Peptide Design Kyle Roberts March 4, 2008. Peptides in Biology Create peptide antibodies Used in mass spec to identify proteins Can probe protein-protein.

Peptide Design

Kyle Roberts

March 4, 2008

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

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)

Peptide Backbone Reconstruction

Adcock SA. Peptide backbone reconstruction using dead-end elimination and a knowledge-based forcefield. J Comput Chem. 2004 Jan 15;25(1):16-27.

Reconstruct an all-atom peptide model from a subset (Cαs or Cβs) of the atomic coordinates

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

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)

Algorithm Overview

Library of amino acid peptide sequences (length 3 )

Cα or Cβ coordinates

Overlay peptides on input coords

Dead EndElimination

Predicted Structure

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

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

Dead-End Elimination Minimize the energy function:

N

r

N

s

jipairi

N

r

total srErEE1 11

single ),()(

Ene

rgy

ri

rj

ri is residue r with the backbone conformation i

Image: Courtesy of Ivelin

Database-Derived Forcefield

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

A BN

i

N

j

BjAiAiAB rrrSrN1 1

)|(|)(

Radial Distribution Function

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

Input Error

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 HelicesHang 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) Science 315 (5820), 1817

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 SIGNALINGM.A. Arnaout, B. Mahalingam, J.-P. Xiong

Annual Review of Cell and Developmental Biology 2005 21, 381-410

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

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

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)

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

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

Platelet inhibitor through signal transduction

ADP scavenger (ADP stimulates plate aggregation)

Inhibits binding to fibrinogen

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

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

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”

Gellman, SH. Foldamers: A manifesto. Acc. Chem. Res.1998, 31, 173-180

http://www.geneticengineering.org/chemis/Chemis-NucleicAcid/Graphics/tRNA.jpg

Creating Foldamers

Find new backbone units with suitable folding propensities

Give the created foldamer interesting chemical functions

Be able to produce foldamers efficiently

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 functionCatherine M Goodman, Sungwook Choi, Scott Shandler & William F DeGrado

Nature Chemical Biology 3, 252-262 (2007)

Monomer Framework Selection

Aliphatic

Aromatic

C10(310)

C14C12

C13(α)

Foldamer Secondary Structure

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

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).

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

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

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

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

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

Questions?