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MOLECULAR MODELING Exercise 1: Homology modeling of the Histamine H1 receptor

David Rodríguez (david.rodriguezdiaz@dbb.su.se), Jens Carlsson (jens.carlsson@dbb.su.se) Stockholm University, 2014

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 (http://gpcr-modsim.org). 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 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.

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

• IMPORTANT NOTE: do not submit MD simulations during this practical! As all publicly

available webservers, GPCR-ModSim has limited resources. MD simulations are very intensive calculations, so if you submit such a job during this session, there is a risk that you would overfill the queue and you and your classmates will not be able to complete the basic steps we need to perform today. We will here focus on learning how to make homology modeling of GPCRs. You may have the chance to run your MD simulations for your drug design project after Christmas, we will advise you in this regard.

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

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1. Login: You have been instructed to apply for an account on the server during the lectures, so please use your account! Log in through the corresponding link in the upper part of the screen. A new (highlighted) button named “Model a GPCR” will appear. Click on it.

• General tips for using GPCR-ModSim:

o Tip 1: If you get lost at any point, just return to the Projects link in the upper part of the screen. All the work you have ever done will always be stored there.

o Tip 2: If at any time you need to go back to the main screen after a new box pops

up, just click on the background of the browser window.

o Tip 3: if you have relevant technical issues related to the server after this practical, always contact the GPCR-ModSim support team first (http://gpcr-modsim.org/feedback/). They are very dedicated and efficient, and will provide you with a solution in an appropriate and timely manner. Unfortunately, teachers of this course will not be able to solve such issues for you. Contact us in this regard only if, after trying the default route, the problem persists for some reason.

2. Retrieve the query sequence: Set the Name of your project as you wish, we recommend “hH1_il3”, for instance. The server allows you to input the sequence of the receptor you aim to model either with its Uniprot ID (introducing the code in the Sequence box, or following the help link), or as FASTA format. 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

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◦ Tip 1: We typically use 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 2: Following a convention in the software MODELLER, if we would like to model a chain

break (e.g. in the IL3, where the T4L will be absent) we must indicate this with a slash (‘/’). See sequence marked in bold above. Despite that it will be inputted, you will have to add it by hand later on, an explanation on how to do it will follow.

We can now proceed to select the templates (active-like GPCRs or inactive-like GPCRs) from the Group drop-down list box, and click on the Submit button. As doxepin is an antagonist of the Histamine H1 receptor, we suggest that you select the GPCRs inactive set. Do not select the “GPCRs inactive 14” nor “GPCRs inactive 19” for this exercise, since they already include hH1. Do not choose the “GPCR active” either, for the reasons explained above.

3. Select your template: The server now generates a multiple sequence alignment (MSA) with

your query sequence, against the available GPCR templates (crystallographic structures).

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You will see two links below your sequence for downloading or editing your MSA. With the latter option, you can interactively modify your alignment through a Jalview applet, we will return to this point later. You will also be provided with some statistics regarding the initial alignment created by the server, including the best template and the corresponding sequence identity in the transmembrane region (“Score”). Moreover, you can click on the Graph link in order to see a chart showing the different homology percentages against the templates of the selected profile (in this case, the first seven crystallized GPCRs in inactive-like conformation):

i) The full query sequence. ii) The sum of the TMH regions. 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. However, you might want to change this selection based on the basis of the other similarity criteria, or simply because of accumulated knowledge on the molecular biology of the system. You will be able to do so in the next step (see point 4). Before running the homology modeling calculations, we should have a look to the alignment. In order to do that, go to the View/edit alignment link. This will open JalView, which is a Java applet that for security reasons requires your permission to run (do not worry, it is safe). Just accept it and click “Run”. You will have to scroll down to the bottom to see the sequence you have inputted. You will see the TM alpha helix profile just above it, and the sequences on top are the possible templates you can use for modeling your sequence. Now you are ready to manually modify the alignment following the instructions below:

▪ Press F2 to enter the edit mode. A black rectangle should appear. ▪ Space inserts a gap, delete or backspace removes one. ▪ Alt + cursor key moves the whole sequence. ▪ Remember to upload ("Save" button below) the edited sequence! Otherwise your

changes will not be recorded. ▪ More info here.

In this example, the alignment to the template that we will use is to the hβ2 adrenergic receptor. It is very important to make sure it is correct, so always double-check the alignment! For instance, is the GPCR-conserved disulfide bridge correctly aligned? Once you think that all is fine in this regard, contact the teacher to verify it, and save it as a FASTA file from the JalView applet. If you have any doubt in any step of this process, do not hesitate to ask. After this, go ahead to the next step. You may need to do more extensive modifications in the MSA for the drug design project you will do after this session. For that case, we would recommend you to use the “GPCR inactive 19” template set, more up to date than the one we are using in this tutorial, and also provides better alignments of relevant motifs like the GPCR-conserved disulfide bridge. If any other disulfide bridge occurs on the template and the involved cysteines are aligned for your target sequence, the disulfide bond will be also modeled without any further information

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from the user. You can also introduce ad-hoc restraints in MODELLER to achieve disulfide bridges that are not present in your template (see below). 4. Generate homology-based models: once your alignment is set up, just click on the “Model!”

button on the right bottom corner.

Up to 50 homology models can 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 (please request only 10, which is enough for this practical), the template to be used (use hβ2 receptor, with PDB code 2rh1), and input any additional disulfide bridge in your query sequence (would you like to do that? Why?). Do not tick the Lennard-Jones restraints box, it is not necessary for this case. Click on “Model” and see the progress of the calculations interactively. If some job(s) takes much longer (minutes) than the others, just refresh your browser. Now you are ready to analyze your models. They are named after their DOPE-HR score (the more negative the better) and the template used. If you click on them you can view and download the resulting PDB file. 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 (hint: “Loop!” button).

◦ Tip: Visual inspection is an important check in this step. Download the tar file with all of

your models and use PyMOL to open the “align.pml” script (you can see the instructions used in that text file). This is useful, i.e. to check the orientation of selected side chains as those in the binding site, within the different models.

5. Refine loops: In the next step, you should try to refine extracellular loop (EL) 2 (which often is a big problem in modeling). In this case we will focus in the region that is important for ligand binding: the stretch that goes from the GPCR-conserved disulfide bridge to TM5. Identify this part locally with PyMOL, avoid to use the JMol applet since its browser window is a bit buggy. Write down the residue numbers delimiting that region (exclude the cysteine of the disulfide). Now use the loop modeling tool to rebuild it! Request 5 models, selecting the 6 residues following the cysteine from the conserved disulfide bridge (excluding the cysteine, again). Do not worry if the residue numbers given by default by the server don’t match yours, just fill the data and click the box to optimize only your residue range. The optimization will take a few minutes, depending on the workload of the server. Check the progress in your project page by refreshing your browser from time to time.

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How to introduce a chain break in GPCR-ModSim

You do not need to do this for this practical, but it is a good practice since you may need to know how to do it for your drug design project. Often GPCRs have divergent sequences for the IL3, so we focus on having a good alignment with the intracellular tips of TM5 and TM6. If you do not tell anything to MODELLER, it will try to artificially join them. This does not affect your modeling significantly, but is always good to be as accurate as possible. In order to test this:

• Create a new project called “hH1_IL3_chainbreak”.

• Edit your alignment as you have done before.

Given that JalView does not allow introducing characters in the main menu, we will have to do a workaround:

• Go to File -> Output to textbox -> FASTA.

• Delete all the sequences except the last one (the one you want to model).

• Insert the ‘/’ character where it was shown in the original sequence (page 2), and delete only one of the gaps (‘-‘) that follow (to compensate the added character). Click on “Add to current alignment”.

• You will see a new sequence added. Double-check the MSA, and see that it matches the previous H1 sequence, as well as the templates. Then delete the old H1 sequence (just below the Mask) by clicking on its name and doing Edit -> Delete.

• Finally you are ready to go, but don’t forget to click on the Save button! Request 10 models. As you will be able to see, they will be divided in two chains (A and B), before and after the chain break that you have introduced. Loop modeling may not work in this case, we will advise you in this regard before the start of your drug design project.

6. Membrane insertion and MD simulations. This step will also be performed in your drug

design project, not in this practical. If you accidentally click the button “Run dynamic”, do not panic, it can be sorted out. Just let the teacher know as soon as possible.

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Using the obtained models, answer the following questions: 1. Explain how homology modeling (or comparative modeling) works. What is the goal of the DOPE-HR score? Briefly, how is it calculated?

2. Compare your models to the template structure (compare backbone using ribbon and, side chains using lines) – how do they differ? How are the models different from each other?

3. Compare your models to the experimental structure of the H1 histamine receptor (PDB ID: 3RZE) using ribbons and lines. Where do the model and experimental structure differ most?

Hint: Use the PyMOL command “align X,Y”.

4. 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!

5. When you do the loop modeling on EL2: 4.1. Which residue range have you optimized? From ___ to ___ 4.2. What is the residue range you have defined for the EL2 region? From ___ to ___ 4.3. How does this region specifically compares to the experimental structure? 4.4. Does it get better after the Loopmodel optimization? 6. Have a look to Fig. 4 of this paper: http://dx.doi.org/10.1021/ci5002235. There you will see a MSA for EL2 of aminergic receptors based on their structures. Does your template-target (β2 ADR/ H1HR) alignment for this region of the protein look the same? If there are differences, how could they have affected the accuracy of your predictions? Would have Loopmodel corrected these? 7. 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 17th of December! If you pass the first time, you get one bonus point to the exam.