1. Bioinformatics 2 -- Lecture 8 More TOPS diagrams Comparative modeling tutorial and strategies.

2. Principles of Comparative modeling Proteins that have common ancestors have the same fold. Changes in structure lead to changes in function : enzyme reaction mechanism, ligand binding specificity, signaling, sub-cellular location, stability, etc. We can infer functional differences from structural differences. We can use energy calculations and simulations to find structural differences. note: Comparative modeling == Homology modeling

3. What can we do by molecular modeling? Structure-based drug design Examples: trimethoprim, HIV protease inhibitors. Protein design Examples: TNT binding protein Function prediction Examples: structural genomics

4. TOPS topology cartoons A simple way to draw a protein beta strand pointing up beta strand pointing down alpha helix A parallel beta sheet An anti- parallel beta sheet connections

5. Reminder: TOPS diagrams number strands and helices draw connections in front (middle) or back (side) 4 3 12 4 2 31 N C 3-layer 2-4-2  sandwich, mixed, up-down-up-up 4312.

6. Draw barrels on a circle all anti-parallel beta-barrel, closed, n=6, 125436 6 5 N C 3 1 2 4

7. TOPS tips Draw beta strands together only if they are H-bonded. Draw beta strands in a circle if they form a barrel. If there are multiple domains, draw them as clearly separated cartoons. If you can’t arrange all secondary structures perpendicular to the screen, find the best approximate solution. (For example, look at helix 3 in the example) Ignore short helices. Sometimes a loop is really a strand. Check H-bonding if in doubt. Beta strands that are close to each other are not necessarilly in the same sheet. Again check H-bonding.

8. Practice drawing TOPS cartoons Draw TOPS cartoons for the following proteins (downloadable in MOE using File-->Protein Database) easy: 1SH11AB1 hard: 1K77.A1FUS harder: 1IKO.P4SBV.A Draw the TOPS cartoon and name the fold SCOP-style.

9. Comparative modeling requires good bookkeeping skills. Proteins are big complicated molecules. Modeling them requires a plan. Alignments must be modified and re-modified. Structurally conserved regions (SCRs) must be identified. Quality of loops must be assessed. Good quality regions can be ‘fixed’ (frozen) while others are modified. Residues known to have functional significance need special consideration.

10. Planning a homology modeling project Choosing a Target: Find out what is known about the sequence you wish to model. Where did it come from? How was it discovered? Is the function known? Choosing templates: Do a database search to get families of known structures, sometimes called a “basis set”. Repeat the search if necessary using parts of the sequence. Study the alignment and edit it. Merge multiple templates into one if necessary. Bookkeeping: Make a simple TOPS model for your template and label it. Refer to this when building the model. Keep track of what is homology-based, and what is not.

11. Planning a homology modeling project Alignment: Start with the automatic alignment from Dynamic Programming. Inspect locations of gaps and insertions. Modify alignment if necessary. Every residue that is aligned is considered to be structurally conserved ! If you do not believe it should be structurally conserved, unalign it or re-align it. Model building: Carry out the loop search, splicing, sidechain rotamer search, energy minimization. Automated model building part: (1) Place aligned sidechains based on identities, similarities. (2) Search for loops to model insertions and deletions. (3) Swap loops, choose lowest energy. (4) Place loop sidechains, (5) Energy minimize

12. Planning a homology modeling project (3) For a detailed description of the automated MOE-Homology method, read the “ promodel.htm ” page under: MOE: Help-->Tutorials-->Homology Modeling.. click on “ Building 3D Protein Models ”

13. Run the MOE Homology Modeling tutorial Help-->Tutorials-->Homology Modeling aka Comparative modeling Run the tutorial word-for-word except... Stop at the points indicated in the following slides and do the exercises.

14. 1st stopping point: After aligning structures Draw a TOPS diagram of the structure. Number the beta strands N to C. Number the alpha helices N to C, independent of the beta strand numbering. Your template sequence is now summarized as:                  

15. Rearrange sequences Open the Command window after running Homology-->Align. You will see the identity table. Move the sequences around to put the most similar together, keeping the query at the top. pro_Align: pairwise percentage residue identity Chains 1 2 3 4 5 6 7 8 1:3014 22.9 22.9 18.4 22.0 20.0 12.0 17.2 2:1KTE 24.7 78.1 15.8 30.5 29.4 15.0 23.0 3:1B4Q.A 24.7 78.1 13.2 32.9 28.2 18.0 21.8 4:1H75.A 14.4 11.4 9.5 15.9 12.9 7.0 18.4 5:1FOV.A 18.6 23.8 25.7 17.1 32.9 19.0 29.9 6:1EGO 17.5 23.8 22.9 14.5 34.1 18.0 25.3 7:1J0F.A 12.4 14.3 17.1 9.2 23.2 21.2 16.1 8:1AAZ.A 15.5 19.0 18.1 21.1 31.7 25.9 14.0 not in order

16. Rearrange sequences Modify gaps if nexessary. Constrain using Edit-->constrain residues Re-run Homology-->Align. pro_Align: pairwise percentage residue identity Chains 1 2 3 4 5 6 7 8 1:3014 21.9 21.9 22.0 20.0 21.1 9.0 16.1 2:1KTE 23.7 78.1 30.5 30.6 18.4 7.0 23.0 3:1B4Q.A 23.7 78.1 32.9 29.4 14.5 11.0 21.8 4:1FOV.A 18.6 23.8 25.7 35.3 21.1 6.0 28.7 5:1EGO 17.5 24.8 23.8 36.6 15.8 11.0 24.1 6:1H75.A 16.5 13.3 10.5 19.5 14.1 4.0 21.8 7:1J0F.A 9.3 6.7 10.5 7.3 12.9 5.3 6.9 8:1AAZ.A 14.4 19.0 18.1 30.5 24.7 25.0 6.0 reordered

17. Evaluate structural conservation in the basis set For each secondary structure and each intervening loop/coil region, look at it carefully. Is it conserved? Are the lengths different? Are any of the loops the same length as the query? Enter a short note for each ss and each loop:    low rmsd, sometimes not present     loop  short, not variable.    SCR (short for Structurally Conserved Region)     loop  variable. Tmpl 2,3,4,5,6 matches.    SCR Bottom line: If you see a SCR, don't put a gap there.

18. SCRs Structurally Conserved Regions (SCR) are assumed to be evolutionarilly invariant. SCRs should be ‘fixed’ during energy minimization. Initially all atoms, and eventually just the backbone atoms.

19. Loops Three types of loops: Designated (conserved) Loop: coordinates derived from homology to the template, not a loop search. May be flexible. If so, don't fix during energy minimization. Variable Loop: Variable from model to model in the basis set. Coordinates derived from a loop search or one of the templates, or constructed by hand. Not fixed during energy minimization Outgap (extension): a variable loop at the end of a chain, usually constructed by hand from a secondary structure prediction.

20. continue tutorial. Now choose a template and run SE:Homology-->Homology model. You may choose minimization=none for faster results, or minimization=coarse for better results if you have time. You may set the number of models to 5 for faster results. Exercise 3. Turn in the MOE file after running the tutorial exactly according to the instructions. Due Feb 14 Exercise 4: Refine your model by re-aligning and manual intervention. (see Lecture 9) Due Feb 19.