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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

“Mirror”-like Protein Dimers Stabilized by Local Heterogeneity at Protein Surfaces Baofu Qiao, Luis G. Lopez, and Monica Olvera de la Cruz J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b01394 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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The Journal of Physical Chemistry

“Mirror”-like Protein Dimers Stabilized by Local Heterogeneity at Protein Surfaces Baofu Qiao, † Luis G. Lopez † and Monica Olvera de la Cruz *,†,‡,§ †Department of Materials Science and Engineering, ‡Department of Chemistry, and § Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208

Abstract: Protein aggregation has been observed inside cells and holds true for membraneless organelles. The precise understanding of protein dimerization is a prerequisite for manipulating protein aggregation, which is promising for elevating enzyme concentration to enhance their catalytic performance. Here, the dimerization of two industrially important enzymes of cytochrome P450 (P450) and organophosphorus hydrolase (OPH) are investigated using all-atoms explicit solvent molecular dynamics (MD) simulations, umbrella sampling, and protein-protein docking calculations. The calculated potentials of mean force of dimer-monomer dissociation demonstrate that the dimeric forms are more stable with the free energy barrier of around 60 kJ/mol for P450 and 101 kJ/mol for OPH. The docking calculations on the OPH dimer evidence the uniqueness of the native orientation. The protein dimers form “mirror”-like orientations with some degree of rotation. Such signature orientations are interpreted based on the predominant polar amino acids in the contact regime. In the dimer conformations the active sites are exposed. This work highlights the crucial roles of the polar and nonpolar protein surface domains to form enzymatically active protein dimer aggregates. Our work will potentially aid the design of molecules that can deliver and protect native protein function in various environments.

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INTRODUCTION Proteins are one of the most important components in biology. Recently, protein phase separation has been discovered inside cells.1-3 Protein aggregation is believed to be of relevance to numerous diseases (e.g., cataract,4 neurodegenerative diseases5). It also drives the formation of spatiotemporal aggregates termed membraneless organelles, 6-7 which enable cellular processes to take place. For example, Nott et al. found that membraneless organelles made of Ddx4N1 proteins selectively stabilized single-stranded nucleic acids.8 Recently, heteropolymer-protein hybrid membraneless organelles were assembled9 by exploiting the local heterogeneity of polar and nonpolar amino acids at protein surfaces and by designing random heteropolymers that form enzymatically active protein-polymer complexes. Interestingly, proteins with given distributions of surface domains were found to selectively interact with amphiphilic random copolymers with similar monomer sequence.10 That is, knowledge of the protein surface domains is essential to manipulate the aggregation behavior of proteins. Another extremely important biochemical process where the surface domain heterogeneity is critically important is protein aggregation into dimers. Protein dimers are common in biology for regulating enzyme activation, for the functions of membrane proteins and for DNA binding.11 The understanding of dimerization, for example, will aid us in manipulating enzyme oligomerization,11 which is promising for advancing catalytic performance by increasing enzyme concentrations. Here, we investigate the homodimerization of two industrially important enzymes, namely P45012 and organophosphorus hydrolase (OPH)13-14 (Figure 1). The dimerization has been reported to affect the catalytic activities of P45015 and OPH.16 The cytochrome P450s (P450s or CYPs for short) are a superfamily of one of nature’s most versatile biocatalysts.17 P450s have been found to play a pivotal role in human steroid metabolism, xenobiotic detoxification and metabolism. These enzymes can catalyze a broad variety of chemical reactions of different substrates (e.g., aromatic hydrocarbon hydroxylation and alkene epoxidation).18 Around 75% of drugs are metabolized by P450s.19 P450s show remarkable versatility with thousands of P450s reported in various organisms.17 Among them, P450BM3 (CYP102A1) is one of the most significant as it shows higher activity and catalytic self-sufficiency, since the HEME and diflavin reductase domains are fused together in one single molecule. 20-22 The aggregation of P450 has been recognized in both crystal and solution.23 Oligomers up to tetramer coexist for CYP126A1.24 The probability of larger oligomers increases with the increase in protein concentration from 10 to 20 µM, . Ligands can also influence the dissociation of CYP126A124 and CYP12125 dimers. The dominant distributions of P450BM3 dimers were supported by a variety of experimental techniques. 26-27 Remarkably, the P450BM3 dimeric form is argued ACS Paragon Plus Environment

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to be catalytically active for fatty acid hydroxylation, where electron transfer occurs between the reductase domain of one subunit and the HEME domain of the other.15

Figure 1. Snapshots of protein dimers (A) P450 (pdb code:1BU7) and (B) OPH (pdb code:1HZY).

In contrast to P450, OPH is much more specific. It is unique in hydrolyzing organophosphates by cleaving their P-X (X = S, O, F) covalent bonds.13-14 Being extremely toxic chemicals, organophosphates are the primary components of pesticides and chemical warfare agents (VX, sarin, soman, etc.). As such, these chemicals pose a huge threat to public safety. For instance, globally, around 38% of pesticides are composed of organophosphorus compounds, which result in around 3 million poisonings and 200,000 deaths annually.14 Therefore, the development of highly efficient methods for degrading organophosphate-based chemicals for on-site bioremediation is critical. 9 The oligomerization state of OPH is in debate. The dimeric form has been extensively supported by X-ray crystallization,28-29 ultracentrifugation28 and gel filtration measurement.16 The monomeric form was claimed in gel filtration measurements at varying ionic strengths (0 to 0.2 M KCl) with up to 2 mM Zn2+ and Co2+ ions.30 Note that the OPH dimer has been shown to be active, which can partially unfold to an intermediate, inactive dimeric form, and then to the dissociated monomeric form. 16 By coupling classical all-atom explicit solvent molecular dynamics (MD) simulations with steered MD simulations and protein-protein docking methods, we investigated the aggregation behavior of P450 and OPH (i.e., the dimer-monomer dissociation). The free energy calculations support the dimeric forms to be energetically stable for both proteins under the conditions investigated. The subunits in the dimeric forms are forming “mirror”-like structures. The protein orientations are explained on the basis of the local heterogeneity of polar and nonpolar amino acids at protein surfaces. These findings improve our ACS Paragon Plus Environment

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understanding of condensing enzymes and they provide guidelines for developing a potential method for increasing enzyme concentration, and consequently enhancing their catalytic performance. COMPUTATIONAL DETAILS All-Atom Explicit Solvent MD Simulations Classical MD simulations were performed at the all-atom resolution using the package GROMACS (version 2016.4).31 The CHARMM36m force field 32 was used along with the recommended CHARMM TIP3P water model33 whose structures were constrained using the SETTLE algorithm.34 The initial structures of P450 and OPH were downloaded from the protein data bank webpage (www.rcsb.org) with the PDB ID of 1BU7 35 for P450 dimer (the HEME domain of P450BM3) and 1HZY 29

for OPH dimer. For both proteins, the ligands required for crystallization were removed. Some

mutations were performed for OPH to be consistent with the experimental sequence from our collaborator (unpublished): (a) Lys169 was carboxylated30, 36 to be Kcx169 by including the atoms of formic acids; (b) Glu159 was mutated to be Lys159; (c) the oxygen atoms which were coordinating two Zn2+ ions simultaneously were modified to be hydroxide ions (OH-);30, 36 (c) the capping amino acids AGSIGTG were added before Asp35. Each protein dimer was dissolved in a water box with the edge length of around 16 nm. The net charge of each monomer is -15e for P450 and +3e for OPH, where e stands for the elementary charge. Consequently, 30 Na+ for P450 and 6 Cl- for OPH were added to neutralize the protein dimers. Then 247 Na+ and Cl- ions were added, corresponding to a salt concentration of approximately 0.1 M. The potential energy of the system was minimized using the steepest descent algorithm, followed by an equilibration of 50 ns. The three-dimensional periodic boundary conditions were applied. The neighbor searching was done for a distance of 1.2 nm using the Verlet particle-based cut-off approach and was updated every 20 time steps. The short-range Lennard-Jones (LJ) 12-6 interactions were switched off from 1 to 1.2 nm. The short-range Coulomb interactions was truncated at 1.2 nm with the long-range interactions calculated by means of the Particle Mesh Ewald algorithm.37-38 By constraining all the covalent bonds using the LINCS algorithm,39-40 a time step of 2 fs was employed. The NTP ensemble (constant number of particles, temperature and pressure) was applied with the temperature coupled via the Nosé-Hover algorithm (reference temperature 298K, characteristic time 1 ps) and the isotropic Parrinello-Rahman barostat (reference pressure 1 bar, characteristic time 4 ps and compressibility 4.5×10-5 bar-1). The protein dimers were stable during the simulation duration of 50 ns based on the calculated density, the potential energy and the root-mean-square-deviation of protein backbone atoms ACS Paragon Plus Environment

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(data not presented). In addition to the steerer MD and umbrella sampling calculations below, an unbiased control simulation of 800 ns also supported the stability of the protein dimer. In this control simulation, an OPH dimer was solvated in a smaller simulation box with the edge length of around 12 nm than the length of around 16 nm in the advanced sampling calculations to decrease the computational cost. Steered MD Simulations and Umbrella Sampling The steered simulations were subsequently employed to pull the protein dimers apart in mimicking the dimer-monomer dissociation process. Based on the generated snapshots, the umbrella sampling was applied to calculate the potential of mean force using the Weighted Histogram Analysis Method (WHAM) method. 41-42 In the steered simulations, the atoms, except hydrogen, of one subunit were restrained using a force constant of 1000 kJ/mol/nm2. The other subunit was pulled away as a function of the protein-protein center-of-mass distance, which approached half of the simulation box (~ 8 nm). A pulling velocity of 5 nm/ns was employed. In the steered simulations, snapshots were saved with an interval of around 1 Å within the distance range of 5.0 - 7.8 nm for P450 dimer, and 4.2 - 7.9 nm for OPH dimer. The umbrella sampling simulations were then performed for all the snapshots. Each system was first equilibrated 100 ps, followed by a production simulation of 20 ns with the first 1 ns abandoned for the subsequent data analysis. The WHAM method was employed to calculate the potential of mean force using the program g_wham.

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The histogram distributions of umbrella sampling are presented in Figure S1, and they support the convergence of the calculations. The umbrella sampling was computationally very expensive due to the large simulations box (~ 16 nm in each dimension). Protein-Protein Docking Calculations for OPH Dimer In order to obtain a free energy landscape of the protein dimers, docking calculations were performed. Package Hex 8.0

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was used to generate protein-protein complexes, and program FastContact

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was

used to estimate both the electrostatic and desolvation energy of each complex. The construction of such a landscape was not possible for the P450 dimer due to the highly lumpy surface as well as the highly negatively charged feature of P450 (i.e., -15e per monomer). Consequently, in this work, the docking analyses were exclusively performed for the OPH dimer. Before the docking calculations, the capping amino acids from Ala28 to Gly34 were deleted from the OPH dimer, since these tails are incompatible with rigid body calculations. Kcx169 was replaced with Lys169 in both subunits due to the absence of the atomistic potential in the programs used. 10,000 ACS Paragon Plus Environment

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protein dimer complexes were generated by means of a rigid docking of OPH subunits A and B. The only criteria to sort the 10,000 “artificial” dimers was the geometric complementarity (also known as surface or shape complementarity). Both the position and orientation of the subunit A (the reference subunit) were kept fixed, while all possible positions and orientations of the subunit B (the neighbor subunit), always in close contact with the subunit A, were scanned. The native OPH complex was used as the reference orientation to specify the relative orientation of the “artificial” complexes. In the reference orientation, the geometric center of the subunit A was at (0, 0, 0) in the Cartesian coordinates, while the geometric center of the subunit B was at (0, 0, 4.2 nm), where 4.2 nm was the average distance between the centers of the two proteins. The energies of the 10,000 complexes were minimized using the program Minimize.x of the Tinker package (version 8.4).45 The free energy of each complex was estimated using FastContact.

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program rapidly estimates both the electrostatic and the desolvation contributions of the free energy. The former is the standard intermolecular Coulomb electrostatic potential, calculated using a distancedependent dielectric constant of 4.5r,

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and the latter is based on an empirical contact

potential developed using a database of structures from the protein databank.47 The desolvation component takes into account the hydrophobic interactions, the change in energy due to the desolvation of polar and charged atoms, and the conformational entropy loss of the side chains. The total free energy is the sum of both contributions. Subsequently, all the protein-protein complexes with repulsive interactions (positive free energy) were discarded. For each position of protein B around protein A, only the complex with the strongest attractive interaction was kept, regardless of its orientation. 409 out of 10,000 complexes remained after this process, and these complexes were used to create the free energy landscape of the protein-protein interaction. RESULTS AND DISCUSSION Free Energy of Protein Dimerization To justify the stability of the protein dimers, we calculated the free energy of protein dimerization. In this respect, the protein-protein docking calculations were performed first, which provided the threedimensional free energy map of the possible protein dimers. Moreover, the potential of mean force of protein dimer-monomer dissociation was calculated for the native complex as a function of the proteinprotein distance. These two calculations combined provide us with a better understanding of the free energy involved in the dimerization. ACS Paragon Plus Environment

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Figure 2. (A) Top and (B) bottom hemispheres of the free energy profile of protein-protein docking for the OPH dimer. Only the attractive interactions (ΔG < 0) are displayed. The center of the reference subunit (in cyan in the top insert) is located at (0, 0, 0), with the neighbor subunit colored in silver. The three strongest orientations are labeled in panel (A), with their free energy values listed in the bottom inset.

Presented in Figure 2 are the three-dimensional distributions of the protein-protein orientations with the attractive free energies projected on the XY plane. It is shown that the free energy attractions are relatively weak (ΔG > -50 kJ/mol) for most of the orientations. The stronger attractions (ΔG < -100 kJ/mol) only occur at some orientations. The three orientations with the strongest free energy attractions are labeled with a, b and c in Figure 2, respectively. Their free energy values are listed in the bottom inset. The orientation at (0, 0, 4.2 nm) shows the strongest attractive free energy, which supports the favorable feature of the native orientation. In complement to the three-dimensional protein dimerization profile in Figure 2, the potential of mean force (PMF, ΔG(ξ)) demonstrates the one-dimensional free energy profile along the protein-protein distance (ξ) (Figure 3A). In what follows, the protein-protein distance refers to the distance between the center-of-mass of the subunits, which include the cofactors (HEME for P450 and two Zn2+ ions for OPH). The calculated results have been shifted so that the average free energy in the distance range of 6.5 ≤ ξ ≤ 7.5 nm is considered as the reference (zero). Consequently, the PMFs of the dimer-monomer dissociation were obtained, with ΔG = 60.9 ± 0.9 kJ/mol for P450 and 101.1 ± 0.7 kJ/mol for OPH, where the error bars refer to the standard deviations obtained from the corresponding PMF values within 6.5 ≤ ξ ≤ 7.5 nm. These results support that the dimeric form is more stable for both proteins. The calculated


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free energy of 101 kJ/mol for OPH dimer reasonably agrees with the docking results (Figure 2). The dimeric form of OPH is energetically more stable than that of P450, which is expected to result from the less net charge of +3e per OPH monomer in comparison with -15e per P450 monomer. The obtained free energies are close to those of insulin dimer (30,48 40,49 50,50 126 kJ/mol 51), trypsin-pancreatic trypsin inhibitor complex (188 kJ/mol) 51 and hemoglobin αβ dimer (> 159 kJ/mol).51 Figure 3. (A) Potential of mean force of the dimer-monomer dissociation for P450 and OPH as a function of the protein-protein distance. Insets are the corresponding simulation snapshots for associated and A

B

dissociated configurations. (B) Solvent accessible surface area of protein dimers of P450 and OPH as a function of the protein-protein distance. The error bars in (B) stand for the standard deviations.

Note that some uncertainties exist in calculating the PMFs, e.g., the accuracy of the force field, the convergence of the umbrella sampling, the numerical integration of the WHAM calculation. In terms of the force field, the CHARMM36m potential is one of the most extensively employed and calibrated allatom potential for protein simulations.32 The simulation duration in the umbrella sampling calculations does affect the obtained values in a quantitative manner. For instance, a shorter simulation of 5 ns for each sampling window results in a slightly weaker value of around 50 kJ/mol for P450 and 91 kJ/mol for OPH, in comparison with the values of 60.9 kJ/mol for P450 and 101.1 kJ/mol for OPH with 20 ns for each sampling window. The convergence of the numerical integration of the WHAM calculations was supported by the overlap of the neighbor occurrence profiles (Figure S1). Additionally, some other approaches were proposed for protein-protein dissociation calculations.52 To further understand the energetic driving force of protein dimerization, we calculated the solvent accessible surface areas (SASA). As demonstrated in Figure 3B the SASAs increase as a function of the protein-protein distance. As the protein dimers dissociate, waters will be adsorbed at the protein surface ACS Paragon Plus Environment

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which was previously buried by the neighbor subunit. The corresponding entropy decreases for the water molecules because of a loss in the degrees of freedom. This kind of solvent entropy contribution is approximately 10 kJ/mol per 1 nm2 of protein surface area.51,

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Therefore, the solvent entropy

contribution (-TΔS) is estimated to be around 190 kJ/mol for P450 and 310 kJ/mol for OPH for the dimermonomer dissociation. Accordingly, the enthalpy change (ΔH = ΔG + TΔS) is estimated to be -140 kJ/mol for P450 and -210 kJ/mol for OPH by ignoring other contributions (e.g., entropy of the protein). It could be suggested that for both proteins, the solvent entropy plays a determinant role in protein dimerization.

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Moreover, the distributions of the SASA and the protein-protein distance in the whole

umbrella sampling process (Fig. S2) also support that the protein dimers are the dominant forms. “Mirror”-Like Protein Dimer Orientation Stabilized by Inter-Protein Hydrogen Bonds The previous section demonstrates that the native orientations of protein dimers are energetically favorable. Thus, a follow-up question is: what structural uniqueness is driving the protein dimer native orientation? To understand the mechanism that is driving the inter-protein orientation, we first calculated the interprotein potential energies. As listed in Table S1, the inter-protein interactions between polar amino acids outcompete the others, evidencing the significant role of hydrophilic interactions in protein dimerization. Moreover, it is shown that the Coulomb interactions dominate between polar amino acids, whereas the LJ interactions dominate when the nonpolar amino acids are involved. In these calculations, a cutoff distance of 1.2 nm was employed, which is smaller than the radius of the P450 monomer of around 2.5 nm and about 2.1 nm for the OPH monomer (Fig. 3). Therefore, these calculations represent the interprotein interactions on the contact regime and adjacent to the contact regime. We then focus on the contact regime exclusively where the two protein subunits are in direct contact with each other. A visualization supports a large amount of inter-protein H-bonds (Figure 4A). In the dimeric form, the P450 proteins are stabilized by around 13 ± 2 inter-protein H-bonds, and 8 ± 1 H-bonds for OPH. The number of H-bonds drops quickly with the increase of the protein-protein distance and disappears beyond around 6 nm. Here, the structural criteria of H-bonds are employed, that is, the donoracceptor distance rDA ≤ 0.35 nm, and the H−donor−acceptor angle θHDA ≤ 30°.54-55


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Figure 4. (A) The number of inter-protein H-bonds as a function of the protein-protein distance. Snapshots of (B) P450 and (C) OPH dimers. Highlighted in the insets of (B, C) are the amino acids which are forming inter-protein H-bonds (yellow rods). The two subunits are colored in silver and cyan, with the O/N/H atoms colored in red/blue/white, respectively.

The energy barrier in breaking one amide H-bond has been estimated to be approximately 17 – 21 kJ/mol.51 Consequently, the energy barrier in breaking all the inter-protein H-bonds is around 221 – 273 kJ/mol for P450 and 136 – 168 kJ/mol for OPH. As demonstrated below, the inter-protein H-bonds play a crucial role in driving the relative orientations of the protein dimers. Based on the last snapshots in the protein dimer simulations, we obtained a list of the inter-protein Hbonds, which are illustrated in Figure 4B and listed in Table 1 for P450, and Figure 4C and Table 2 for OPH. It is found that the two subunits are roughly the “mirror” to each other. For instance, for P450 dimer Asp121 on subunit A forms an H-bond with Arg132 on subunit B; meanwhile, Arg132 on protein A forms an H-bond with Asp121 on protein B. Similar observations have been obtained for the OPH dimer. For example, Ser280 and Ser35 on subunit A form H-bonds with Ser110 on subunit B; meanwhile, Ser280 and Ser35 on protein B form H-bonds with Ser110 on protein A. The majority of the contacting amino acids (6 out of 8 for P450; 2 out of 3 for OPH) on protein A are shown to have their “mirror” amino acids on protein B located in the contact regime. Such “mirror”-like structures strongly demonstrate the uniqueness of the contact regime in protein dimerization. ACS Paragon Plus Environment

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Also listed in Tables 1 and 2 are the types of the amino acids (P0: polar and neutral; Pn: polar and negatively charged; Pp: polar and positively charged) which are forming inter-protein H-bonds. The contacting amino acids are exclusively polar amino acids (neutral or charged). It is thus concluded that the local hydrophilic interactions between the neighbor proteins are playing a predominant role in the orientation of protein neighbors.

Table 1. H-bonds Between the Two Subunits of P450 Dimer in Figure 4B. Subunit A Subunit B number a type b

Asp 121 Arg 132 1 Pn - Pp

Arg Asn Arg Asp Gln Asp Gln Asn 132 134 161 168 169 168 125 163 Asp Arg Asn Gln Asp Asp Gln Arg 121 161 134 169 168 168 128 132 3 2 2 1 1 2 1 1 Pp - Pn P0 - Pp Pp - P0 Pn - P0 P0 - Pn Pn - Pn P0 - P0 P0 - Pp

a) Number of H-bonds b) Pn: polar and negatively charged; Pp: polar and positively charged; P0: polar and neutral

Table 2. H-bonds Between the Two Subunits of OPH Dimer in Figure 4C. Subunit A Subunit B number a type b

Ser Ser Ser 35 110 280 Ser Ser Ser 110 35 110 2 2 1 P0 - P0 P0 - P0 P0 - P0

Ser 110 Ser 280 1 P0 - P0

Lys 132 Glu 44 1 Pp - Pn

a) Number of H-bonds b) Pn: polar and negatively charged; Pp: polar and positively charged; P0: polar and neutral

In fact, it has been shown that proteins rarely work alone. Around 50% of the reported structures in the Protein Data Bank are protein complexes, most of which are composed of four subunits or less, and are homomeric.56 The homodimeric protein complexes can form symmetric57-59 or asymmetric60-61 structures. To explore if our findings above are universal, another protein dimer is explored, too. Remarkably, the “mirror”-like orientation is also observed for the XoxF dimer (Table S2). XoxF is an enzyme that efficiently catalyzes methanol oxidation in the exclusive presence of light lanthanide metal ions,62-63 and is therefore an important enzyme for applications in creating low-carbon chemicals. Condensing XoxF ACS Paragon Plus Environment

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in aqueous solution provides a promising method to efficiently separate lanthanides, and importantly, an alternative means for recycling rare earth elements. 64-66 Local Heterogeneity at Protein Surface Drives Protein Dimer Orientation

Figure 5. Electrostatic potential at OPH dimer surface: (A) lower and (B) upper subunits. Negative and positive potentials are colored in red/blue, respectively. The snapshots are rotated to display the active sites (cave regions, blue ellipses) of the two subunits.

To understand the mechanism driving protein dimerization, it is a prerequisite to calibrate the local heterogeneity at protein surface. We first calculated the electrostatic potential at the surface of the OPH dimer. In these calculations, the lower and the upper subunits were saved separately, both of which were converted to the .pqr format using the PDB2PQR program.67 The recommended parameters were employed: the implicit solvent PARSE force field and the PROPKA program68 at pH = 7 for the ionization states of amino acids. The Adaptive Poisson–Boltzmann Solver (APBS)69 plugin of the package VMD 70 was then employed for the electrostatic potential calculation and visualization. The obtained results are demonstrated in Figure 5. First of all, it is demonstrated that the active sites are not buried by the neighbor protein. Such organization is essential for preserving their enzymatic activity when dimerized. This finding holds true for the P450 dimer. It may thus be feasible to synthesize enzyme dimer aggregates for advanced catalytic performance by elevating their concentrations. The negative (red) and positive (blue) electrostatic potentials coexist on both the lower and the upper subunits. Figure 5 also shows that the two subunits rotate against each other by some degree. The rotation ACS Paragon Plus Environment

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angle was calculated to be 103 ± 6 degree for the P450 dimer and 60 ± 3 for the OPH dimer. These stand for the angles between the normal vectors of two planes. The plane of each subunit was created using three coordinates: the metals in the active site (Fe in P450, one Zn in OPH), the geometric center of the subunit backbone atoms, and the corresponding center of the neighbor subunit. The error bars refer to the standard deviations calculated over the simulation time. The small error bars indicate that the orientations of the protein dimers are quite stable.

Figure 6. Distribution of surface domains for proteins P450 (top) and OPH (bottom). The distributions of the amino acids in the contact regimes (Tables 1 and 2) are labeled by yellow crosses.

To further understand the uniqueness of the proteins’ contact regime, we calculated the distributions of the polar and nonpolar protein surface domains (Figure 6). The spherical coordinates were employed. To display the protein-protein contact regime at the centers of the plots, the reference subunit was translated to the center of the simulations box, the dimer was rotated so that the neighbor subunit was aligned in the opposite of the X-dimension, as shown in the upper inset. The translation and rotation of the protein dimers were removed over the simulation duration using the first configuration. These calculations were done using an in-house script, which is based on our previous work

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and was

improved to handle the lumpy surface of protein P450. See the Supporting Information for more details. It is demonstrated in Figure 6 that small polar surface domains coexist with small nonpolar surface domains at the surfaces of both proteins, similar to what we previously found for other proteins.9-10 Also presented in Figure 6 using yellow crosses are the locations of the amino acids which are distributed in the contact regime (Tables 1 and 2). These contacting amino acids are exclusively located on (charged ACS Paragon Plus Environment

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or neutral) polar surface domains. They are distributed within the region of 50⁰ < θ < 130⁰ and 150⁰ < φ < 220⁰. Such broad distributions favor multiple inter-protein contacts, which cooperatively drive the specific orientations of protein dimers. Note that for some proteins the hydrophobic interactions are argued to be crucial in protein dimerization, where the contacting regions are occupied by the nonpolar amino acids.71 For instance, the aromatic side groups on phenylalanine, tyrosine and tryptophan amino acids are abundant in the contact region of STAT5 protein.72 Conclusions Within the context of quantifying protein aggregation, we calculated the free energy profile at the protein surface using protein-protein docking, and the potential of mean force of protein dimer-monomer dissociation using the steered MD and umbrella sampling approaches. The dimeric forms are found to be more energetically favorable for proteins P450 and OPH. Calculations of the solvent accessible surface areas supported that the solvent entropy contribution plays a determinant role in stabilizing the protein dimers. Moreover, the protein-protein docking results for the OPH dimer evidenced the uniqueness of the native orientation of protein dimers. The inter-protein H-bonds were shown to stabilize the relative orientation of the protein dimers. In particular, there exist around 10 inter-protein H-bonds for P450 and OPH dimers. Inspection of the contact regime demonstrated that the protein dimers are forming “mirror”-like structures with some degree of rotation. The protein surface heterogeneity is shown to play a crucial role in stabilizing the “mirror”-like protein dimer orientation. These findings support that the local heterogeneity of polar and nonpolar amino acids on the protein surface, though small in size (nm), is strong enough to drive the recognition of ligands. In our previous work, the local heterogeneity at a protein surface was shown to form stable protein-polymer complexes, which consequently preserved the enzymatic activity in non-native environments.9-10 Moreover, the unique fingerprint of protein-lipid interactions73 are likely related to the local heterogeneity at a protein surface. In the dimeric forms the enzyme active sites are exposed, suggesting that it is feasible to fabricate P450 and OPH dimer aggregates for elevated catalytic performance by increasing the concentrations of the enzymes, for example, by using suitable random heteropolymers

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or by designing specific

polyampholytes. 74 The aggregates will be structurally similar to membraneless organelles. 8 That is, the P450 and OPH dimers, which are the active forms, will be employed as the building units to design the


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molecules. The molecules can connect neighbor building units, while preserving the stability and the enzymatic activity of P450 and OPH dimers in aqueous and organic conditions.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/XXX. Supporting figures and tables, and details in calculating protein surface domains.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected], Phone: +1-8474917801 ORCID Baofu Qiao: 0000-0001-8870-5985 Luis G. Lopez: 0000-0002-1587-6043 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS. The research was supported by Department of Energy Award DE-FG0208ER46539 and the Sherman Fairchild Foundation.

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