Our case study: NMDA receptor. Use electrostatic surfaces and normal mode analysis to predict how glutamate and a drug bind to the glutamate receptor in the human brain.

The ionotropic glutamate receptor is a multi-chain, multi-domain transmembrane protein that is expressed in neurons in the brain. It gates cations (K+, Na+, Ca2+) in response to both trans-membrane voltage and glutamate+glycine binding. Glutamate receptors are named based on whether they stimulate ion flux via direct (ionotropic, iGluR) or indirect (metabotropic, mGluR) interaction with the ion channel, and based on agonist molecules that cooperatively bind and amplify the glutamate+glycine stimulus. One such

Molecular modeling, Homework 12! ! Due Apr 25, 2014

agonist is N-methyl D-aspartic acid (NMDA). Calcium flow through the NMDA iGluR has been linked to gene regulation, and the NMDA iGluR is thought to be responsible for memory and forgetting, via gene expression that causes dendritic growth or retraction.

The binding of Glu to the ligand binding domain of NMDA iGluR probably triggers a conformational change in the transmembrane domains (there are four), opening the channel and allowing ions to pass. How does Glu bind and what might be the pathway for binding?

(1) Retreive 3GBA (NMDA Ionotropic glutamate receptor ligand binding core domain) from the MOE protein database. Delete all chains except A, including ligands and waters.

(2) Run Compute/Structure preparation, and Protonate3D. Then calculate the molecular surface with electrostatic coloring. Save the MOE file.

(3) Retrieve the structure 2ZNS from the PDB. This structure shows the Glu-bound state of the same domain. Superpose. Be sure to synchronize by chain tag so the Glu goes with the 2ZNS protein chain when superposing.

(4) Delete the 2ZNS protein chain, keeping the docked Glu and 3GBA. Save the MOE file.

(5) Create the 1st powerpoint slide showing the docked Glu in spacefilling atoms, with the Z-clipped, semi-transparent electrostatic molecular surface. In thin lines, show the residues near the Glu. Show only the non-hydrogen atoms. Label them. Display using Left-right stereo.

(6) Look at the structure. Predict where Glu enters the NMDA iGluR receptor.

(7) Create a 2nd powerpoint slide, showing the electrostatic surface with the Glu docked approximately at the point of entry to the binding site, still outside the protein. Add a caption in Powerpoint.

(8) Energy minimize 3GBA with the backbone atoms tethered. Then minimize again without tethering any atoms. Save this MOE file. Return to it if and start again if you feel you have pushed the structure out of its native state too much!

Molecular modeling, Homework 12! ! Due Apr 25, 2014

(9) Turn on Minimize (run 'gizmin.svl'), and Colorforce (run 'gizcolorf.svl'). Also turn on Contacts | H-bond, but only display ligand H-bonds.

(10) Dock, as follows.................

Grab the Glu (alt-shift-middlemouse to translate, alt-middlemouse to rotate) and guide it through protein from the point of entry to the binding site along what you think will be the most reasonable path. Note that atoms turn red as they collide, signaling not to go that way. On the other hand, side chain atoms will move out of the way if it is energetically possible.

Find a path from the outside to inside ending at the binding site.

(10.1,10.2, 10.3) At three points along the path, (1. just outside, 2. halfway through, and 3. all the way in) stop and take a picture, as described in this box:

Un-select all atoms.

Turn off Minimize and Colorforce.

Recolor all atoms by element.

Display only the Glu and atoms near the Glu.

Select the Glu. (double-click on it)

Run Compute/Simulations/Dock. [ Select the induced fit method. This will allow slight movements in the receptor. For receptor, use Receptor. For site, using selected atoms. For ligand, use selected atoms. For other settings, use the defaults. Save the mdb file with a new name. Run Dock! Watch!

When Dock is done. Go to the database window. Select the Mol (Glu) with the lowest E_refine. Send to MOE (right-click menu). Delete the old Glu.

Leave the electrostatic surface displayed. Use Zclip to show only the surface around the Glu, as shown in the examples. Show all non-hydrogen atoms as ball-and-stick, element colors, with white background and white carbons. Use Zclip to restrict the view as shown in the example. Use Left-Right stereo.

Adjust your viewpoint. Save an image file for 3rd powerpoint slide.

Then turn Colorforce and Minimize back on and repeat the process in this box. Push the Glu from just outside to mid-channel, mid-channel to fully inside.

Molecular modeling, Homework 12! ! Due Apr 25, 2014

(11) Make a 3rd powerpoint slide using the three saved images. Did you use the same point of view for each image? I hope so. Add a caption. I made these examples,... but you should zoom in more and add captions.

A good pathway will have favorable charge-charge interactions and hydrogen bonds as it moves to its binding site. Does it? The Glu should not flip its orientation between snapshots. Does it? Answer using a caption. Label a few key residues (You may do this in Powerpoint rather than on MOE if you wish)..

Molecular modeling, Homework 12! ! Due Apr 25, 2014

(12) For 25% extra credit, dock the drug dysiherbaine in the same way. Create the drug using Builder. Be sure to run Compute | Structure preparation, Protonate3D, and energy minimize the drug molecule first, before docking.

Molecular modeling, Homework 12! ! Due Apr 25, 2014