Comparative Modeling for Protein Structure Prediction

1. Biology 5412Structural Bioinformatics IIDemonstrationProtein Structure Prediction by Comparative Modeling Roland L. Dunbrack, Jr. Institute for Cancer Research Fox Chase Cancer Center Roland.Dunbrack@fccc.edu http://dunbrack.fccc.edu

2. http://dunbrack.fccc.edu/kinases/DunbrackLecture1_2017.pptx http://dunbrack.fccc.edu/kinases/DunbrackLecture2_2017.pptx http://dunbrack.fccc.edu/kinases/DunbrackLecture3_2017.pptx

3. Sequence Analysis For a protein of interest, e.g. human AKT3 Get sequence from Uniprot (under Format tab, select FASTA). http://uniprot.org On Uniprot page: Domains, variants, PTMs, isoforms, PDBs, interacting proteins Disorder prediction. http://iupred.enzim.hu/ Pfam: http://pfam.xfam.org/search ProtCID for these PDBs/Pfams: http://dunbrack2.fccc.edu/protcid Prediction with: Hhpred/Modeller, SwissModel, Robetta, Multicom, I-Tasser Build complex with ligands/DNA/other proteins with SCWRL4

4. Websites Wiki https://en.wikipedia.org/wiki/List_of_protein_structure_prediction_software Hhpred/Modeller http://toolkit.tuebingen.mpg.de/hhpred(Modeller password: MODELIRANJE) Robettahttp://robetta.bakerlab.org/ Phyre2http://www.sbg.bio.ic.ac.uk/phyre2/ SwissModel http://swissmodel.expasy.org/ Multicom http://sysbio.rnet.missouri.edu/multicom_cluster/ I-TASSER http://zhanglab.ccmb.med.umich.edu/I-TASSER/ SCWRL4 http://dunbrack.fccc.edu/scwrl4/ BioAssmblyMdlr http://dunbrack.fccc.edu/BAM/ CAMEO http://www.cameo3d.org/sp/3-months/ ProtCID http://dunbrack2.fccc.edu/protcid/ Uniprot http://uniprot.org Pfamhttp://pfam.xfam.org/ IUPred http://iupred.enzim.hu/ Exacs http://exac.broadinstitute.org/ (exome sequences) Kinome http://kinase.com/human/kinome/ Kinase families http://dunbrack.fccc.edu/kinases/humankinome.txt Kinase alignment http://dunbrack.fccc.edu/kinases/humankinases.mfa Kinase secstr http://dunbrack.fccc.edu/kinases/0features.txt Kinase pse files http://dunbrack.fccc.edu/kinases/index.html Jalview http://www.jalview.org/

5. Accuracy of Models vs. Purpose of Modeling Baker and Sali, Science294:93-6(2001)

6. SwissModel http://www.uniprot.org/uniprot/AKT3_HUMAN.fasta http://swissmodel.expasy.org/

7. HHPred/Modeller http://toolkit.tuebingen.mpg.de/hhpred/results/AKT3_HUMAN

8. Modeller Good at multiple templates Bad side chains https://salilab.org/modeller

9. Modeller Input Multimers: https://salilab.org/modeller/manual/node29.html

10. Hhpred/Modeller http://www.uniprot.org/uniprot/AKT3_HUMAN.fasta http://toolkit.tuebingen.mpg.de/hhpred http://toolkit.tuebingen.mpg.de/hhpred/results/AKT3_HUMAN http://toolkit.tuebingen.mpg.de/modeller/results/AKT3model Modeller Password: MODELIRANJE

11. Overall AlgorithmsI-TASSER (Zhang, U. Michigan) http://zhanglab.ccmb.med.umich.edu/I-TASSER/output/S316711/

12. RaptorX http://raptorx.uchicago.edu/StructurePrediction/myjobs/97137846_213404/

13. Phyre2 http://www.sbg.bio.ic.ac.uk/phyre2/webscripts/jobmonitor.cgi?jobid=f4f4c22086e10837

14. Robetta Server http://robetta.bakerlab.org/results.jsp?id=72797&p=1

15. SCWRL4 1. Model monomer(s) with other programs 2. Superpose models into complex from template that contains binding partners, e.g. heterodimer – model both monomers, superimpose onto monomers of template heterodimer. Complex with DNA – superpose onto template protein in protein-DNA complex 3. Write out coordinates of transposed protein(s) to be modeled = input.pdb 4. Put ligands and nucleic acids into separate PDB file = frame.pdb 5. Run SCWRL4 on command line: % Scwrl4 –i input.pdb –f frame.pdb –o output.pdb > logfile.txt 6. Concatenate output model and frame file into model of complex % cat output.pdbframe.pdb > ERCC3withDNA.pdb

16. Structural Bioinformatics of Protein Kinases Manning G et.al. Science (2002)298: 1912-1934.

17. Kinase Structures C-helix ATP A-loop Mg ion Catalytic loop Substrate 3BU5

18. Active Kinase Structures Salt Bridge Hydrophobic spine INSR Kinase 1IRK 3BU5 Regulatory and activation spines: Kornev and Taylor, PNAS, 2008

19. Active and Inactive Kinase Structures Inactive form Active form Hydrophobic spine 1IRK 3BU5 All active kinases are alike. Each inactive kinase is inactive in its own way. Tolstoy, Anna Karenina

20. Catalytic HRD motif DFG (act. loop start) APE (act. loop end)

21. DFG (ASP-PHE-GLY) Motif C-helix C-helix PHE PHE ASP ASP ATP Pocket 3BU5 3ETA DFGin ACTIVE DFGout INACTIVE

22. Type I and Type II Inhibitors DFGin DFGout 2GQG 2HIW Type I Type II

23. Gleevec is a Type II inhibitor 1IEP 2OIQ Inactive loop Strong binding to ABL Weak binding to c-SRC

24. BUT, there are more than two states… Kinase activation is not a binary process "DFGup" is an intermediate orientation between DFGin and DFGout Found in different kinases like AURKA, BRAF, IGF1R and SRC A variety of names in literature, not discussed as a group (SRC-like, etc.) Adistinct conformational state in kinases, not well characterized or exploited for inhibitor design DFGup DFGout DFGin AURKA

25. Conformational states in BRAF DFGout DFGin DFGout DFGup Cluster 1 & 2 Cluster 3, 4 & 5

26. Conformational states in AURKA DFGup DFGup DFGout DFGin Cluster 1, 2, 3 and 6 Cluster 5, 7 and 8

27. DFGin-Active – Phe under C helix and pointing downwards, loop to the right, salt-bridge and spine intact DFGin-Inactive – Phe under C helix and pointing downwards, loop disordered or pointing towards the viewer, salt-bridge and/or spine broken DFGup1-Inactive – Phe under C helix or adjacent to it, and pointing upwards; loop disordered or towards viewer DFGup2-Inactive – Phe out of pocket and some distance from C helix, usualy pointing upwards; loop disordered or towards viewer DFGout-Inactive – Phe completely away from pocket, and switched places with Asp which is not in pocket; loop disordered and/or in front of kinase

28. http://dunbrack.fccc.edu/kinases