B. Comparing the interactions of different ligands with the same target

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i) Return to the ligand entry of 1f0r. Select Similar Binding Site Search at the top of the page. In the resulting Binding Site Analysis page, type 100% in the Minimum Sequence Identity box and 2.5 in the Lowest Resolution box. Click on Submit.

ii) A total of 45 structures are found. If this was a drug discovery project we would select all of the structures in order to include as much diversity of the ligands as possible. For a clearer picture we will limit our selection to 5 structures. Click on Invert Selection in order to de-select all entries. Select the following entries: 1nfy, 1nfx, 1nfw, 1ksn, and 1xka by ticking the boxes next to the structure names. A total of 5 entries. Type 8.0 in the Radius of sphere around Ligand box, and click Submit.

iii) In the Superposition Analysis page that comes up there is a lot of information about differences between the reference structure and the other structures that is presented in table form. Click on Show in ReliView to study all of the structures.

iv) Use the Protein Explorer to switch off the displaying of entries 1nfy, 1nfx, and 1nfw this is done by un-ticking the boxes next to the structure names in protein explorer. Compare the interactions of the remaining ligands in 1f0r, 1xka and 1ksn. Find waters 12 and 170 in the reference structure (1f0r) through the protein explorer (expand the ‘water-tree’) – are the waters retained in 1ksn and 1xka? What is the difference between the interaction of 1f0r and 1ksn in the S1 pocket?

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v) Invert your selection – de-select 1xka and 1ksn (keep the reference structure 1f0r displayed) and select 1nfy, 1nfx, and 1nfw. How has the ligand-protein interaction changed? Check waters 12 and 170, are they retained in all of the structures? Study the interaction of the ligands in 1nfy, how do they differ from the reference structure 1f0r?

vi) The ligands in 1nfy, 1nfx and 1nfw are known as reverse binders. The reversal of the binding pose can be explained by a number of ‘new’ interactions. The two waters in the S1 pocket are now completely displaced, which is likely to be thermodynamically favourable. The S4 interaction of the (cationic) basic moiety is explained by cation-pi interactions. There is also an unusual interaction between the chloride and the phenyl ring of Tyr228. Create a centroid of Tyr228 in 1nfy by selecting the menu Calculate -> Centroid -> New centroid. Pick the atoms in the phenyl ring of Tyr228 then click OK. A red dot should appear in the center of the phenyl ring. Measure the distance between the chloride of the ligand and the centroid of Tyr228 – this done by selecting Measure Distances in the Picking Mode box at the top of the ReliView window and picking the red dot in the center of the phenyl ring (the previously created centroid) and the chloride atom on the ligand.

This ends Part2B - Part2C - Back to Start