proteins STRUCTURE O FUNCTION O BIOINFORMATICS

Lipid-associated aggregate formation of superoxide dismutase-1 is initiated by membrane-targeting loops Choon-Peng Chng1* and Richard W. Strange2 1 Biophysical Modeling Group, Bioinformatics Institute, A*STAR (Agency for Science, Technology and Research), Singapore 138671, Republic of Singapore 2 Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, Life Sciences Building, Crown Street, The University of Liverpool, Liverpool L69 7ZB, United Kingdom

ABSTRACT Copper-Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo-SOD1 aggregates may be due to their ability to cause defects within cell membranes by co-aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane-bound copper chaperones. But how apo-SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc-binding loops in apo-SOD1 dimers play a critical role in the binding of 1-octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo-SOD1 monomer also associates with phospholipid bilayer via its zinc-binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane-bound copper chaperone. The orientation also suggests how membrane-bound monomers could act as seeds for membrane-associated SOD1 aggregation. Proteins 2014; 82:3194–3209. C 2014 Wiley Periodicals, Inc. V

Key words: amyotrophic lateral sclerosis; motor neuron disease; membrane-targeting loops; protein–lipid co-aggregation; 1-octanol.

INTRODUCTION Superoxide dismutases (SODs) are enzymes that catalyze the conversion of superoxide anions into dioxygen and hydrogen peroxide. They thus protect cells from oxidative damage. There are three forms of SOD in humans: homodimeric Cu–Zn SOD1,1 located in the cytosol,2 the intermembrane space of mitochondria3 and the nucleus4; homotetrameric Mn-SOD2 found in mitochondria3 and homotetrameric glycosylated Cu-ZnSOD3, located in extracellular space,5 SOD1 is mutated in 20% of hereditary amyotrophic lateral sclerosis (ALS), a progressive, lethal neurodegenerative disease leading to loss of motor neurons, paralysis and death.6 First described in 1869, ALS has since claimed the lives of 1–2 per 100,000 people worldwide annually. According to the ALS Association in the USA, approximately 5600 people in the US are diagnosed with ALS each year, with varying life expectancies. Although the majority of ALS cases

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(90–95%) are sporadic, they are clinically similar to SOD1-related familial ALS (FALS).7 Each monomer of the SOD1 homodimer contains one Cu and one Zn ion and a disulphide-bridge, formed between residues Cys57 and Cys146. Reduction of this Additional Supporting Information may be found in the online version of this article. Abbreviations: ALS, amyotrophic lateral sclerosis; CCS, copper chaperone protein; FALS, familial ALS; GRP1, general receptor of phosphoinositides 1; MD, molecular dynamics; PH, pleckstrin homology; PME, Particle-MeshEwald; SASA, solvent accessible surface areas; SOD1, superoxide dismutase; VMD, visual molecular dynamics; WT, wild-type Grant sponsor: A*STAR Computational Resource Centre through the use of its high performance computing facilities; Grant sponsor: Wellcome Trust ISSF nonclinical Fellowship (to R.W.S.). *Correspondence to: Choon-Peng Chng, Bioinformatics Institute, 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Singapore. E-mail: [email protected] Received 21 May 2014; Revised 25 August 2014; Accepted 7 September 2014 Published online 11 September 2014 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.24688

C 2014 WILEY PERIODICALS, INC. V

Apo-SOD1 Binds Lipids Through Flexible Loops

bond in metal-depleted forms of the enzyme is known to destabilise the dimer and promote monomerization,8 an event that may induce self-aggregation.9 Monomerization of the FALS A4V mutant was abolished by engineering a disulphide bond between the two monomers.10 These observations and others have led to the widespread belief that all cases of ALS share some common mechanism of pathology: metal-depleted reduced SOD1, through inherited mutations or other destabilizing causes, could become toxic through misfolding, unfolding, and aggregation.11,12 SOD1 was found to associate with cell membranes, possibly related to the radioprotective function of the enzyme.13 Kelly and co-workers found that both active, holo-(metallated) and inactive, apo-(demetallated) forms of the enzyme are embedded within phospholipid bilayers,14 but spin labelling data suggested that the apoform disorders the bilayer to a larger extent. This was suggested to be due to a structural change upon loss of copper and zinc ions that exposes additional hydrophobic residues on the enzyme surface, and that the structural change is reversible upon reconstitution of the ions.14 On the other hand, there is evidence that amphiphilic molecules could induce the cytotoxic aggregation of SOD1.15,16 Takahashi and co-workers found that long chain unsaturated fatty acid binding may change SOD1 conformation to favour aggregation. Both wild-type (WT) apo- and mutant SOD1 showed high propensity to bind fatty acids but not WT holo-SOD1.15 It is suggested that fatty acid binding alters SOD1 conformation to form oligomers. Yi and co-workers suggested that neutral phospholipid molecules might act as a promoter to accelerate SOD1 aggregation under physiological conditions, dependent on SOD1 stability and lipid-binding affinity.16 Both apo-SOD1 dimers and SOD1 aggregates showed a higher propensity for aggregation with lipids than holo-SOD1 or control protein. Circular dichroism spectra indicated that apo-SOD1 retains a b-sheet structure whereas apo-SOD1 incubated with POPC lipid vesicles is amorphous in structure, implying that lipids accelerated apo-SOD1 unfolding.16 The authors further showed that SOD1 aggregates caused defects within cell membranes by co-aggregation with lipids.17 providing an explanation for the cellular toxicity of SOD1 aggregates. Patch clamp measurements of membrane permeability found that both WT and A4V mutant apo-SOD1s slightly perturbed the membrane, possibly through binding to membrane and co-aggregation with lipids, but with no significant changes for holo-SOD1. The A4V apo-SOD1 aggregate produced the largest changes to the permeability, possibly also by formation of defects. Structural disorder and surface hydrophobicity, which increases upon partial unfolding, of the SOD1 aggregates were suggested to be responsible for the membrane perturbation.17

Although partial unfolding likely accelerates amphiphile binding to buried hydrophobic residues, several surface-exposed hydrophobic groups on SOD1 dimer might also bind amphiphiles. Furthermore, polar residue side-chains on the surface (Lys and Arg) may also attract amphiphiles through electrostatic interactions. These observations led us to examine if holo-SOD1 could bind amphiphiles to some degree and whether there is a difference in amphiphile binding propensity between holoand (natively folded) reduced apo-SOD1. Furthermore, could the reduced apo-SOD1 monomer, known to be prone to aggregation,9 bind to amphiphiles like fatty acids or phospholipids while being essentially natively folded? Recently the cell membrane was proposed to play a scaffolding role to facilitate interaction between copper delivery chaperone and apo-SOD1 for the transfer of copper ion to SOD1.18 The copper chaperone protein (CCS) catalyses SOD1 maturation in a stepwise manner, via Cu11 loading and subsequent formation of the Cys57–Cys146 disulphide bond. The efficiency of the Cu transfer is increased when SOD1–CCS interaction occurs with the Zn21 loaded reduced SOD1 monomer.19 The full structure of human CCS is unknown but comprises three distinct domains, one of which, domain II (residues 86–234), has 50% sequence homology to SOD1 and has been solved to 2.75A˚.20 Domain II is involved in formation of the SOD1–CCS heterodimer and is involved in copper loading to SOD1. Membrane-binding assays indicated that apo-SOD1 and preformed SOD1–CCS complex could associate with liposomes, reducing the search space of CCS for SOD1 and greatly increasing the efficiency.18 Hence, there is a clear functional relevance to understand how apo-SOD1 monomers associate with phospholipid membranes, as well as with CCS alone, for which there have been a number of studies.21,22 In this article, we focus on the common pathogenic form of SOD1, the disulphide-reduced apo-enzyme whose structure is not easily accessible to experiment, as well as the NMR-derived23 oxidised apo-monomer. We seek to answer the above questions by using molecular dynamics (MD) simulations to reveal detailed, atomiclevel interactions between apo-SOD1 dimer/monomer and 1-octanol molecules (a typical amphiphile). We also investigate the interaction of an apo-SOD1 monomer with a DMPC-cholesterol lipid bilayer. We found that natively folded apo-SOD1 monomer could bind to octanol clusters at the hydrophobic dimerization interface exposed after dissociation of the apo-SOD1 dimer, which is not surprising. What is unexpected is that flexible electrostatic and zinc-binding loops (characterized previously by NMR23,24 and MD simulation25,26 studies) in apoSOD1 dimer also showed an ability to bind octanol clusters and the lipid bilayer surface. Restriction of the loop flexibility to mimic holo-SOD1 abolishes amphiphile binding. Disordered electrostatic and zinc-binding loops PROTEINS

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may thus function as membrane-interacting loops allowing apo-SOD1 dimers/monomers to associate with the cell membrane to co-localize with copper chaperone on for the transfer of copper.18 The same loops may also orientate apo-SOD1 monomers/aggregates on the membrane surface to act as nucleation seeds to accelerate SOD1 aggregation.

MATERIALS AND METHODS Reduced apo-SOD1 dimer structure

The starting point for all simulations in this article is the reduced apo-SOD1 dimer structure prepared by Dr Chin W. Yong (unpublished data) from the Computational Science and Engineering Department, Daresbury Laboratory, United Kingdom. Briefly, all metal ions from PDB structure 2VOA were removed. Disulphide-bridges in the PDB structure were omitted for apo-form. After energy minimization and solvation with TIP3P water, the system was equilibrated under NVE ensemble MD for 160 ps and then switched to the NPT ensemble using the Nose-Hoover formalism, with temperature increased from 150 K to 300 K over 300 ps at 1 atm. The system was then further equilibrated at 300 K for 300 ps. CHARMM22 force-field was used with the DL_POLY 4 simulation package. The resultant reduced apo-SOD1 dimer conformation is taken as the reference structure in this article. MD simulations of SOD1 dimer in 1-octanol solvent

To search for plausible amphiphile binding pockets on the SOD1 dimer surface, we have simulated SOD1 dimer in pure 1-octanol solvent. But rather than obtaining a simulation box of equilibrated octanol molecules and try to create space for the dimer, we took the approach of adding octanol molecules into the simulation box until the SOD1 dimer surface was wrapped. The SOD1 dimer was placed into a periodic, octahedron box with the minimum distance between the protein and edges of the box being 1.5 nm. Three hundred octanol molecules were placed randomly in the empty space around the dimer, in addition to 12 sodium ions for charge neutralization. Positional restraints were imposed on the heavy atoms of the protein during steepest descent energy minimization, followed by NVT-MD for 100 ps at 100 K using Velocity-Rescale thermostat27 with time-constant of 0.1 ps. Most of the octanol molecules were observed to adsorb onto the SOD1 dimer surface, as would be expected in a vacuum simulation. NPT-MD was next performed for 100 ps at 100 K and 1 atmospheric pressure using Berendsen barostat28 NPTMD was performed to allow the simulation box to contract and reduce the amount of empty space. The system

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temperature was then increased in stages: 200 K for 100 ps and 300 K for 500 ps. The process was then repeated by enlarging the system box and adding 300 more octanol molecules (Stage 2), 485 more (Stage 3), and 400 more (Stage 4). The production run (Stage 5) was then performed under NPT-MD at 300 K with the ParrinelloRahman barostat29 (time constant of 1 ps; compressibility assumed to be 4.6 3 1025 bar21 as for water at 300 K and 1 atm) for 10 ns. Positional restraints on the SOD1 dimer were turned off during 300 K simulations of stages 4 and 5. The system density during stage-5 simulation was stable at 877 kg/m3 (1-octanol and SOD1 dimer). This is close to the experimental value of 825 kg/m3 at 298 K provided by ChemSpider webserver and Generalized Amber Force-field value of 834 kg/m3 (VirtualChemistry.org). The Particle-Mesh-Ewald (PME) sum method30 was used for all electrostatic calculations with a cutoff distance of 1.4 nm during NVT-MD and NPT-MD runs for stage 1. Subsequent stages used different values of cutoff during 300 K NPT-MD simulations: 2.2 nm (stage 2), 1.8 nm (stages 3 and 4), and 1.0 nm (stage 5). van der Waals interactions were calculated using a Lennard-Jones potential with potential switching to zero between 0.8 and 0.9 nm (stages 1 and 2) but truncated to zero at the same PME distance cutoff for stages 3 to 5. Neighbourlist cut-offs were the same as the PME cut-offs. The simulation time-step was 2 fs, with bonds constrained by the LINear Constraint solver.31,32 The AMBER99SB-ILDN force-field33 was used to describe interactions involving SOD1. Topology files for 1-octanol for AMBER99SB force-field in GROMACS were generated using the AnteChamber PYthon Parser interfacE34 that used AnteChamber in AmberTools.35,36 The Gromacs 4.6.x package37,38 was used for all simulations and analysis (unless otherwise stated) in this article. At the end of the 10 ns production run, SOD1 dimer together with octanol molecules that are found to be within 0.9 nm of the dimer interface residues Lys9, Ile113 and Val148 were extracted from the simulation snapshot for follow-up simulation in water. MD simulations of SOD1 dimer in water with 1-octanol clusters

SOD1 dimer with bound 1-octanol molecules (49 molecules) from 1-octanol solvent simulation above was solvated with 13,785 TIP3P39 water molecules and charge-neutralized with 12 sodium ions in the same periodic simulation box. With SOD1 heavy atoms restrained (harmonic potential with force constant of 1000 kJ/mol), energy minimization using steepest descent was performed, followed by 1 ns of NPT-MD. Restraints were then removed for production run of 100 ns. PME sum method30 was used for all electrostatic interactions with cut-off distance of 1 nm. van der Waals (vdW)

Apo-SOD1 Binds Lipids Through Flexible Loops

interactions were also cut-off at the same distance for using Verlet cut-off scheme. For the simulation of SOD1 dimer with 1-octanol molecules dispersed in water, 60 octanol molecules were randomly placed in an octahedron simulation box in the space between reference apo-SOD1 dimer and simulation box edges. The simulation box volume is 767 nm3, which translates to a molar concentration of 0.13 M for the octanol molecules. Inter-atomic vdW distances were temporarily increased from default of 0.105 nm to 0.2 nm to avoid overlaps. The box was filled with 23386 TIP3P water molecules and charge neutralized. Positional restraints were imposed on heavy atoms of SOD1. After energy minimization, NVT-MD at 100 K was performed for 100 ps. PME cut-off of 1.4 nm was used, and vdW interactions switched to zero from 0.8 to 0.9 nm. Next, NPT-MD at 100 K and 1 atm was performed for 100 ps. The temperature was increased to 300 K in steps of 100 K over 100 ps intervals. For the 300 K simulation, both electrostatic and vdW interactions were cut-off at 1 nm. A production run without positional restraints on the SOD1 dimer was then performed for 30 ns with the same parameters. To test for any force-field dependence of the observed binding of SOD1 to octanol, the interaction between freely dispersed octanol molecules and the unrestrained SOD1 dimer was simulated using the OPLS-AA force-field.40 Gromacs-compatible parameters for 1-octanol created by Caleman and co-workers40–42 were used, downloaded from the Lipidbook repository43 hosted at the University of Oxford. The parameters have been validated against experimental values (http://virtualchemistry.org/molecules/111-87-5/ index.php). The same equilibration protocol as for the AMBER force-field was used. Octanol clusters were observed to bind to electrostatic and Zn loops as for the AMBER force-field, though taking longer simulation times. For the simulation with one monomer restrained, the positional-restrained dimer structure before the production run above was taken as the new reference structure. Positional restraints were then imposed on the Ca atoms of one of the monomers during production NPT run at 300 K and 1 atm for 52 ns. The unrestrained monomer of the 52 ns structure was then extracted together with a 0.3 nm neighbourhood of bound water for the simulation with lipid bilayer below. Simulations were also carried out with a single apoSOD1 monomer in an octahedron simulation box with 60 octanol molecules randomly placed around it. The same protocol as for unrestrained dimers above was used. In all simulations above, a Velocity-rescale thermostat27 with time-constant of 0.1 ps was used. The Berendsen barostat28 with time-constant of 1 ps maintained the system pressure. The simulation time-step was

2 fs, with all bond lengths constrained by the LINear Constraint solver.31,32 MD simulations of SOD1 monomer with DMPC-Cholesterol lipid bilayer

The coordinates of an equilibrated DMPC-cholesterol bilayer patch (45 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 19 cholesterol molecules in each monolayer, with 30% fraction of cholesterol) were obtained from the Stockholm Lipids database (http://people.su.se/jjm/ Stockholm_Lipids/Downloads.html). AMBER-compatible DMPC and cholesterol parameters from Stockholm Lipids force-field are provided by Joakim J€ambeck.44–46 Compatibility with AMBER amino acid force-fields have been tested in simulations of WALP23 peptide with DLPC and DOPC bilayers.45 The SOD1 monomer with bound water obtained above was placed in a water box and heated from 100 K to 300 K following the same procedure with positional restraints. The structure at 500 ps of the 300 K simulation was extracted with 0.3 nm of bound water for placement just above the DMPC-cholesterol lipid bilayer, using the visual molecular dynamics (VMD) program. The minimum separation between SOD1 monomer atoms and lipid bilayer head-group phosphate atoms is 1.3 nm. Positional restraints were imposed on the SOD1 monomer heavy atoms during 100 ps NVT-MD at 100 K, and 100 ps NPT-MD at 100 K, 200 K and 300 K. With the temperature at 300 K, positional restraints were then slowly relaxed by first only imposing them on protein Ca atoms for 100 ps, and then reducing the restraints on Ca atoms to 100 kJ/mol for a further 100 ps before being turned off completely for the production run. Pressure of 1 atm was maintained using a Berendsen barostat with semi-isotropic coupling (Z separate from X–Y directions). Both electrostatic and vdW interactions were cut-off at 1.2 nm. VMD was also used for visualization and rendering of simulation trajectories as well as computation of hydrogen-bonds.47 Solution structures of apo-SOD monomers (with F50E/G51E/E133Q triple mutation) determined by NMR spectroscopy (PDB ID 1RK7) were also used for simulation with the DMPC-cholesterol lipid bilayer. Solvent accessible surface areas (SASA) of each of the 30 conformers were computed using VMD’s “measure sasa” function and conformers number 3 and 11 with low and high SASA respectively were selected as representative structures for simulation. The NMR monomers were least-squares-fitted onto the holo-dimer derived monomer structure, placed above the DMPC-cholesterol lipid bilayer to have the same starting point as before. Reverse amino acid substitutions (E50F/E51G/Q133E) were carried out to obtain the WT monomers, using MMTSB48 mutate.pl program. Identical simulation procedures as above were then followed. PROTEINS

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Figure 1 Overview of SOD1 dimer 3D structure. (Top) Cysteine residues are represented as spheres on the cartoon representation (left). Amino acid residue side-chains are colour coded on the surface representation (middle) as follows: white for hydrophobic, green for polar, blue for basic and red for acidic. Electrostatic potential map calculated by APBS is shown on the right. (Bottom) Electrostatic loops (residues 121–142) are coloured red. Zn loops (residues 50 to 83) are coloured green. Hydrophobic, basic, and acidic side-chains are shown as spheres. Basic and acidic side-chains on Znbinding loop are coloured in lighter shades. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

RESULTS Reduced apo-SOD1 dimer binds 1-octanol molecules via hydrophobic and basic surface residues

An overview of the SOD1 dimer 3D structure and the distribution of hydrophobic, basic and acidic residue side-chains on its surface is shown in Figure 1. Each SOD1 monomer consists of a b-barrel with long electrostatic and zinc-binding loops (hereafter Zn-loops) between b-strands. Solvent-exposed hydrophobic residues are found not only at the dimer interface but also on the exposed face of the b-barrel and (to a lesser extent), on the electrostatic and Zn-loops. Basic (or positively charged) side-chains are found on the flanks of the b-sheet (see blue areas on the surface representation in Fig. 1) as well as concentrated in the area between the electrostatic and Zn-loops. Acidic (negatively charged) side-chains are generally located close to basic side-chains for charge neutralization. Basic sidechains slightly outnumber acidic ones on the Zn-loop, whereas the numbers are similar on the electrostatic loop. Nevertheless, we could not deduce from the charge distribution or electrostatic potential (calculated using APBS49 Plugin v1.3 in VMD47 with default parameters) (see Fig. 1) that SOD1 would associate with phospholipid bilayers because of the lack of a positively charged surface patch.

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Simulation of demetallated and disulphide-bridge reduced apo-SOD1 was carried out in 1-octanol solvent to detect amphiphile binding pockets on the SOD1 dimer surface. In particular, we wish to test if the solvent-exposed surfaces of the hydrophobic dimerization interface in the intact dimer could bind amphiphiles through hydrophobic interactions. Octanol molecules were added in stages to allow condensation onto the SOD1 dimer surface (Fig. 2). After 10 ns of MD simulation, octanol molecules within 0.9 nm of the dimer interface residues (Lys9, Ile113 and Val148) were extracted from the simulation snapshot and used for the subsequent simulations of SOD1-octanol complexes. The “top” dimer interface opening (in the 0 ns snapshot in Fig. 2) is larger than the “bottom” one, which explains the asymmetric octanol clusters obtained. Such clusters would also be obtained for metallated, disulphidebridged holo-SOD1, as protein backbone flexibility is very limited in the concentrated 1-octanol solvent. The extracted SOD1-octanol complex was then surrounded by water molecules and simulated for 100 ns (Fig. 2), to determine if the octanol cluster could remain bound to SOD1 in an aqueous environment. During the simulation, the SOD1 dimer remained intact and Cys57–Cys146 distance (the reduced disulphide-bridge location) did not become significantly larger (data not shown). The simulation showed that the cluster remained bound but shifted away from the dimer interface to overlap with the exposed b-sheet of one monomer. This suggests that the

Apo-SOD1 Binds Lipids Through Flexible Loops

Figure 2 Molecular dynamics simulation snapshots of disulphide-reduced, apo-SOD1 dimer with bound octanol clusters at the dimer interface in water. Largest cluster shifted during the simulation to associate with the beta-barrel surface of one of the monomers through surface-exposed hydrophobic and positively charged Lys residues. Water molecules are omitted for clarity. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

indentations surrounding the dimer interface would likely not be the primary binding site for amphiphiles in the intact dimer. Hydrophobic residues Val5, Val7, Ile17, and Trp32 are present on the face of the b-sheet that could bind the aliphatic chains of octanol molecules. In addition, basic residues flank the two edges of the b-sheet: Lys9, Lys36 on one edge (“top” blue spheres in the 100 ns snapshot, Fig. 2); and Lys3, Lys23, and Lys30 on the opposite edge (“bottom” blue spheres). Compared with the “top,” the “bottom” edge of the b-sheet also contains acidic residues Glu21 and Glu100, thus reducing the electropositive nature of the “bottom” edge. Hence, the cluster remains bound to the “top” edge of the b-sheet with hydrogenbonds formed between AOH groups of octanol molecules and ANH3 groups of Lys residues. Besides polar interactions, the four-carbon aliphatic side-chains of Lys residues also interacted favourably with the aliphatic chains of octanol molecules through hydrophobic packing. This indicates that lysines are key residues in binding amphiphiles/amphiphile clusters onto the SOD1 surface. Electrostatic and Zn loops of reduced apoSOD1 dimers are important in the binding of 1-octanol clusters in aqueous solution

To locate other amphiphile binding sites on the apoSOD1 dimer surface, we have performed simulations of SOD1 with 60 molecules of 1-octanol dispersed in water

(Fig. 3). Such self-assembly simulation would indicate whether octanol molecules in low concentration could still bind to SOD1, as individual molecules or clusters. It took only 5 ns of simulation to observe clustering of octanol molecules onto the electrostatic and Zn-loops of each monomer (Fig. 3). Both loops contain hydrophobic and basic (Lys, Arg, His) side-chains that interacted favorably with the hydrophobic and polar portions of octanol molecules, suggesting that amphiphiles preferentially bind at the loop regions relative to the core b-barrel surface. Although acidic side-chains are also present on the electrostatic and Zn-loops, most of the polar interactions with octanol molecules were observed to involve the basic side-chains. Nevertheless, it is again a combination of hydrophobic and polar interactions that bind octanol molecules to the SOD1 surface. This was confirmed not to be force-field sensitive, with similar findings from simulations using the OPLS-AA force-field (Supporting Information Fig. S1). The Cys57–Cys146 separations for each monomer remained close to starting values of 0.6 nm but could reach 0.9 nm (for monomer B) during the later parts of the simulation (Supporting Information Fig. S2). Such fluctuations can be attributed to thermal agitation of the free Zn-loop. The observation that both monomers bind to octanol clusters suggest that the reduction of the Cys57–Cys146 disulphide bond is likely not important to the flexibility of the electrostatic and Zn-loops. This is corroborated by findings by Ding and Dokholyan using discrete MD simulations.26 PROTEINS

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Figure 3 Molecular dynamics simulation snapshots of reduced, apo-SOD1 dimer with 60 1-octanol molecules dispersed in water. Octanol molecules cluster rapidly onto electrostatic and Zn loops (5 ns snapshot). Periodic boundary images are shown in 30 ns snapshot to display complete SOD1-octanol clusters. Octanol molecules are omitted in zoomed-in insets for the 30 ns snapshot. SOD1 side-chains on electrostatic and Zn loops interacting with octanol molecules are shown as sticks. Electrostatic loop of the left (L) monomer interacts with two octanol clusters, hence the two subfigures. Lys136 on the electrostatic loop of left monomer forms a hydrogen bond to octanol molecule (right subfigure). His63 and His71 on Zn loops on the left and right (R) monomers hydrogen-bonds to octanol molecules in the respective clusters. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Flexibility of electrostatic and Zn loops drives reduced apo-SOD1 dimer binding to 1-octanol clusters

Simulations were performed to establish whether the disulphide-bridged holo-SOD1 dimer would also bind octanol molecules. Previously, simulations of disulphide-

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bridged holo-SOD1 dimers with Cu and Zn ions covalently bonded to ligating amino acid residues were carried out by Strange and co-workers using the CHARMM force-field. These showed that the loops are tightly constrained in presence of metals.25 Rather than repeat this step and commit to additional simulation time, we note

Apo-SOD1 Binds Lipids Through Flexible Loops

Figure 4 “Two-face” simulation of reduced, apo-SOD1 dimer with restrained flexibility on one monomer. Selected snapshots along MD simulation trajectory are shown. The 60 1-octanol molecules randomly dispersed around the SOD1 dimer formed two clusters bound on the flexible monomer. On the 52 ns snapshot: (i) hydrophobic residues within 0.3 nm of octanol molecules are shown as gray spheres, (ii) positively-charged residues within 0.3 nm of octanol molecules are shown as blue spheres. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

that restraining the backbone of the whole subunit in a manner know from the previous modeling achieves a similar effect at much lower computational cost. From the simulations, we found that almost all octanol molecules clustered onto the unrestrained monomer within 10 ns (Fig. 4). Specifically, a cluster first formed on the flexible, electrostatic and Zn loop regions of the unrestrained monomer within 3 ns. Another cluster near the dimer interface on the b-sheet face (but centred on the unrestrained dimer) appeared within the next 7 ns.

In contrast, only single octanol molecules were bound to the restrained monomer. Both restrained and unrestrained monomers showed similar SASA (Supporting Information Fig. S3), indicating that the core b-barrel of the monomers remained native-like during the simulations. A repeated simulation with a different initial velocity distribution produced similar outcomes, with octanol clustering occurring on the electrostatic and Zn- loops of the unrestrained monomer (Supporting Information Fig. S4). In contrast to the first simulation, no clustering PROTEINS

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occurred on the b-sheet surface. A floating octanol cluster was observed to first associate transiently (contact lasting for 6 ns) with the restrained monomer b-sheet before migrating over to the b-sheet of the unrestrained monomer (50 ns snapshot in Supporting Information Fig. S4). Taking the 52 ns snapshot from the first simulation and swapping the conformational restraints of the two monomers, we observed detachment of the octanol cluster from the newly restrained electrostatic and Zn- loops (Supporting Information Fig. S5). On the other hand, the cluster on the b-sheet face remained intact for the simulation duration of 40 ns. Side-chain, rather than backbone, flexibility is likely sufficient to engage the octanol clusters in this case. The electrostatic loop of the unrestrained monomer was observed to approach the octanol cluster and interact with it via a Lys side-chain from 27 ns. The configuration remains similar at 40 ns, though the octanol cluster had spread slightly over to the unrestrained monomer to interact with hydrophobic residues on the Zn-loop. Together with the data presented above, our simulations strongly suggest that backbone flexibility in the electrostatic and Zn loops (and the loops flanking the b-sheet to a smaller extent) is required to drive loop binding to amphiphilic molecules. Reduced apo-SOD1 monomer adsorbs onto lipid bilayer surface via electrostatic and Zn loops

Although 1-octanol might be a good representation of an amphiphilic molecule, to understand SOD1 binding to cell membranes requires studying SOD1 binding to phospholipid bilayers. We wish to determine whether a SOD1 monomer would adsorb onto the lipid bilayer surface using the hydrophobic dimer interface, hydrophobic groups on the b-sheet surface or hydrophobic/polar groups on the electrostatic and Zn loops. Costa and coworkers have found that SOD molecules could partition into the interior of a mixed DPPC-cholesterolstearylamine (6:3:1 or 7:2:1) bilayer using a modified Langmuir monolayer relaxation study.50 As DPPCcholesterol topologies were not available from the Stockholm Lipids database, DMPC (same head-group as DPPC)-cholesterol bilayer with 30% cholesterol was used instead. Attempts to obtain a stable DPPC-cholesterol bilayer created by the CHARMM-GUI webserver (http:// www.charmm-gui.org/) were not successful (data not shown). However, it turned out that no interaction of SOD1 with the embedded cholesterol was observed in our simulations, possibly due to the relatively short, nanosecond time-scales of our simulations. By performing the simulation at a slightly elevated temperature of 330 K, the lipids are in the fluid phase and protein dynamics occurs faster than at 300 K (where no adsorption events were observed within 30 ns). We found

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that the SOD1 monomer could adsorb onto DMPC surface (after free tumbling above the bilayer) using its Zn loop (and electrostatic loop to a lesser degree) [Fig. 5(A)]. The Zn-loop contains a positively charged side-chain (Arg69) that forms up to three strong H-bonds to a negatively charged DMPC phosphate ion [Supporting Information Fig. S6(A)], and the neighbouring hydrophobic side-chain (Leu67) attracts the aliphatic -CH3 groups on the DMPC head group. Transient H-bonds to lipid head groups involving Lys136 on electrostatic loop is responsible for bringing the loop closer to the bilayer surface [Supporting Information Fig. S6(A)]. The b-sheet with solvent-exposed hydrophobic residues such as Trp32, did not face the lipid bilayer surface any time during the simulation. These calculations show that the Zn loop is mechanistically important for the observed co-aggregation of SOD1 with phospholipids.16,17 Increasing the simulation temperature by 30 K expanded the SOD1 dimer (mean radius of gyration increased to 1.44 nm from 1.42 nm) and made hydrophobic residues more solvent accessible (an increase of 0.7 nm2) (Supporting Information Fig. S7). It is thus possible that the increase in conformational sampling of the electrostatic and Zn- loops and thus larger solvent accessibility enabled the SOD1 monomer to interact more favorably with hydrophilic and hydrophobic parts of phospholipids in the bilayer. However, adsorption is stable for only around 15 ns out of total simulation time of 60 ns, probably due to diffusive forces acting on the monomer from the solvent at the elevated temperature. Temperature-quench simulations at 300 K starting from the 14.7 ns snapshot from the 330 K simulation suggests that the monomer could be stably adsorbed on the lipid bilayer surface via the Zn-loop [Fig. 5(C)] for at least 44 ns. Both Arg69 and Leu67 on the Zn-loop remained within 0.4 nm of the phosphate ion during periods when the Zn-loop is within 0.25 nm of the DMPC head-groups [Fig. 5(D)]. The H-bonds between SOD1 monomer and DMPC head-group phosphate switches from those mediated by Arg69 side-chain on the Zn-loop (0–20 ns, 30–35 ns, 40–44 ns) to those mediated by Ser25 and Asn26 side-chains on one of the loops flanking the beta-barrel (20–30 ns, 35–44 ns) [compare Fig. 5(C) with Supporting Information Fig. S6(B)]. The radius of gyration during T-quench did not reduce to 300 K values and the hydrophobic side-chains actually became slightly more solvent exposed (Supporting Information Fig. S7). These seem to (weakly) suggest that SOD1 monomer became slightly denatured during its time on the bilayer surface. The relatively transient nature of the adsorption is expected because the monomer could not embed into the bilayer interior without extensive hydrophobic surfaces. Perhaps a cluster of apoSOD1 monomers/dimers or higher-order oligomer might be able to maintain the adsorption for longer simulation times.

Apo-SOD1 Binds Lipids Through Flexible Loops

Figure 5 Adsorption of reduced, apo-SOD1 monomer onto lipid bilayer patch. (A) Minimum separation between SOD1 monomer (centre of mass of whole monomer, flexible loops or single residue) and head-group phosphorous atoms of a DMPC-cholesterol lipid bilayer during 330 K simulation. (B) Simulation snapshot at 14.7 ns [marked by X in (A)], where SOD1 monomer adsorbs onto the DMPC-cholesterol bilayer. Cholesterol molecules are shown as orange spheres. Electrostatic loop is shown in red and Zn loop in green. (C) Minimum separation during temperature-quench (Tquench) simulation at 300 K starting from 14.7 ns snapshot at 330 K. (D) Side-chain atoms of residues Leu67 and Arg69 on Zn-loop mediates the interaction of SOD1 with DMPC head-groups during T-quench simulation. Arg69 side-chain is within hydrogen-bonding distance to polar DMPC head-group atoms. Leu67 side-chain packs against apolar DMPC head-group atoms.

Root-mean-squared-fluctuation plots suggest that both electrostatic and Zn loops gained considerable flexibility at 330 K relative to 300 K (Supporting Information Fig. S8). In particular, the N-terminal section of the Zn-loop showed a drastic increase in flexibility, due to Cys57–Cys146 backbone Ca separation before 25 ns (Supporting Information Fig. S9). The range of this separation (0.5–0.6 nm) for 300 K, 330 K (before 25 ns) and T-quench simulations is similar to that seen for each monomer in the dimer simulation with free octanol molecules (Supporting Information Fig. S2). T-quenching the 14.7 ns monomer from 330 K to 300 K maintained the Cys57–Cys146 separation at 300 K value and reduced the corresponding RMSF (Supporting Information

Figs. S8 and S9). Nevertheless, the RMSFs of Zn- and electrostatic loops are still slightly larger than in the unbound 300 K simulation, especially for the Zn loop (Supporting Information Fig. S8). This was due to fluctuations of the loop segment involving residues Ala55 to Cys57. NMR derived apo-SOD1 monomers also adsorb onto the lipid bilayer surface via electrostatic and Zn loops

The SOD1 monomer structure used as the starting point in the above simulations was extracted and rebuilt as disulphide-reduced from the holo-dimer. To confirm PROTEINS

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that it is representative of the experimental apo-SOD1 monomer, and show that the loop conformations are adequately sampled in our MD simulations, additional simulations were carried out using the experimental NMR structures of monomeric apo-SOD1, which was engineered with F50E/G51E/E133Q mutations.23 The first two mutations substituted residues at the dimer interface to create soluble monomers, while the Glu133Gln mutation was carried out to partially restore the enzymatic activity. In these NMR structures, the Cys57–Cys146 disulphide bond was intact. We chose two representative conformers with small (denoted “compact”) and large (denoted “expanded”) SASA (Fig. 6). These structures were reverted to the WT sequence via amino acid substitutions. Simulations carried out at 330 K suggest that both conformers could adsorb onto the lipid bilayer after 25 ns (“compact”) or 30 ns (“expanded”). No correlation was found between the radius of gyration and SASA of the monomers and their location relative to the bilayer surface (Supporting Information Fig. S10). Essentially no significant structural changes occur to the monomers upon bilayer surface binding. Nevertheless, both the radius of gyration and the SASA (hydrophobic, hydrophilic and total) are larger than values obtained above using holo-dimerderived monomer (Supporting Information Fig. S7), indicating that the conformations are more “open.” The simulations thus confirm that both Zn and electrostatic loops contribute to the binding of apo-SOD1 monomers to lipid head-group atoms (Fig. 6). Of particular note is the positively charged Arg69 on the Zn-loop that can form multiple H-bonds with lipid head groups. The side-chain contributed to lipid binding in “compact” monomer simulation even though the Zn-loop is not as close to the bilayer surface as the electrostatic loop. Lys128 on the electrostatic loop was found to contribute to lipid binding in the “compact” monomer simulation (Supporting Information Fig. S11). But in the case of the “expanded” monomer, Lys136 is closer to the bilayer surface. Both the radius of gyration and SASA of the “compact” and “expanded” monomers are larger than the monomer extracted from the apo-dimer as presented in the section above (compare Supporting Information Fig. S10 with Supporting Information Fig. S7 middle plots). This is no surprise given the more disordered loops of the NMR monomers. We have also performed additional simulation runs for both NMR monomers (Supporting Information Fig. S12) and found that although the overall orientations of the monomers on the bilayer surface vary from the first set of runs, the electrostatic and/or Zn loops still contribute significantly to lipid binding, as shown by the interaction energy plots (Supporting Information Fig. S13). These orientations (shown as insets to Supporting Information Fig. S12) are metastable states with lower interaction energies between protein and lipid head-group phosphate atoms.

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DISCUSSION SOD1 electrostatic loop (loop VII, residues 121–142) guides the negatively charged superoxide substrate toward the catalytic copper site, whereas the Zn-loop (loop IV, residues 50–83) contains the zinc-binding residues. Our MD simulations suggest that both holo- and apo-SOD1 dimers could bind octanol molecules (a typical amphiphile) as individuals or clusters through polar and hydrophobic residues on the solvent-exposed b-barrel face. But it is the “activation” (increase in flexibility) of the electrostatic and Zn-loops in each apo-SOD1 monomer that allows the dimer to bind to additional octanol clusters, as restraining the backbone atoms of the loops to the (holo-) structure made the loops unattractive to octanol molecules/clusters. It appears that enhanced flexibility enables the loops to adapt readily to the constantly changing topography of amphiphile cluster (and phospholipid bilayer surface, see below), thus maintaining favorable interactions. NMR studies23,24 and MD simulations25,26 have observed increased flexibility of these two loops in WT apo-SOD1 relative to holo-SOD1. In particular, Ding and Dokholyan used enhanced conformational sampling methods on discrete MD simulations to reveal enhanced flexibility for electrostatic and Zn- loops in apo-SOD1 monomers with and without the Cys57–Cys146 disulphide bond. On the other hand, flexibility of the Znloop is diminished in the presence of bound Cu and Zn ions regardless of the disulphide-bond.26 These findings agreed with our observations that the Cys57-Cys146 separation does not affect octanol cluster binding by the electrostatic and Zn-loops. The preference of amphiphiles for the disordered loops over the b-barrel face observed in our simulations signifies that the rate of amphiphile binding for natively folded apo-SOD1 dimers would be higher than that of holo-SOD1 dimers. The 3D “epitope” of the disordered loops (on apo-SOD1) possibly allows for binding of larger clusters compared with the 2D “epitope” of the bbarrel surface (on apo- and holo-SOD1). These amphiphile clusters may then act as nucleation seeds to attract more apo-SOD1 dimers via their disordered loops and apo-SOD1 monomers via exposed dimerization interface (see below), eventually forming large and dispersed SOD1-amphiphile aggregates as reported in experiments.15,16 Future experiments and/or coarse-grained MD simulations involving multiple copies of SOD1 may be able to test the above hypothesis. The separation of destabilised, disulphide-reduced apo-SOD1 dimers into monomers8 would be expected to increase the hydrophobic surface area (by exposing hydrophobic residues at the dimerization interface to solvent). No separation of apo-SOD1 dimers into monomers was observed during our MD simulation, possibly due to the short, nanosecond time-scale studied.

Figure 6 Simulations of apo-SOD1 monomer adsorption onto lipid bilayer patch using F50E/G51E/E133Q engineered NMR conformers from PDB ID 1RK7. Two conformers denoted “compact” and “expanded,” were chosen from the 30 available models in the PDB file based on their SASA, and then reverted to the WT sequence. Minimum separations between centres-of-mass of protein, flexible loops or Trp32 and the DMPC head-group phosphorous atoms were monitored during elevated temperature simulations. Zoomed-in snapshots show SOD1 side-chains interacting with the lipid bilayer (within 5A˚ of DMPC lipids). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

C.-P. Chng and R.W. Strange

Simulation of the apo-SOD1 monomer in solution with amphiphiles was carried out and amphiphiles were indeed attracted to the dimerization interface (Supporting Information Fig. S14). This suggested that a natively folded apo-SOD1 monomer could bind amphiphile clusters at the hydrophobic dimerization interface, which is not that surprising. However, in dimer form with the interface buried, the disordered electrostatic and Zn loops becomes the preferred site of amphiphile binding. Also, under extremely high amphiphile concentration that is likely to be non-physiological, the disordered loops might become the preferred amphiphile cluster binding sites (Supporting Information Fig. S15), though more studies are needed to investigate further this potential switch in binding site preference. We thus propose that in the case of fatty acid binding, both apo-SOD1 monomers and apo-SOD1 dimers could bind to fatty acid clusters in solution via exposed dimerization interface and disordered electrostatic and Zn loops, respectively. But in the case of holo-SOD1, no monomers are present and ordered electrostatic and Zn loops do not bind to fatty acid clusters, thus explaining the difference in binding affinities for apo- versus holo-SOD1 reported.15 We also studied the interaction of the reduced apoSOD1 monomer with a phospholipid bilayer (with neutral phosphatidylcholine lipids, as in experiments14,16,17). To accelerate the adsorption process, we have performed our simulation at a slightly elevated temperature of 330 K. The monomer was observed to adsorb onto the phospholipid surface primarily through favourable electrostatic and hydrophobic interactions involving the Zn-loop (primarily Arg69 side-chain) and to a lesser extent the electrostatic loop (Lys136 side-chain). The elevated temperature might have further enhanced the conformational flexibility of the electrostatic and Zn- loops, enabling the loops to interact more favourably with hydrophilic and hydrophobic parts of phospholipid head-groups in the bilayer. It is possible that more of the basic side-chains on the Zn-loop as well as basic sidechains on the neighbouring electrostatic loop would participate in membrane binding if acidic lipids are present in the bilayer, as in the inner cell membrane. Both the hydrophobic dimerization interface and the small solvent-exposed hydrophobic patch on the b-barrel surface are not involved in bilayer surface binding. Although a partially unfolded apo-SOD1 with extensive hydrophobic surfaces might embed into the bilayer interior as suggested by various research groups,14,16,17 here we argue that the disordered, membrane-interacting loops on apoSOD1 would allow a (mostly) natively-folded monomer to associate with the phospholipid bilayer surface without any significant unfolding. Simulations initiated from NMR apo-SOD1 monomers, which better sampled the conformational flexibility of the electrostatic and Znloops, showed a similar outcome, with monomers bind-

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ing to the bilayer surface via the membrane-targeting loops. This observation strongly suggests that despite the rather different conformations of the loops among the apo-SOD1 monomers used in our study, the disordered loops are the preferred lipid bilayer-binding surface. The association of monomeric SOD1 with the bilayer surface may also be the initial event leading to eventual membrane disruption following the penetration of monomers or aggregates into the membrane interior.14,16,17 Various examples of membrane-interacting/targeting loops have been reported in the literature, the most studied of which is the phosphatidylinositol lipid binding pleckstrin homology (PH) domain of the general receptor of phosphoinositides 1 (GRP1). Sansom and coworkers combined MD, NMR spectroscopy and monolayer penetration experiments to discover that loops (containing both basic and hydrophobic residues, similar to SOD1’s electrostatic and Zn-loops) flanking the PIP3 binding site interacts with the lipid bilayer in conjunction with PIP3 binding.51 Further modelling work by Voth and co-workers found that the membrane-targeting loops bind negatively charged phosphatidylserine lipid head groups to create transient lipid defects allowing a hydrophobic Val side-chain (next to Arg and Lys sidechains) to penetrate deeply into the bilayer head-group region, stabilizing the association.52 Both Arg69 and Leu67 on the Zn-binding loop of SOD1 might play a similar membrane-anchoring role for apo-SOD1. Sitedirected mutagenesis and binding assay experiments would shed light on the importance of these two residues to the membrane-targeting ability of SOD1. Our finding that reduced apo-SOD1 monomers bind lipid bilayers through the Zn loop (and the electrostatic loop to a lesser extent) also provides a timely mechanistic explanation for how it could engage the cell membrane for the purpose of copper transfer from the membrane-bound copper chaperone, suggested by Unger and co-workers.18 The heterodimeric structure of yeast SOD1-CCS complex53 suggests that the SOD1 Zn-loop is far from the membrane surface in the complex. We therefore propose that natively folded, apoSOD1 monomers first associate with the membrane via their (membrane-interacting) loops and upon encountering the membrane-bound CCS, SOD1 monomers dissociate from the membrane and change orientation to present the dimerization interface for binding to the chaperone. The homology between CCS domain II, where the putative membrane-binding site is located, and monomeric apo-SOD1 suggests that membrane binding of CCS may occur in a similar fashion, via its charged loops rather than involve hydrophobic regions. Indeed, Unger and co-workers have shown that mutating positively charged Lys and Arg residues to Ala on the loops forming the putative membrane-binding interface of yeast CCS significantly reduced lipid binding.18

Apo-SOD1 Binds Lipids Through Flexible Loops

There have been suggestions that SOD1 binds Zn ions before Cu.19 However, to-date the source of Zn for apoSOD1 has not been identified. To rationalize our findings with existing literature, we propose the following mechanism: (i) apo-SOD1 first localizes to the inner cell membrane using flexible Zn-binding and electrostatic loops, (ii) a Zn chaperone (yet unknown) for SOD1 is assumed to exist at the inner cell membrane to transfer Zn ions to membrane-bound SOD1, (iii) this stabilizes the Znloop but does not affect the flexibility of the electrostatic loop,25 which maintains the binding of SOD1 on the membrane surface, (iv) upon encountering CCS, Cu ion transfer to SOD1 stabilises the electrostatic loop, (v) with both membrane-targeting loops stabilized, the SOD1 monomer dissociates from the inner membrane, and (vi) CCS gets replenished by association with copper transporter 1 for the next SOD1 monomer. A plausible Zn chaperone for SOD1 might be DJ-1. DJ-1 (a dimer) has been identified as a copper chaperone for SOD1 that acts through a backup, CCS-independent pathway.54 Similar to CCS, DJ-1 has also been found to associate with the cell membrane, in particular to lipid rafts.55 Furthermore, DJ-1 was also found to bind Zn(II) ions in the same binding pocket as Cu(II) ions with a higher binding affinity.56 Together, these latest findings suggest that DJ-1 might act to transfer Zn and/or Cu to SOD1 independent of or in conjunction with CCS at the cell membrane. The orientation of the SOD1 monomer to the bilayer surface leaves opens the possibility that further unfolding of the b-barrel core at strands 4 and 5, which are normally covered or protected by these loops,57 might expose more hydrophobic surface for binding phospholipid tails. Significantly, the solvent-exposed hydrophobic dimerization interface of the monomer and its b-barrel surfaces, comprising b-strands 1–3, 6, 8 and loops I, II, VI and VII, face away from the bilayer surface and thus are available for binding to additional apo-SOD1 monomers or dimers, leading to lipid-associated SOD1 aggregate formation. In support of this aggregation mechanism, we note that these same exposed structural elements (Supporting Information Fig. S16) correspond exactly to the core regions of SOD1 protein fibrillar aggregation that have been experimentally identified.58 Furthermore, the solvent-exposed Trp32 on b-strand 3, that can promote SOD1 self-aggregation,59,60 is on a region of the b-barrel that exhibits partial unfolding in ALS mutants61 and has already been confirmed as a small molecule binding site in intervention strategies directed against protein aggregation in ALS.62,63 The cell membrane surface has been proposed to facilitate formation of aggregates/fibrils from amyloidogenic proteins or peptides (see review by Relini et al.64). A paralog of the prion protein has been shown to bind to and aggregate on anionic lipid vesicles, leading to vesicle disruption and formation of supramolecular lipo-protein

complexes.65 Thus, the MD simulations suggest that apo-SOD1 monomers on the lipid bilayer surface may form nuclei that would accelerate the self-assembly of larger protein aggregates. Pre-formed cytosolic SOD1 aggregates that have exposed and flexible electrostatic and Zn-loops should also bind to a cell membrane in a similar manner to that suggested by the MD simulations. Essentially the bilayer surface acts to increase the local concentration of apo-SOD1 and increases the chance of protein-protein encounters (2D membrane surface versus 3D cytosol volume). However, it is as yet unclear from experimental investigations whether the bilayer surface indeed acts to promote aggregation of SOD1. Further studies using coarse grained or longer time scale MD simulations approaches in conjunction with experimental methods may shed further light on the mechanisms of lipid-associated aggregation of SOD1.

CONCLUSIONS Using MD simulations, we found that reduced apoSOD1 binds to amphiphiles on its highly flexible (upon metal loss) electrostatic and zinc-binding loops. These loops enable apo-SOD1 to bind strongly to amphiphilic clusters before any unfolding of the b-barrel. Furthermore, during the simulations the Zn-binding loop helped target the apo-SOD1 monomer to associate with the phospholipid bilayer surface. The proximity of positively charged Arg69 side-chain and hydrophobic Leu67 sidechain on the Zn-binding loop is important to its membrane-targeting role. The adsorbed monomers may then seed SOD1 aggregation on the bilayer surface. Disordered loops in apo-SOD1 thus play a critical role in cell membrane targeting as well as co-aggregation of the apo-enzyme with phospholipids, events resulting in cytotoxicity.16 These simulations therefore provide a mechanism for how reduced apo-SOD1 interacts with and binds to amphiphiles and lipid bilayers in the biochemical processes of lipid-associated SOD1 aggregation and co-localization with membrane-bound copper chaperone for efficient acquisition of copper ions.

ACKNOWLEDGMENTS C.P.C. acknowledges the use of BII in-house GPU computing facilities in the form of a NVIDIA Tesla C2075 provided by Mr Tai Pang Yong. The authors thank Dr. C.W. Yong (STFC Daresbury Laboratory UK) for his contribution to initial stages of this work. REFERENCES 1. McCord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969;244: 6049–6055.

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