1 Å) to S139 (Figure 7C), with which we suggest it could H bond, directly or water-mediated, during the reaction cycle of the channel. Membrane receptors that have in their TM domain interhelical carboxylate-hydroxyl H-bond motifs include the μ-opioid receptor. This is a member of the large protein of the G-protein coupled receptor family, which are seven-helical membrane-embedded proteins that mediate cell signaling. The recent cryo-EM structure of the μ-opioid receptor bound to a Gi protein194 indicates that D114, a group whose mutation to Asn largely reduces binding and receptor activation by morphine,195 has interhelical H bonds with S154 and S329 (Figure 7F). In the crystal structure of protein from the same family, the δ-opioid receptor, D95 is part of the coordination shell of the allosteric sodium ion,196 and it further engages in an interhelical H bond with S311 (PDB 4N6H196) (Figure 7G); the hydroxyl group of S311 can further H bond to the backbone carbonyl group of Y308, whose side chain H bonds to the D128 carboxylate (Figure 7G). That is, in the structure of the δ-opioid receptor196 S311 helps mediate an interhelical connectivity between two carboxylate groups. The examples discussed above suggest that interhelical carboxylate-hydroxyl motifs can be present at functionally important sites of membrane proteins. Our analysis here is based on a small subset of proteins, a more conclusive analysis would need to be performed to include representatives of the classes of membrane proteins for which three-dimensional structures have been solved. In spite of this caveat, the examples illustrated here and elsewhere104,186 highlight interhelical carboxylate-hydroxyl motifs as sites important for the conformational dynamics of membrane proteins and for coupling proton-transfer reactions with changes in protein and water dynamics. According to this model,104 changes in the dynamics of the interhelical H bond between the carboxylate and hydroxyl group (for example, due to a change in the protonation state of the carboxylate) couple to changes in the local dynamics of the helix hosting the Thr group, which can compete with amide groups for H bonding to a backbone carbonyl.197−199 An interhelical H bond might also facilitate insertion into the lipid bilayer of a TM helix that contains a carboxylic group. Because membrane insertion of a carboxylate group is energetically unfavorable,200 pairing carboxylates with a Ser of Thr hydroxyl group could provide a stabilizing interhelical H bond. Pairing a carboxylate with a hydroxyl group might provide weaker H bonding than salt-bridging with Arg/Lys and could allow for closer local packing of the helices.

the time that the proton spends bound to the proton antenna region.204 Proton antennas have been discussed, for example, for bacteriorhodopsin,203,206 cytochrome c oxidase,202,204,206 and for the soluble PsbO subunit of photosystem II.205,207,208 Molecular dynamics simulations of PsbO in water revealed that closely spaced negatively charged groups on the surface of PsbO can bridge via H-bonded water molecules and that the mobility of waters close to the surface of the protein is significantly reduced relative to bulk water.205 Persistent H bonding via short water chains was observed for a cluster of carboxylates that could be involved in proton transfer.205 The functional role of the water-bridged carboxylate clusters on the surface of PsbO remains, however, an open question; bioinformatics analyses and simulations of PsbO mutants led to a more nuanced view of the potential role of the surface carboxylates, whereby these groups might be important for ensuring solubility of the protein in the cell and for binding to the photosystem II complex.208 We suggest that observations made on the dynamics of H bonding at interface of PsbO205 are relevant to other proteins that might bind protons and expose to the bulk negatively charged interfaces, including the SecDF channel of the Sec protein secretion machinery, the ectodomain of the amyloid precursor protein substrate of the γ-secretase, and the soluble ectodomains of PD-1 and PD-L1. The crystal structure of SecDF,59 for example, indicates that TM helices hosting essential carboxylates D340 and D637 (see ref 185) contain additional charged groups and help delineate a cavity that appears exposed to bulk water (Figure 3B). In the case of the ectodomains of PD-1 and PD-L1, the presence of numerous carboxylates groups on the surface of the complex (Figure 4B) could lead to carboxylate−water interactions similar to those discussed above for PsbO, potentially with additional involvement of hydroxyl groups from Ser and Thr groups (Figure 4B). The important open question here is whether, as hypothesized for PsbO,205,207,208 carboxylate groups of the PD1−PD-L1 complex might bind a proton when the local pH of the cancer cell is acidic and how would such binding of a proton influence the overall structural stability and dynamics of the PD1−PDL1 complex.

7. FUTURE DIRECTIONS As discussed above, membrane proteins that function in the bilayer as single entities are valuable model systems for examining the complex effect of lipids on reactions at biomembranes using experiments and computations. Combining experiments with computations has the potential to reveal valuable insights into how lipids shape reaction coordinates of membrane proteins, but this approach can be challenging. For some membrane proteins, multiple structural snapshots have been solved using X-ray crystallography or cryo-EM, yet less is known about their conformational dynamics in the lipid bilayer and, further, how specific lipids impact dynamics and function. We know that membrane proteins have various degrees of flexibility: Some are rigid, for example, channels like aquaporin, whereas other proteins, such as the ATP driven CaATPase transporter that has been crystallized in at least six different structures209 has multiple conformational states and structural changes within the membrane domain. The activity of the Ca-ATPase is known to vary in the presence of different lipids,210 and a recent analysis showed lipids bind to region of known regulatory or inhibitor sites.211 Sensitivity to the lipid membrane composition might also be a characteristic of other

6.2. H-Bond Dynamics at Negatively Charged Protein Interfaces

Solvent-exposed regions of a membrane protein that are enriched in carboxylate groups could bind protons. The question of whether and how protons might bind at the protein interface is important for proteins and protein complexes that function in conditions of acidic pH (e.g., the PD-L1 ligand, Figure 2) and for proteins whose functioning involves proton uptake and release (e.g., SecDF, Figure 3B). Negatively charged regions of such proteins might function as proton antennas, which consist of closely spaced carboxylates and could also involve histidine groups (see, e.g., ref 202−205). The close spacing of the carboxylates increases M

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suggest that the reaction coordinate of intramembrane proteases might include, in addition to the typical stationary points of a protease (Figure 2), additional intermediary states given by structural rearrangements of the protein, substrate and, potentially, the lipid bilayer that hosts enzyme and substrate. An atomic-level description of the reaction coordinate of intramembrane proteases will require QM/MM computations of reaction coordinates with the protein embedded in a hydrated lipid membrane environment and validation of the reaction energetics by comparison with experiments. Given its relatively small size, GlpG is an ideal model system to perform systematic QM/MM reaction pathways in bilayers with different lipid composition. The results of such computations will inform on mechanisms by which lipids shape reaction coordinates of intramembrane proteases and facilitate description of the reaction mechanism of the much more complex γ-secretase. A remaining challenge for γ-secretase will be assessing reaction coordinates for the numerous TM substrates it cleaves. The importance of such studies is underlined by the disappointing results of recent clinical studies testing the usefulness of the drug semagacestat, which is an inhibitor of γsecretase.217 The hypothesis was that semagacestat would inhibit the cleavage by γ-secretase of amyloid-precursor protein substrates and thus be useful for treating Alzheimer’s disease.217 The trial was terminated due to severe side effects experienced by the patients, including skin cancers,217 and it has been suggested that semagacestat might have interfered with cleavage by γ-secretase of another of its substrates, Notch.17,218 The very recent high-resolution cryo-EM structures of the γ-secretase in complex with Notch24 and amyloid-precursor protein substrate25 are an important step forward to enable computations aiming to solve the reaction coordinate of substrate cleavage. We suggest that knowledge derived on the role of lipids in the reaction coordinates of rhomboid proteases has the potential to inform on more general mechanisms by which lipids impact membrane protein function. An example here is TM substrate interactions of the SecY protein translocon. It has been noted that GlpG and SecY display an intriguing symmetry in their mode of action, as they need to open lateral helical gates toward the membranes to dock and, respectively, release TM substrates.152 A challenge specific to the Sec protein machinery is deriving an atomic-level description of the entire reaction coordinate of the machinery; this includes deriving a high-resolution threedimensional structure of the complete machinery, probing its motions in a hydrated lipid membrane environment, and combining experiments with computations to describe the conformation-coupled changes in electronic structure−bond breaking and forming during ATP hydrolysis by SecA and, potentially, during protonation changes of SecDF. Knowledge of the role of lipids in reaction coordinates of membrane proteins such as rhomboids and the translocon might also facilitate our understanding of significantly more complex membrane reactions involving two different cells, as is the case of the PD-1−PD-L1 pathway. Issues that are directly relevant here include lipid-mediated interactions between proteins and proton binding reactions at the membrane interface and the functional role of H-bonding motifs in interhelical and solvent-exposed regions of the membrane protein. Future studies combining experiments with theory of model systems of increasing complexity will be needed to

understudied membrane proteins. Lack of data from experiments and computations represents a large gap in knowledge given the important roles that the lipid bilayer can have in the folding, assembly, and functionality of membrane proteins. Lipid compositions at distinct membrane compartments can be diverse including stereoisomers, adding further complexity.2 Experimental in vivo studies on the role of lipids will be essential to truly assess the role of lipids in reactions occurring at membrane interfaces. Precise single cell measurements are needed in physiologically relevant cell lines. Moreover, experimental studies need to account for cell type when conducting experiments with exogenously expressed proteins because protein folding, trafficking, and lipid composition varies from cell to cell.2 For some membrane protein families, only one representative structure may exist and molecular dynamics is needed to explore conformations sampled by the protein along its reaction coordinate. When the protein can have several isoforms and the structure of only one isoform has been solved, a fundamental question is how representative is the existing experimental structure for other isoforms of that protein. For example, the rhomboid family has several isoforms, yet crystal structures have been solved for only two bacterial forms, both having a 6-TM helix topology; there is no structural information about rhomboid isoforms that have a 7TM helix topology form such as the human rhomboid RHBDL2 found at the plasma membrane. Although homology models of 7-TM rhomboid isoforms could be derived using 6TM rhomboids as templates, the validity of the homology models, especially for interactions of the seventh TM helix, might be difficult to validate computationally. Individual lipid molecules can function at active sites to provide regulatory elements. Cryo-EM is providing a wealth of knowledge for large complex membrane protein structures with insight into lipid binding sites (see example in Figure 2B).212 However, this is just the beginning in a long journey to understand how membrane proteins function and communicate with lipids in their functionality. With technology advances in the field of cryo-EM, and with a concomitant increase in resolution, individual lipid molecules are being solved with membrane proteins structures (see, for example, γsecretase,24,213 the mycobacterial respiratory supercomplex,214 and proton-translocating vacuolar-type ATPases (V-ATPases)).215 With more structures of membrane proteins being solved by cryo-EM, motifs for lipid recognition, for which there is a paucity of data, may become clear. The main challenge in this analysis remains to be the generation of the membrane protein sample in a homogeneous form and in a native conformation with endogenous lipids copurified. For some membrane proteins, expression and purification can be a challenge, but advances in extraction protocols using lipid mimetics and amphiphols is providing more options.216 For the specific protein complexes discussed here as examples, the challenge will be to build upon knowledge derived from studies on simpler model systems, or on isolated components of the system, and derive a description of the reaction coordinate of the complex in bilayers whose lipid composition resembles that of the region of the biological cell where the complex locates in vivo. The reaction coordinates for substrate cleavage by the γsecretase and by GlpG remain unclear at the atomic level of detail. An important open question is why is their activity slow and why are intramembrane proteases inefficient enzymes. We N

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derive a general framework for reactions at membrane interfaces in realistic cell membranes.

AUTHOR INFORMATION Corresponding Authors

*M.J.B.: phone, (780)492-3586; E-mail, mlemieux@ualberta. ca. *A.-N.B.: phone, +49-30-838-53583; E-mail, nbondar@zedat. fu-berlin.de. ORCID

Ana-Nicoleta Bondar: 0000-0003-2636-9773 M. Joanne Lemieux: 0000-0003-4745-9153 Author Contributions

A.-N.B. and M.J.L. contributed equally to the writing of this review. Notes

The authors declare no competing financial interest. Biographies Ana-Nicoleta Bondar obtained a Diploma in Physics from the “Alexandru Ioan Cuza” University of Iasi, Romania, and a doctoral degree from the University of Heidelberg, Germany. She worked at the Interdisciplinary Center for Scientific Computing of the University of Heidelberg, the German Cancer Research Center (DKZF) Heidelberg, and the University of California, Irvine, USA. Since 2010, she leads a research group in Theoretical Molecular Biophysics at the Freie Universität Berlin, Germany. Her research is focused on understanding membrane protein function. M. Joanne Lemieux obtained a BSc and MSc from the Biochemistry and Neuroscience program at Dalhousie University, where she worked on membrane proteins involved in demyelination. Her PhD at New York University School of Medicine in Cell Biology in the Structural Biology Program under the supervision of Dr. Da Neng Wang focused on the three-dimensional structure of gradient-driven transporters. Her postdoctoral training in enzyme biochemistry was conducted at the University of Alberta under the supervision of Dr. Michael James. In 2007 she obtained a faculty position in the Department of Biochemistry at the University of Alberta. She is currently a Professor in the Department of Biochemistry, and Director of the Membrane Protein Disease Research Group at the University of Alberta, with a research focus on the structure/function of several classes of membrane proteins that include lipid biosynthetic enzymes, intramembrane proteases, and immune checkpoint inhibitors.

ACKNOWLEDGMENTS Research was supported in part by the Freie Universität Berlin within the Excellence Initiative of the German Research Foundation, the DFG, and by the DFG Collaborative Research Center SFB 1078 “Protonation Dynamics in Protein Function,” Project C4 (to A.-N.B.). This work was also supported by Canadian Institute for Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada (NSERC) (to M.J.L.). We thank Konstantina Karathanou for the coordinate files of the POPC:POPG bilayer. REFERENCES (1) van Meer, G.; Voelker, D. R.; Feigenson, G. W. Membrane Lipids: Where They Are and How They Behave. Nat. Rev. Mol. Cell Biol. 2008, 9, 112−124. O

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DOI: 10.1021/acs.chemrev.8b00596 Chem. Rev. XXXX, XXX, XXX−XXX