Structural Bioinformatics: Comparative Modeling

1. Structural Bioinformatics: Comparative Modeling Target T0205 5th Best in world-wide CASP5 experiment sb.nrbsc.org

2. Towards a High-Resolution Understanding of Biology Structural Biological Analyses can provide the ultimate insight into the mechanism behind a biological function, understand how biological function follows structure.

4. Aliphatic, Hydrophobic, Important for binding hydrophobic substrates, modulating binding sites

6. Hydrogen bond donors and acceptors Can lose a proton to act as nucleophiles

7. Hydrogen bond donors and acceptors Assist in catalyzing reactions

8. Positively charged amino acids, Can donate a proton as part of enzymatic reactions, general acids, electrophilic

9. Negatively charged, hydrogen bond acceptors, can abstract a proton as part of enzyme mechanism, general bases, nucleophiles

10. Can participate in cation-pi interactions, hydrogen bonding, hydrophobic interactions. Tyrosine can donate or accept protons.

11. Rotation around Ca-C = psi Rotation around C-N = phi Ramachandran plot Tertiary structure of MsrA

12.  helix: 3.6 amino acids per turn, with H-bonds between every 4th residue Secondary Structure Elements Secondary structure refers to the interactions that occur between the C=O and NH groups on amino acids in a polypeptide chain, and form helices, sheets, turns and loops.  sheet: Formed by H-bonds between 5-10 consecutive amino acids in one section of the chain with another 5-10 in another section

13. Tyrosine 7 Glutathione S-Transferase backbone structures from 100 picosecond MD simulation. Lys121

14. Structure Determination by x-ray crystallography or NMR is still relatively difficult and expensive Synchrotron in Grenoble, France. At present there are only 57 synchrotrons in the world 750MHz NMR

15. Cryo-Electron Microscopy • Allows visualization of structure and dynamics of biological assemblies at resolutions spanning from molecular (20-30 Ǻ) to near atomic (3 Ǻ). • Near atomic models can be built by combining information from high resolution structures of individual components in the complex with low resolution structure of entire assembly.

16. Sequence-Structure Disparity More than 2.5 million proteins have been sequenced (Maroon). Only 45,000 structures have been experimentally solved (Turquoise). Structure prediction methods can provide a method to address this disparity

17. Basis of Comparative Protein Modeling • Predicts the three-dimensional structure of a given protein sequence (TARGET) based on an alignment to one or more known protein structures (TEMPLATES) • If similarity between the TARGET sequence and the TEMPLATE sequence is detected, structural similarity can be assumed. Structural superposition of ALDH Family members Comparative Protein Models will be increasingly utilized to help solve biological problems

18. Basic Comparative Protein Modeling Procedures Start End Yes Model ok? Identify templates No Select templates Evaluate the model Align target with template Build the model

19. Identifying Templates by Sequence-based methods • BLAST, PSI-BLAST • Use MEME program to identify motifs • Increase the signal-to-noise ratio by using patterns called “motifs” as the query. Motifs describe only a small portion of the query sequences which reduce chance similarities. • MAST (Motif Alignment and Search Tool) • http://meme.sdsc.edu/meme/website/mast.html

20. Basic Comparative Protein Modeling Procedures Start End Yes Model ok? Identify templates No Select templates Evaluate the model Align target with template Build the model

21. Factors to Consider in Selecting Templates • Phylogenetic tree construction can help find the subfamily closest to the target sequence • Consider Multiple Templates if possible. Other concerns: Ligands present? Environmental conditions? pH • Some structures have been solved at multiple resolutions.

22. ALDH2(blue)/ALDH1(yellow) overlay ALDH2/ALDH3(red) overlay • Sequence Identity w/ ALDH2RMSCD over domains • ALDH1 67% < 1.0 Ǻ • ALDH3 27% > 2.0 Ǻ • RMSCD (root mean square coordinate difference) over Cα atoms.

23. Selection of the Correct Template Critical Assessment of Structure Prediction (CASP6) Results Target T0282: Rank and Name: GDT (% correct): 1. Ginalski 70.6 2. Skolnick 70.4 3. Venclovas 68.2 93. Wymore (1PQ3) 57.09 (3 ) Wymore (1GQ6) 70.1 Would have been 3rd best

24. Basic Comparative Protein Modeling Procedures Start End Yes Model ok? Identify templates No Select templates Evaluate the model Align target with template Build the model

25. Initial 3D-Model Construction • Essentially copy coordinates of equivalent atoms from the template to the target structure. • Internal coordinates are used for remaining unknown coordinates • Generate stereochemical and homology derived restraints Template: -GGMG- Target: -GGKG- Template: -GGSG- Target: -GGTG-

26. Errors in Homology Modeling a) Side chain packing b)Distortions and shifts c) no template ---template Actual Model

27. Errors in Homology Modeling d) Misalignments e) incorrect template Marti-Renom et al., Ann. Rev. Biophys. Biomol. Struct., 2000, 29:291-325.

28. Comparative Modeling Tutorial Determining the Basis for Stereoselectivity in R- and S-HPCDHs ? R-HPCDH S-HPCDH

29. Tutorial Philosophy • Tutorial is currently appropriate for students that have completed either an organic chemistry or biochemistry undergraduate class. (??) • Goal is to integrate it into a sequence-based bioinformatics course • Attempting to teach bio-molecular structure through the web without having to learn “any” technical details about visualization or modeling software. Can choose to be more intimate with programs. • Tutorial progresses in a logical manner from learning about the properties of the substrate to the binding interactions between enzyme and substrate and finally to understanding the enzyme mechanism. • Comparative modeling is the tool by which to construct a structure-function relationship. We are striving to weave structure-function relationships throughout all the tutorials.

30. C C O C S C C O S O R-Hydroxypropylthioethanesulfonate {R-HPC}

32. The enzymatic oxidation of HPC is accomplished through a proton transfer from the substrate to Tyr155 and a hydride transfer from the substrate to NAD.

35. Enhancing the tutorial in the future • Students could read the journal article that reports on the kinetic characterization of wild-type and site-directed mutants before the tutorial to see what was thought about the enzyme prior to the structure being solved. Biochemistry, 2004, 43:6763-6771 • What did the authors correctly predict? What details were probably surprising to the authors upon solving the structure? • Start off examining the deposited PDB id: 2CFC. The deposited structure contains a co-crystallized product molecule. How would the structure differ if we could examine the enzyme bound to the reactant? Compare between x-ray model and computational model. • Examine the NAD binding residues. Are they conserved? • Docking of the substrate into the S-HPCDH binding site. • Would this tutorial work in reverse? (i.e. If the structure of S-HPCDH had been solved and we were asked to model R-HPCDH)