LoopGrafter: a web tool for transplanting dynamical loops for protein engineering

Abstract

The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering is the unique dynamism that enables them to exert allosteric control over the catalytic function of enzymes. Thus, when engaging in a transplantation effort, such dynamics in the context of protein structure need consideration. A second practical challenge is identifying successful excision points for the transplantation or grafting. Here, we present LoopGrafter (https://loschmidt.chemi.muni.cz/loopgrafter/), a web server that specifically guides in the loop grafting process between structurally related proteins. The server provides a step-by-step interactive procedure in which the user can successively identify loops in the two input proteins, calculate their geometries, assess their similarities and dynamics, and select a number of loops to be transplanted. All possible different chimeric proteins derived from any existing recombination point are calculated, and 3D models for each of them are constructed and energetically evaluated. The obtained results can be interactively visualized in a user-friendly graphical interface and downloaded for detailed structural analyses.

Graphical Abstract

LoopGrafter calculates loop properties on homologous input structures informing the choice of regions to transplant. Exhaustively exploring the recombination space and evaluating 3D structures facilitate selecting successful protein chimeric designs.

Figure 1.

Scheme of LoopGrafter workflow. Scaffold and insert 3D input structures can be fetched from the RCSB PDB or uploaded from a local file system by the user. Secondary structures and loop geometries are calculated on input structures using DSSP and Archer definitions. Flexibility and cross-correlations are calculated by elastic network models using ProDy-GNM and ProDy-ANM. Flexibility analyses (Figure 2) are followed by structural superimposition using CE and pairing of loops in the two input proteins. After systematic exploration of possible grafting boundaries (recombination points), the server provides a list of designed sequences. Finally, a local structural superimposition is used to guide the generation of 3D models for each of the designed sequences, and each of the models is evaluated with MODELLER and Rosetta scores.

Figure 2.

Graphical user interface of LoopGrafter server at the ‘Flexibility evaluation’ step. In the central part of the top banner, a diagram of the grafting pipeline shows the current grafting progress on the workflow. On the 3D view (upper left), the two input proteins are superimposed, and loops can be zoomed for inspection. On the main panel, the flexibility evaluation of the scaffold protein is presented. Previously defined loops are represented as rectangles below the 2D representation of the protein, and are coloured according to the B-factors either provided by the crystal (pdb) or calculated by elastic network models (anm and gnm). A plus (+) sign on each of the boxes allows for easily including the corresponding loop to the list of loops to be grafted. In the lower part of the panel, a graphical comparison of how the different methods used for assessing flexibility correlate among themselves is provided. In the lower left part, below the 3D view box, the server lists the loops in the scaffold protein indicating which of them are selected for grafting.