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Binding Site Decomposition


 1.  GOAL

 2.  Introduction

 3.  Input

 4.  Chain and heteroatom selection

 5.  Box selection

 6.  Residue selection

 7.  Probe and algorithm selection

 8.  Runtime estimation

 9.  Output visualization

10.  Output interpretation

11.  Reference

1.  Goal

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- To access contribution of specific sidechains to protein-ligand interaction. - To provide information to guide mutagenesis.


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   Basically, the user could select up to 10 residues, excluding alanines and glycines, to be replaced by alanines. Point-to-point comparison is conducted on the molecular interaction fields (MIFs) of the wildtype and mutant structures. Difference clusters are reported as regions showing change in molecular interaction properties upon sidechain removal, and thereby provide some insights into the structural and functional role of the altered residues.

  Adenylate kinase facilitates the transfer of a phosphoryl group from ATP to AMP. PDB entry 1aky is the structure of yeast adenylate kinase ligated with inhibitor AP5P and IMD. Occupying both the ATP site and the AMP site, AP5P mimicks the structure of an ATP molecule coupled to an AMP molecule. AP5P is negatively charged in the middle region for phosphate transfer. [1] This region is surrounded by the sidechains of several positively charged residues, suggesting that electrostatic interactions might play a crucial role in protein-ligand interaction.

  This tutorial illustrates how SiteComp serves as a tool to analyze contribution of specific sidechains and identify residues crucial to ligand binding.

  The example output page used in this tutorial can be found here.


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On the input page:

(1) Set task as "analyze one protein ".

(2) Enter a protein PDB code or upload a PDB file. For example, enter "1aky ".

(3) Click on "Submit ".

Note: Alternatively, one could start by using SiteHound-web to identify binding sites of one protein, or by using SiteComp's Binding site comparison option to identify subsites showing different ligand-binding properties. Either way, there will be buttons on the output page for single cluster analysis, which links to the Box Selection step with a default box enclosing the indicated region of interest.


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(1) If there are more than 1 chain in the protein, select chains for calculation.

(2) If there are heteroatoms, select the ones you intend to display on the output page. In this example, select AP5P.

(3) Click on "Submit ".

Note: This step is skipped if the protein contains only 1 chain and no heteroatoms. Unselected chains and selected heteroatoms could be displayed at later steps only for the purpose of visual reference, but they will be excluded for calculation.


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Define a box-shaped region within which comparison will be performed. To ensure informative results, use a relatively small box which encompasses the region of interest. In this example, this could be the binding site of AP5P. It is recommended that a margin of a few angstroms is allowed in each dimension.

(1) For better visualization, use simple mouse actions and Section 1 to manipulate the molecule in Jmol applet.

(2) Box center could be set in Section 3 in three different ways: 1. clicking on an atom in Jmol applet; 2. specifying a residue number; 3. specifying center coordinates. Box center will be moved to the specified atom/residue/coordiantes.

(3) Box dimensions could be set in Section 4 (Unit: angstrom).

(4) Click on "Binding site decomposition " in Section 2.

Initial view:

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Manipulated View (obtained with the following steps: 1. hiding protein backbones; 2. showing and labeling heteroatoms; 3. moving box center to <10.771, 26.991, 18.374> by clicking on AP5P in Jmol applet; 4. resizing box to 18*26*18 in order to encompass AP5P; 5. rotating the molecule by dragging in Jmol applet):

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This page might take up to a minute to load while SiteComp is collecting information for residues participating in the calculation box.

(1) For better visualization, use simple mouse actions and buttons Section 1 to manipulate the molecule in Jmol applet.

(2) Select up to 10 residues to be replaced by alanines, check the corresponding checkboxes in Section 2. In this example, five arginine residues surrounding the phosphate transfer region are selected: R40, R93, R132, R165, R176.

(3) Click on " >> ".

Initial view:

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Manipulated View (obtained with the following steps: 1. hiding protein backbones; 2. showing and labeling AP5P; 3. selecting five arginines: R40, R93, R132, R165, R176.):

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(1) Select a probe for EasyMIFs calculation. Phosphate Oxygen(OP) probe is selected in our case to inspect the effect of arginine -> alanine mutations on MIF.

(2) Select a clustering algorithm. For example, select "Average Linkage".

(3) To go back to the previous step, click on " << ".

(4) Click on " >> ".

Note: Please refer to EASYMIFs & SITEHOUND User's Guide for details about the different probes and clustering algoritms.


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Upon submission, SiteComp provides a runtime estimation and a link to the output page. You will be redirected to the output page when calculation finishes (usually within a few minutes). The output page will be kept on the server for 30 days, during which period it can be revisited as many times as desired.


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(1) Job information, including the altered residues, is listed in Section 1.

(2) For better visualization, use simple mouse actions and buttons in Section 3 to manipulate the molecules in Jmol applet. The size of Jmol applet could also be altered.

(3) Use Section 2 to visualize each set of difference clusters. Available options include:
  - visualize and zoom in to each difference cluster;
  - visualize and label protein sidechains surrounding each difference cluster;
  - show surfaces of difference clusters;
  - color difference cluster points by relative or absolute energy to visualize distribution of interaction energy difference.
If colored by relative energy, the points with lightest and darkest colors are the ones with least and most favorable energy values within each difference cluster. If colored by absolute energy, the points with lightest and darkest colors are the ones with least and most favorable energy values from each set of difference clusters.

(4) Check the "Highlight " box in Section 1 to highlight the mutation(s).

(5) Information of the difference clusters, including energy, energy range, volume, center coordinates and participating residues, is shown in Section 4. Residue information could be shown or hidden using the "Show/Hide residue information " button.

(6) By default, the information about the difference clusters favorable for the wildtype struture is displayed. If there are any difference clusters favorable for the mutant structure, use the "Show details " buttons in Section 2 to display the relevant information. This button is hidden if there is no such difference cluster.

Initial View:

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(5) Use the "DOWNLOAD " link in Section 3 to download output files.

* Explanation of files in the downloaded package   

Note: The .pdb files can be used to display the proteins, heteroatoms and difference clusters in molecular visualization softwares such as PyMol or Jmol. Please refer to EASYMIFs & SITEHOUND User's Guide for detailed description of each file type.

(6) To go back to the previous step, click on " << ".


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   Our interest is to identify regions in the ligand binding site wildtype structure which are more favorable for binding, as compared to the corresponding regions in the mutant structure. In the Manipulated View, the first difference cluster favorable to the wildtype structure(1aky) is displayed in a large Jmol applet. This difference cluster suggests that, in the wildtype structure, the five arginine sidechains (R40, R93, R132, R165, R176) mostly contribute to protein-ligand interaction in this region. Difference luster points close to AP5P are more likely to have darker colors which indicate more favorable energy values. In other words, the signals are stronger around the phosphoryl groups than elsewhere.

   It should be mentioned that, even though the shape of the binding site is about symmetric, the predicted difference cluster is biased to the right side, which is the AMP site based on the orientation in this figure. This is compatible with the existing knowledge that phosphate transferring takes place closer to the AMP binding site [1].

Manipulated View (obtained with the following steps: 1. hiding protein backbones, showing heteroatoms; 2. zooming in to the first difference cluster favorable for the wildtype structure; 3. coloring this difference cluster according to relative energy; 4. highlighting mutations; 5. hiding other difference clusters, hiding boundbox; 6. setting applet size to "large;" 7. rotating the molecule by dragging in Jmol applet):

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   Next, single mutation analyses for each of the 5 arginines illustrate the contribution of each individual arginine to ligand binding and phosphate transferring. R40 probably stabilizes the phosphoryl group connected to the adenosine group in the AMP site. R132 mostly stabilizes the center of AP5P. R93, R165, and R176 mostly contribute to the ATP-AMP interface and therefore are likely to be directly responsible for phosphate transferring.

   In this example, SiteComp provides useful insights into single residue contribution to protein-ligand interaction. Such information could be helpful for prioritizing and planning mutations in a detailed way.

Single mutation analyses (Each of the five alanine residues (R40, R93, R132, R165, R176) is mutated alone, and each resulting structure is compared with the wildtype. Shown below are the first difference cluster detected by SiteComp favorable for the wildtype structure in each mutation analysis.)

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Abele, U. and Schulz, G.E. (1995) High-resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer, Protein Sci, 4, 1262-1271.

For help and questions, please email sitecomp@sanchezlab.org