Molecular modeling 2012: Exercise 1: Homology modeling of the Histamine H1 receptor

The goal of this exercise is to you will go through the different stages covered by the GPCRModSim server for automated GPCR modeling and simulation. The server uses homology modeling with the program Modeller to predict structures of G protein-coupled receptors, which is also the focus of the structure-based drug design project.

• We will work on the human histamine H1 receptor (hH1R). This receptor was recently crystallized in complex with the antagonist doxepin (PDB entry 3rze). The T4L crystallization strategy was employed, thus the third intracellular loop is not observed in the structure. In addition, a fragment of the N-terminus and C-terminus, as well as the most solvent exposed section of the long second extracellular loop are not determined in this crystal structure.

• Work flow: A brief flowchart of the whole procedure is shown in the following chart.

• Proceed! The next screens will show you step by step what to do and, most important, why you do it.

1. Login: You have been instructed to apply for an account during the lectures, so please use your account! Click on the “Model your GPCR” button on the left panel.

2. Retrieve the query sequence: The server allows you to input this information either as or as

fasta format. The best way is to retrieve the sequence from uniprot (http://www.uniprot.org/). The header line (optional, starting with the sign: >) designates the title of the project that will be created on the server, in the current example 3rze_A (see figure 3). In this case, we will supply you with a (modified) sequence of the human H1 histamine receptor.

>H1 histamine receptor, modified in ICL3 TTMASPQLMPLVVVLSTICLVTVGLNLLVLYAVRSERKLHTVGNLYIVSLSVADLIVGAV VMPMNILYLLMSKWSLGRPLCLFWLSMDYVASTASIFSVFILCIDRYRSVQQPLRYLKYR TKTRASATILGAWFLSFLWVIPILGWNHFMQQTSVRREDKCETDFYDVTWFKVMTAIINF YLPTLLMLWFYAKIYKAVRQHC/LHMNRERKAAKQLGFIMAAFILCWIPYFIFFMVIAFC KNCCNEHLHMFTIWLGYINSTLNPLIYPLCNENFKKTFKRIL

◦ Tip 1: If a “>” line does not exist, the project will be named literally “your sequence”. ◦ Tip 2: We typically use only the fasta sequence corresponding only to the regions observed

in the crystal structure of the receptor. This will facilitate a proper comparison between the obtained models and the experimental crystal structure. In the above sequence we have deleted intracellular loop three (because it does not exists in the template structure).

◦ Tip 3: Following a convention in the software Modeller, if we want to model a chain break (i.e. in the IL3, where the T4L will be absent) we must indicate this with a backslash (see fig 2). See sequence marked in bold below.

We can now proceed to select the templates (active-like GPCRs or inactive-like GPCRs) from the top right window, and click on the “submit” button. As doxepin is an antagonist of the Histamine H1 receptor, we suggest that you select the inactive set:

3. Select your template: The server now generates a multiple sequence alignment (MSA), which can be inspected and manually edited through the Jalview applet. A chart is provided, showing the different homology percentages, corresponding to: i) the whole query sequence, ii) the sum of the TMH regions and iii) each of the 15 regions of a GPCR structure (i.e., 7TMHs, 3ELs, 3ILs, plus amino- and carboxy-terminus). This allows the identification of regions that influence the total sequence similarity. Without any additional information, we recommend that you use the 7TMH sequence identity as a major indicator of the suitability of a template. If you want to modify this selection, on the basis of the other similarity criteria or simply because of accumulated knowledge on the molecular biology of the system, just click on a different template on the “templates” window.

◦ Tip 1: You can manually modify the alignment using the JalView applet following the instructions bellow: ▪ Click the above applet and press F2 to enter the edit mode (a black rectangle should

appear). ▪ The red square is the area you are editing. Click anywhere to reset it to the whole

sequence. ▪ Space inserts a gap, del or backspace removes one. ▪ Alt + cursor key moves the whole sequence.

▪ Remember to upload ("Save" button below) the edited sequence. ▪ More info here.

In this example, the alignment to the template that we will use the hβ2 adrenergic receptor and the alignment to this protein is essentially correct, so you can just go ahead to the next step. However, in your drug design project, the sequence may need modifications. 4. Generate homology-based models: Up to 10 homology models will be generated on the basis

of the pairwise alignment with the selected template. This step makes use of the software modeller, thus you must provide the modeller key in the corresponding box. Then you select the number of desired models (we recommend 10) and input any additional disulfide bridge in your query sequence. Click on “Model” and wait for a few minutes. ◦ Tip 1: Please note that the conserved disulfide bridge between EL2 and TM3 will be

maintained, provided that the two cystein residues involved are well aligned with the template (this occurs in all cases examined to date). If any other disulfide bridge occurs on the template and there is homology in the alignment with the corresponding cystein

residues, the bridge will be also modeled without any further information from the user.

Now you are ready to analyze your models. They appear sorted by their most favorable value of the scoring function (DOPE-HR). In addition, a Ramachandran plot is provided as PDF file, so you can check the stereochemistry quality. There is not a magical rule of the thumb to select the most appropriate model, but a combination of these two values is usually a good idea. In this case, the best model according to DOPE-HR has also good stereochemical quality, so we will select this one for the next step (check box selected by default on the best DOPE-HR model). ◦ Tip 1: Visual inspection is an important check in this step. Download the tar file with all of

your models and use the pymol to align them (type “align X,Y” on the command line, where “X” and “Y” are two models). This is useful, i.e. to check the orientation of selected side chains as those in the binding site, within the different models.

Refine loops: In the next step, you should try to refine extracellular loop two (which often is a big problem in modeling). Identify this part of the structure and use the loop modeling tool to rebuild it! Don’t forget to specify the disulfide bridge! Membrane insertion and MD simulations (optional, this may take 24 hours)

Using the obtained models, answer the following questions (the report should be about 1 page long): 1. Explain how homology modeling (or comparative modeling) works! How is the DOPE score calculated? 2. Compare your models to the template structure (compare backbone using ribbon and, side chains using lines) – how are they different? How are the models different from each other? 2. Compare your models to the experimental structure of the H1 histamine receptor (PDBid: 3RZE) using ribbons and lines. Where do the model and experimental structure differ most? Hint: Use the pymol command “align X,Y”. 3. Compare the orthosteric (binding site) of the model and crystal structure (show Doxepin in sticks). Would the ligand fit in the models? Include some nice figures! 4. When you do the loop modeling – does it get better? 5. As a last exercise, I’d also like you to repeat the above with a different template. This time you should select a really bad one. Which one did you choose? Then compare this model to the experimental structure – are the models good?

This is also the kind of questions you should ask yourself when you are carrying out the drug design project!

The final report should be handed in before the 4th of January! If you pass the first time, you get one bonus point to the exam.