Automated scaffold selection for enzyme design

A major goal of computational protein design is the construction of novel functions on existing protein scaffolds. There the first question is which scaffold is suitable for a specific reaction. Given a set of catalytic residues and their spatial arrangement, one wants to identify a protein scaffold that can host this active site. Here, we present an algorithm called ScaffoldSelection that is able to rapidly search large sets of protein structures for potential attachment sites of an enzymatic motif. The method consists of two steps; it first identifies pairs of backbone positions in pocket-like regions. Then, it combines these to complete attachment sites using a graph theoretical approach. Identified matches are assessed for their ability to accommodate the substrate or transition state. A representative set of structures from the Protein Data Bank ( approximately 3500) was searched for backbone geometries that support the catalytic residues for 12 chemical reactions. Recapitulation of native active site geometries is used as a benchmark for the performance of the program. The native motif is identified in all 12 test cases, ranking it in the top percentile in 5 out of 12. The algorithm is fast and efficient, although dependent on the complexity of the motif. Comparisons to other methods show that ScaffoldSelection performs equally well in terms of accuracy and far better in terms of speed. Thus, ScaffoldSelection will aid future computational protein design experiments by preselecting protein scaffolds that are suitable for a specific reaction type and the introduction of a predefined amino acid motif

Identification of Protein Scaffolds for Enzyme Design Using Scaffold Selection

The identification of suitable protein structures that can serve as scaffolds for the introduction of catalytic residues is crucial for the design of new enzymes. Here we describe how the automated and rapid scaffold search program ScaffoldSelection can be used to find the best starting points, namely protein structures that are most likely to tolerate the introduction and promote the proper formation of a specific catalytic motif

Binding pocket optimization by computational protein design.

Engineering specific interactions between proteins and small molecules is extremely useful for biological studies, as these interactions are essential for molecular recognition. Furthermore, many biotechnological applications are made possible by such an engineering approach, ranging from biosensors to the design of custom enzyme catalysts. Here, we present a novel method for the computational design of protein-small ligand binding named PocketOptimizer. The program can be used to modify protein binding pocket residues to improve or establish binding of a small molecule. It is a modular pipeline based on a number of customizable molecular modeling tools to predict mutations that alter the affinity of a target protein to its ligand. At its heart it uses a receptor-ligand scoring function to estimate the binding free energy between protein and ligand. We compiled a benchmark set that we used to systematically assess the performance of our method. It consists of proteins for which mutational variants with different binding affinities for their ligands and experimentally determined structures exist. Within this test set PocketOptimizer correctly predicts the mutant with the higher affinity in about 69% of the cases. A detailed analysis of the results reveals that the strengths of PocketOptimizer lie in the correct introduction of stabilizing hydrogen bonds to the ligand, as well as in the improved geometric complemetarity between ligand and binding pocket. Apart from the novel method for binding pocket design we also introduce a much needed benchmark data set for the comparison of affinities of mutant binding pockets, and that we use to asses programs for in silico design of ligand binding

'Where is the sun?' The sun is 'up' in the eye of the beholder

Barnett-Cowan M, Ernst MO, Buelthoff HH. 'Where is the sun?' The sun is 'up' in the eye of the beholder. Perception. 2010;39:146

Comparison of the energy scores versus the affinities of the mutations show how well the programs reproduce the differences.

<p>For each test case with more than two mutations, we plotted the top binding scores of CADDSuite, Vina, and Rosetta designs for each mutation on each scaffold structure together with the logarithm of the affinity. Here we show plots for Carbonic anhydrase II, HIV-1 protease, and Streptavidin test 1. All other plots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052505#pone.0052505.s001" target="_blank">Information S1</a>. Values are scaled to fit in the same range. Shown on the x-axis of a plot are the mutants in order of affinity to the ligand (the leftmost has the lowest affinity, compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052505#pone-0052505-t001" target="_blank">Table 1</a> for the actual values). The y-axis measures predicted binding scores for the designs, and the log affinities, scaled between 0 and 1. Both are proportional to the binding free energy, and can therefore be compared when scaled to the same range. The lowest predicted binding score or log affinity is set to 0, the highest respective value to 1. Each plot contains a line for the affinity logarithm (solid, black no marker). This line represents the goal, if a method predicts binding well, the binding score lines should closely follow the log affinity line. The other markers and lines show the scaled predicted binding scores. One line represents the designs calculated for all available mutants, calculated by using one crystal structure as the scaffold. (Crystal structure 1: dashed, blue, circle markers; structure 2: red, dotted, square markers; structure 3: green, dash-dot pattern, diamond markers; structure 4: cyan, two dashes one dot pattern, star markers). We chose to use lines for representation, because this makes it easy to visually compare the shape of the black log affinity line to the lines representing the design binding scores. Each row has plots for one test case, in parentheses the order of scaffold structures is listed: <i>CA</i>: Carbonic anhydrase II (1ydb, 1yda, 1ydd), <i>HP</i>: HIV-1 protease (1met, 1meu, 1mes), <i>S1</i>: Streptavidin test 1 (1swe, 1n43).</p

Two-dimensional structures of benchmark set ligands.

<p>The ligands of the test cases of our benchmark sets. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052505#pone-0052505-t001" target="_blank">Table 1</a> for which ligand belongs to which test case.</p

Differences of the ligand poses and pocket side chains in the benchmark designs compared to the crystal structures.

<p>The upper graph shows the average RMSDs and standard deviation between the ligand pose in the designs and in the crystal structures. The lower graph shows the average RMSD and standard deviation between the binding pocket side chain heavy atoms of designs and the corresponding crystal structure. The RMSDs are calculated after superimposing the structures using the backbone to make sure that the differences come from pocket/ligand pose differences only. RMSD from PocketOptimizer CADDSuite score designs are plotted in blue, from PocketOptimizer vina designs in green, and from Rosetta designs in red. Each point marks the average RMSD for all designs of a test case usign one score. The number of designs that contribute to a value depends on the number of mutations with a crystal structure, it is the square of this number (because each structure is used as a design scaffold for each mutation). Test cases are: <i>CA</i>: Carbonic anhydrase II, <i>ABP</i> D7r4 amine binding protein, <i>ER</i>: Estrogen receptor , <i>HP</i>: HIV-1 protease, <i>KI</i>: Ketosteroid isomerase, <i>L</i>: Lectin, <i>MS</i>: Methylglyoxal synthase, <i>N1</i>: Neuroaminidase test 1, <i>N2</i>: Neuroaminidase test 2, <i>PNP</i>: Purine nucleoside phosphorylase, <i>S1</i>: Streptavidin test 1, <i>S2</i>: Streptavidin test 2, <i>TS</i>: Thymidylate synthase, <i>T</i>: Trypsin.</p

Workflow of PocketOptimizer.

<p>The input specific for a design is depicted in circles, parts of the pipeline are shown in pointed rectangles, and output components in rounded rectangles. The output is stored in standard file formats (SDF and PDB for structural data, csv for energy tables). This allows the easy replacement of a component with another that solves the same task (e.g. replacing the binding score function).</p