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Mutagenesis, Site-directed, Molecular docking simulation, Molecular dynamics simulation, What&,rsquo s Known At physiological pH, ocriplasmin is very autolytic and proteolytic, which restricts its activity duration. Proteolytic activity can lead to photoreceptor damage and vision loss in more serious cases. Ocriplasmin suffers autolytic degradation after injection into the vitreous, resulting in its fast inactivation. What&,rsquo s New Three ocriplasmin variants were designed via site-directed mutagenesis, and the mutational analysis of the variants was performed through homology modeling, molecular docking, and molecular dynamic simulations. Our in silico mutational analysis of ocriplasmin revealed that A59T and K156E mutagenesis could be used to develop new ocriplasmin variants with higher therapeutic efficacy. IntroductionOcriplasmin (formerly &,ldquo microplasmin&,rdquo and trade name &,ldquo Jetrea&,rdquo ) is a novel FDA-approved pharmacological agent for the treatment of various vitreoretinopathies such as symptomatic vitreomacular adhesion and vitreomacular traction. Ocriplasmin, with a molecular weight of 27 kDa, is a recombinant truncated form of human plasmin with serine (Ser) protease activity. 1, Before the FDA approval of ocriplasmin, the only treatment for vitreomacular adhesion was the surgical method of vitrectomy. 2, This surgical modality carries the risk of retinal damage, which explains why some plasmin-containing proteolytic enzymes have been trialed for the enzymatic release of retinal traction. 3, While ocriplasmin might avert the risk of surgical treatment, pharmacologic vitreolysis is not devoid of risk. 4, The proteolytic and autolytic nature of this enzyme at physiological pH limits its activity duration. Proteolytic activity can lead to photoreceptor damage and even vision loss in more serious cases. 5, , 6, Specific plasmin inhibitors such as &,alpha 2-antiplasmin, &,alpha 2-macroglobulin, and &,alpha 2-antitrypsin can be found in the vitreous under normal and disease conditions, and they can influence its function. 6, It should, therefore, come as no surprise that the vitreous may sustain extensive damage from an enzyme with wide substrate specificity and high proteolytic activity. 3, Moreover, ocriplasmin suffers autolytic degradation after injection into the vitreous, which leads to its fast inactivation. 7, Molecular dynamic (MD) simulations, as a computational predictive method, can be used successfully to analyze the motions of ions, water, macromolecules, and further complex systems. Specifically, structure/function relationships such as those that are reliant on the solute/solvent and temperature are essential to the determination of the paradigm of protein-protein or ligand-protein complexes. Indeed, the fact that these motions can be modeled by MD simulations attests to the utility of this predictive method. 8, Amino acid mutation analysis has rapidly evolved into dynamic research in the computational studies of proteins. This is mainly related not only to the availability of growing levels of protein sequences throughout the post-genomic era, which confers a rapid collection of data on protein variations, but also to many new bioinformatics tools for the investigation of the consequences and effects of amino acid mutation. Mutated proteins are crucial both to a more in-depth understanding of protein functions and genotype-phenotype relationships and to a more rational design and engineering of therapeutic proteins. 9, Computational mutagenesis revealed that some mutations may have an important role in diminishing the autolytic and proteolytic activities of ocriplasmin and producing an optimized enzyme. Previously, the structure of microplasmin (ocriplasmin), in conjunction with its active sites, proteolytic sites, and autolytic sites, has been determined. 7, , 10, The key amino acids in the active site/proteolytic site of ocriplasmin comprise the catalytic triad of aspartic acid (Asp), histidine (His), and Ser, whose hydroxyl sidechain is responsible for nucleophilic attacks to the peptide substrate in the catalytic mechanism. In the active form of ocriplasmin, the catalytic triad of Ser-His-Asp (S199, H61, and D104 in ocriplasmin) forms a compact unit with the His sidechain that is centrally located and hydrogen-bonded to both Asp and Ser. 10, The major autolytic cleavage sites are limited to three positions, K156&,ndash E157, K166&,ndash V167, and R177&,ndash V178. All of them align with the cleavage-site specificity of ocriplasmin, which is prone to cleavage after being positively charged with lysine (Lys) or arginine residues. The first cleavage occurs at position 156&,ndash 157, as it is faster and after this cleavage, the sensitivity of the other sites to cleavage is greatly enhanced. 7, Therefore, the substitution of glutamic acid (Glu) for Lys is more effective than that for other amino acids in the reduction of autolytic activity. 3, In dysplasminogenemia, the functional activity of plasminogen is diminished. The mutation of Ala601Thr has been described in dysplasminogenemia, and the substitution of threonine (Thr) for alanine 601 plays an important role in the reduction of the functional activity of plasminogen. (Alanine 601 in plasminogen is equivalent to alanine 59 [Ala59] in ocriplasmin). 11, In this study, three variants of ocriplasmin, each containing one mutation, were designed. In the first variant, termed &,ldquo the autolytic variant&,rdquo , Lys156 was mutated to Glu to reduce autolytic activity. In the second variant, termed &,ldquo the proteolytic variant&,rdquo , Ala59 was mutated to Thr to lessen proteolytic activity. The third variant, termed &,ldquo the mixed variant&,rdquo , comprised both mutations (A59T and K156E). In addition, the wild-type variant lacked any mutation. For the bioinformatics analysis of the different features of ocriplasmin, the 3D structure of ocriplasmin was modeled by I-TASSER and Swiss Model webservers. 12, , 13, Subsequently, the position of these mutations was studied by Swiss-PdbViewer and PyMOL software tools. 14, , 15, Thereafter, molecular docking simulations were performed to study the interactions between the Chromogenix S-2403 substrate (L-pyroglutamyl-L-phenylalanyl-L-lysine-p-nitroaniline hydrochloride, Chromogenix, Milano, Italy, cat. 822254-39 as a chromogenic substrate for plasmin, ocriplasmin, and streptokinase-activated plasminogen) and the ocriplasmin variants. Afterwards, the binding capability was evaluated by calculating free binding energy values. Finally, for the assessment of the conformational features of protein-substrate complex systems for the wild-type variant and all the mutant variants of ocriplasmin, MD simulations at 100 nanoseconds were performed.MethodsSequence AvailabilityThis study was performed in Tabriz University of Medical Sciences, Tabriz, Iran, 2019. The amino acids and the accession number (DB08888 [DB05028]) of ocriplasmin were retrieved from the DrugBank database and saved in FASTA format for further analyses.Three- and Two-dimensional Structure Prediction Using Homology ModelingThe 3D structure of the ocriplasmin was predicted via an automated homology modeling approach in the (PS)2v2 server (http,//ps2.life.nctu.edu.tw/). 16, A combination of PSI-BLAST, IMPALA, and T-Coffee methods was used by the server to perform the necessary template selection and target-template alignment. In addition to the modeling procedure on this server, the 3D structure of the ocriplasmin enzyme was modeled in the I-TASSER and Swiss Model webservers. 12, , 13, In the I-TASSER webserver, through the application of the crystal structure of similar enzymes, 3D models were built by LOMETS and iterative template fragment assembly simulations. 17, The I-TASSER webserver produced five models of ocriplasmin. Models with the best confidence score (C-score) and standard score (Z-score) were chosen, and the locations of these mutations were visualized and studied using the Swiss-PdbViewer and PyMOL software tools. 14, , 15, The secondary structure of ocriplasmin was predicted in the SYMPRED webserver. 18, The secondary structure of this protein was also predicted in the PSIPRED and SOPMA webservers. 19, , 20, Energy minimization was performed using the YASARA force field (http,//www.yasara.org/minimizationserver.htm). For the mutant variants, the mutated amino acid was first changed in the linear sequence of amino acids. Then, this changed sequence was introduced to the I-TASSER webserver, and the structure of the mutant enzyme was modeled. Subsequently, other studies similar to that on the wild-type enzyme were performed on these mutant enzymes.Physicochemical Properties of the ModelsThe physicochemical properties of the native and mutant proteins, comprising the molecular weight, the theoretical isoelectric point (pI), the amino acid composition, the whole number of negatively and positively charged residues, the instability index, and the aliphatic index, were determined. Additionally, hydrogen bonds were estimated using the WHAT IF and PIC webservers. 21, , 22, Molecular Docking Simulation StudyModels of all the variants of ocriplasmin with the S-2403 substrate were evaluated using molecular docking simulations to study the interaction manner of this substrate in the 3D structure of ocriplasmin and its binding capability by calculating free binding energy values. Therefore, the freely available packages of AutoDock 4.2.6 and AutoDock Tools 1.5.6 were used. 23, The 2D chemical structure of the S-2403 substrate was achieved using the ChemDraw Ultra 10.0 software, 24, and the molecular energy optimization of this structure was performed via molecular mechanics (MM+) and semi-empirical (AM1) approaches for the optimization of the structure. The Fletcher&,ndash Reeves algorithm was applied during the optimization procedure. The HyperChem 8.0.8 software was utilized for the aforementioned optimization procedure. 25, The molecular structures of all the variants of ocriplasmin (wild, proteolytic, autolytic, and autolytic/proteolytic mutant types), which were used as macromolecules in the molecular docking simulation study, were prepared via the homology modeling knowledge-based protein tertiary structure prediction method. Molecular docking simulations were performed in the proteolytic site/active site (Ala59, His61, Asp104, and Ser199) and the autolytic site (Lys156 and Glu157) of all the ocriplasmin variants to assay their substrate-binding potency and to assess the efficacy of this binding in decreasing autolytic and proteolytic activities. In the first step, autolytic and proteolytic sites were defined for the molecular docking simulations of the substrate in all the variants independently. Then, according to the obtained results of docking in the first step, a high-number conformations cluster-binding site was selected for the focus docking approach in the second step. In this step, the size of the grid box dimensions at grid points in x&,times y&,times z directions was set to 62&,times 62&,times 62 &,Aring 3 (0.375 &,Aring grid spacing) for ocriplasmin in all the variants. The center of the grid box in x, y, z centers was fixed to -42.95, 31.70, and -12.78. During the preparation of the substrate and macromolecule structures for docking, polar hydrogen, charges of the Gasteiger-type and Kollman were added using the AutoDock Tools software. The Lamarckian genetic algorithm approach with 100 runs of the genetic algorithm was performed in this molecular docking simulation study for the substrate-wild-type, substrate-proteolytic-type, substrate-autolytic-type, and substrate-autolytic/proteolytic-type ocriplasmin systems. 26, Simulation SetupFour different protein-substrate complex systems were provided. Then, MD simulations at 100 nanoseconds were performed for all the systems using the GROMACS 2016 package. All the simulations were directed with GROMOS96 54A7 via the simple-point-charge water model. 27, The topology and parameter files of the designed substrates were prepared with the PRODRG webserver. 28, The solvated systems in explicit water molecules extended 10 Ao from each edge of the cubic box to the solute atoms. 29, These systems were energy-minimized via the steepest descent approach and equilibrated under constant pressure (NPT) and volume (NVT) ensemble conditions, for 200 ps. For the visualization of the simulation results, the UCSF Chimera and VMD 1.9 software tools were utilized. 30, , 31, With the aid of the UCSF Chimera package, total molecular graphics were produced. 30, Trajectory snapshots were saved every two fs through the simulation time, and 3D coordinate files were collected every two nanoseconds for post-dynamic evaluation. With the use of NaCl counterions, the systems were neutralized. After the addition of a proper number of the ions of sodium (Na+) and chloride (Cl&,minus ), the NaCl concentration in all the systems was 100 mM. Short-range non-bonded interactions, in all the systems, were truncated at 1.2 nm through a long-range correction to pressure and energy terms to account for the truncated Van der Waals forces. Additionally, the particle-mesh Ewald (PME) approach was applied for the calculation of the electrostatic energy of the interactions. Moreover, the LINear Constraint Solver (LINCS) algorithm was utilized for all the constraints, providing an integration time-step of 2 fs. 32, In all the directions, periodic boundary conditions were used. The temperature of the system was conserved at 300 &,deg K with the Berendsen weak-coupling approach, and the pressure was preserved at 1 bar through the application of the Parrinello&,ndash Rahman barostat in the constant-pressure ensemble. 33, Additionally, the root mean square deviation (RMSD), the root-mean-square fluctuation (RMSF), Dictionary of Secondary Structure for Proteins (DSSP), and hydrogen bonds were analyzed throughout the MD simulation. Comparison of the Center-of-Mass Distance between Ocriplasmin and the SubstrateFor the investigation of the affinity between ocriplasmin and the substrate, the center-of-mass distance between the ocriplasmin active site and the substrate during the MD simulation was calculated. His61, Asp104, and Ser199 in the active site residues had an outstanding role in the interaction between the substrate and the ocriplasmin variants. The center-of-mass distance between the ocriplasmin active site and the substrate in the wild-type variant and all the mutant variants (A59T, K156E, and the mixed form) was also calculated during the simulation. Finally, the center-of-mass distance was compared between the active site residues and the substrate in all the variants.ResultsSequence Availability and Amino Acid CompositionThe amino acid sequence of ocriplasmin, containing 249 amino acids, was stored in a FASTA file format, and its feature was deposited in table 1,. ResiduesNumberPercentageResidue MassSpecific VolumeAla145.6290.100.74Asx00.00133.610.61Cys124.82122.160.63Asp72.81134.110.60Glu166.43148.130.66Phe93.61166.200.77Gly2510.0476.070.64His72.8156.160.67Ile104.02132.180.90Lys135.22147.190.82Leu218.43132.180.90Met20.8150.220.75Asn83.21133.120.62Pro197.63116.130.76Gln93.61147.150.67Arg135.22175.210.70Ser166.43106.100.63Thr135.22120.120.70Val249.64118.150.86Trp62.41205.230.74Unk00.00138.150.72Tyr52.01182.190.71Glx00.00147.640.67Total249100.0027232.47 DaTable 1.The features of the amino acid sequence of ocriplasminSecondary StructureThe secondary structure components in ocriplasmin encompassed &,alpha -helix 15.26%, extended strand 29.72%, &,beta - turn 0.00%, and random coil 55.02%. The situation of each amino acid in the secondary structure of ocriplasmin, and its plot showed that this sequence had one helix-turn-helix (HTH) motif, which is a DNA-binding motif. Three-dimensional Structure Prediction and Physicochemical Properties of the ModelsFor the investigation of the 3D structure of ocriplasmin, the I-TASSER webserver was employed. The server produced five models. The best model displayed a total C-score of 1.60, a template modeling score (TM-score) of 0.94&,plusmn 0.05, and an estimated RMSD of 3.4&,plusmn 2.4. Table 2, illustrates the physicochemical features of the ocriplasmin model, which were estimated using the ProtParam software. Thereafter, the structural models of ocriplasmin were precisely studied for the location of the A59T and K156E mutations. The results showed that these mutations were distributed in different regions of the ocriplasmin structure. The 3D structure of ocriplasmin, which was modeled in the I-TASSER webserver, along with A59 and K156, is depicted in figure 1A,. Subsequently, the location of A59 and K156 was precisely evaluated in this model. The analysis of this 3D model showed that the Ala59 residue formed a hydrogen bond with His61 and Cys62 (figure 1B[a],). However, not all of these hydrogen bonds could be shown in the structure viewer software, which is a limitation defined for this software. The computation of non-covalent interactions was performed with the WHAT IF and PIC webservers. The results of these evaluations are presented in table 3,. The substitution of Thr for Ala59 had a significant role in the reduction of the proteolytic activity of the enzyme. The mutation of Ala59 to Thr59 was followed by a change in some non-covalent interactions and the creation of new interactions (figure 1B[b],). No.ParametersWild TypeA59TK156EA59T and K156E1Theoretical pI8.278.277.677.672Molecular weight27231.3427261.3627232.2827262.313Sequence length2492492492494Extinction coef&,#64257 cient41200-4045041200-4045041200-4045041200-404505 Asp+Glu232324246 Arg+Lys262625257Instability index48.3548.3550.0050.008Grand average of hydropathicity-0.14-0.15-0.14-0.159Aliphatic index82.1381.7382.1381.73* The first value is according to the hypothesis that both cysteine residues are oxides and form cystine, and the second value is based on the hypothesis that supposes, which of the cysteine residues is decreased. pI, Isoelectric point |