Peptide Design

1. Peptide Design Kyle Roberts March 4, 2008

2. Peptides in Biology • Create peptide antibodies • Used in mass spec to identify proteins • Can probe protein-protein interactions • Function as protein ligands • Antimicrobial Peptides http://employees.csbsju.edu/hjakubowski/classes/ch331/protstructure/phipsi.gif

3. Motivation for Peptide Design • Understand how the basic components of proteins function and interact • Abstract out general rules that can be applied to understand protein folding • Design useful and novel protein binders and inhibitors • Utilize the growing pdb structures to refine structures and build novel ones • Apply knowledge to chemicals similar to peptides to create novel structures (foldamers)

4. Peptide Backbone Reconstruction • Reconstruct an all-atom peptide model from a subset (Cαs or Cβs) of the atomic coordinates Adcock SA. Peptide backbone reconstruction using dead-end elimination and a knowledge-based forcefield. J Comput Chem. 2004 Jan 15;25(1):16-27.

5. Uses of Backbone Reconstruction • Enhancing low-resolution structures • Conversion of coarse grain structures into all-atom models • Ab initio folding • *Comparative modeling techniques* • Normal mode analysis

6. Current Methods • Use fragment libraries and construct the backbone with energy, homology or geometric criteria • Perform de novo construction with geometric or energy criteria • Statistical positions and frequency tables • Molecular dynamics and Monte Carlo • Maximize peptide dipole alignments (max H-bonds)

7. Algorithm Overview • Input: Cα or Cβ coordinates • Generate a library of peptide sequences of length 3 • Overlay the peptides on the input coordinates • Use dead-end elimination to find the global energy minimum conformation Library of amino acid peptide sequences (length 3 ) Overlay peptides on input coords Dead End Elimination Predicted Structure Cα or Cβ coordinates

8. Peptide Backbone Fragments • Selected three-residue backbone fragments from 1336 random nonredundant PDB structures • All the fragments were aligned into a standard frame • Fragments were clustered by RMSD and duplicates were discarded

9. Fragment Overlap • Fragments were overlapped with the input coordinates by minimizing the sum of squared distances • Kearsley’s method: state the minimization problem as an eigenvalue problem with quaternion algebra • not iterative • improper rotations aren’t produced • no special cases • RMSD is easy to calculate

10. Dead-End Elimination • Minimize the energy function: ri ri is residue r with the backbone conformation i Energy rj Image: Courtesy of Ivelin

11. Database-Derived Forcefield Radial Distribution Function P(occurance) = e-E/RT EAB = -RT ln(gAB(r)) gAB(r) NAB(r) http://www.nyu.edu/classes/tuckerman/stat.mech/lectures/lecture_8/node1.html

12. Results • Generally structures are obtained at 0.2-0.6Å RMSD to crystal structure • When compared to a well used server (MaxSprout) the server was about 20% less accurate • An alternative algorithm worked “better” but author claims training set was biased • Phi-Psi angle correlation was on average 0.95 and 0.88 respectively • Computation can be completed in minutes

14. Input Error

15. Peptides that Target Transmembrane Helices • Methods exist for the design or selection of antibodies for water soluble proteins • Different methods must be developed for membrane proteins due to our lack knowledge • Idea: Develop a peptide alpha helix that can insert into membrane and bind target membrane α-helix • Computational Design of Peptides That Target Transmembrane Helices • Hang Yin, Joanna S. Slusky, Bryan W. Berger, Robin S. Walters, Gaston Vilaire, Rustem I. Litvinov, James D. Lear, Gregory A. Caputo, Joel S. Bennett, and William F. DeGrado (30 March 2007) Science315 (5820), 1817

16. Transmembrane Proteins • Embedded in lipid bilayer • Difficult to crystallize • Underrepresented in PDB • Allow communication from outside to inside of cell INTEGRIN STRUCTURE, ALLOSTERY, AND BIDIRECTIONAL SIGNALING M.A. Arnaout, B. Mahalingam, J.-P. Xiong Annual Review of Cell and Developmental Biology 2005 21, 381-410

17. Design Overview • Choose the target alpha helix sequence • Find matching templates in the pdb database to native binding structure of target helix • Thread the target sequence onto one of the template helices • Choose proximal positions to mutate on the other template helix • Mutate those positions to all hydrophobic residue rotamers and repack

18. Find Templates • The integrin alpha helices that were chosen as targets contain a small-X3-small motif and a right handed crossing angle • Membrane proteins in the pdb were searched for a helix-helix dimer with this motif and crossing angle • Note: Among the few crystallized membrane helix-helix pairs they seem to fall into a few well defined motifs

19. Threading and Allowable Mutations • Change the amino acid identities of one of the alpha helices to that of the target sequence (αIIB) • Align the small-X3-small motif • Allow mutations (for design of “anti” helix) at positions close to helix-helix interface (pink)

20. Repacking • “Anti” peptide designed with Monte Carlo simulated annealing • At each step one residue identity is changed, and then the rotamers are optimized with DEE • The new energy is then calculated with a linearly damped Lennard-Jones potential and membrane depth-dependent knowledge based potential • Accept structure based on a Boltzman coin flip

21. Testing the Design • Target membrane was integrin αIIb alpha helix • Integrins are inactive when the α-subunit helix is bound to the β-subunit helix and active when not bound • αIIb causes the aggregation of platelets through binding with fibrinogen

22. Platelet inhibitor through signal transduction ADP scavenger (ADP stimulates plate aggregation) Inhibits binding to fibrinogen

23. Extensions • Currently this method is restricted to dimers and helices that are non-polar • Could include motifs with polar side chains • Design for multispan bundles rather than dimers • Use negative design to avoid amyloid formation or binding to undesired targets • Improve scoring function to account for more interaction types http://sb.web.psi.ch/images/amtb_in_membrane.png

24. Membrane Targeting Helices • Probe TM helix binding and function by targeting different membrane helices • Characterize folding of membrane proteins by blocking alpha helices as they form • Requires novel testing methods in order to determine whether helix is actually binding and affecting function

25. Moving Past Peptides: Foldamers • Proteins and RNA are unique in that they adopt specific compact, stable conformations • Biology has been fairly constrained so there should be much potential for other compactly folded polymers • Foldamer: “any polymer with a strong tendency to adopt a specific compact conformation” http://www.geneticengineering.org/chemis/Chemis-NucleicAcid/Graphics/tRNA.jpg Gellman, SH. Foldamers: A manifesto. Acc. Chem. Res.1998, 31, 173-180

26. Creating Foldamers • Find new backbone units with suitable folding propensities • Give the created foldamer interesting chemical functions • Be able to produce foldamers efficiently

27. Foldamer Uses • Test our understanding of protein function • Since all our analysis has been on only α-amino acids, have we “overfit” our understanding • Develop new building blocks and molecular frameworks for the design of pharmaceuticals, diagnostic agents, nanostructures, and catalysts Foldamers as versatile frameworks for the design and evolution of function Catherine M Goodman, Sungwook Choi, Scott Shandler & William F DeGrado Nature Chemical Biology 3, 252-262 (2007)

28. Monomer Framework Selection • Aliphatic • Aromatic

29. Foldamer Secondary Structure C10(310) C13(α) C12 C14

30. Predictability of Secondary Structure • Adding salt bridges spaced one turn apart introduce stability • Charged groups at helix ends stabilize according to their polarity • α-amino acid knowledge can be transferred about stabilization by disulfides, covalent bridges, and binding of metal ions

31. Aromatic Oligomers • Size of monomer and substitution of aromatic ring provide reliable determination of helical radius Jiang, H., Leger, J.M. & Huc, I. Aromatic -peptides. J. Am. Chem. Soc. 125, 3448–3449 (2003).

32. Designing Foldamer Function • Foldamers can interrupt Tat/TAR binding • Penetrate bacterial cells in a passive process • Have antimicrobial properties dependent on the length and hydrophobicity • Mimics to interrupt protein-protein interactions with Ki up to 0.8 uM and 7.1nm • By using an α/β sequence a ten-fold higher affinity was found than the native peptide ligand

33. Foldamer Tertiary Structure • A zinc finger-like motif was recently built consisting of β-peptides with a β hairpin and 14-helix • An octomer consisting of β-peptides was created with only non-covalent interactions

34. Benefits of Foldamers • Foldamers are more resistant to enzymatic attack then peptides • Fewer monomeric units are needed to adopt a well-defined secondary structure • Can be used as a strategic method to downsize peptides to small molecules Natural Peptide 14-helix β-peptide Arylamide foldamer Phenylalkylnyl

35. Summary • Peptide design can be used in a variety of ways • Backbone reconstruction • Antibodies for membrane proteins • Foldamers • All of these methods help us understand how proteins fold and the underlying rules, which will allow better models and hopefully better functional designs

36. Questions? “It is not clear to the author why LYS-59 is reported as such, because the crystal structure contains a valine at position 59.”