Undergraduate Exercises with Trp Cage

Paula Evans, Chet Fornari, Jeff Hansen,Jennifer Inlow, Larry Merkle

Background

GLP1R Glucagon-like peptide 1 receptor

GLP-1 natural peptide ligand of GLP1RExendin peptide from gila monster venomTrp cage synthetic peptide similar in sequence and structure to Exendin

GLP-1 increases glucose-dependent insulin secretion,decreases glucose-dependent glucagon secretion, andand decelerates gastric emptying.

Questions and Hypotheses for Students to Explore1. How do single-site mutations affect polypeptidestructure?If we change specific amino acids, then detectableStructural and Functional alterations will occur.

2. Can we predict general ligand-receptor interactions from structural comparisons, models, and MSA’s?If residues are conserved in the receptors and ligands then these residues are critical for ligand-receptor interactions.

3. Which ligand residues interact with which receptorresidues? The chemical properties of the amino acids wherethe structures of the three ligands overlap will be similar.

4. What are the phylogenetic relationships among thesetypes of receptors?Ligands with similar structures will bind to commondomains and motifs.

We can examine and analyze each question/hypothesis interms of the BioQuest philosophy:

How do single-site mutations affect polypeptidestructure?

If we change specific amino acids, then detectablestructural alterations will occur.

Biological Principleschemical properties of amino acidsmutationssequence/structure/function

Analysis ToolsCACHEDeep View and PyMol

Data SetsPDB files (Trp Cage, Exendin, GLP-1, receptors)

Explore Deep View as a tool for predicting structural changes that might result from mutation of a given residue.

Mutation of Trp to Met in Trp cage

Installed CACHE, loaded PDB file of Trp cage

Future Work:

Explore CACHE as a tool for predicting structural changesthat might result from mutation of a given amino acid residue,and compare these results with the results obtained fromDeeP View, MaTras (this study) and from NCBI’s Vast.

Can we predict general ligand-receptor interactions from structural comparisons, models, and MSA’s?

If residues are conserved in the receptors and ligands then these residues are critical for ligand-receptor interactions.

Biological Principlesreceptor-ligand interactionsintermolecular forces of interaction

Analysis ToolsCLUSTALW, Bioquest WorkbenchCACHE, Chimera

Data Setsprimary sequences of Trp Cage, Exendin, GLP-1

Multiple Sequence Alignment (MSA) of primary sequences of Trp cage, exendin, and GLP-1 reveal conserved residues

MSA generated using CLUSTALW (default parameters) athttp://www.ebi.ac.uk/clustalw/index.html

Trp_cage -------------------NLYIQWLKDGGPSSGRPPPS Exendin --EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKG-----R---- . :* ** .*

Which ligand residues interact with which receptorresidues?

The chemical properties of the amino acids wherethe structures of the three ligands overlap will be similar.

Biological Principleschemical properties of amino acidsreceptor-ligand interactions

Analysis ToolsProtein ExplorerMatras (Kawabata, T. MATRAS: A program for protein 3D structure comparison. Nucleic Acids Res. (2003) 31:3367-9.)

Data SetsPDB files of 3 ligands

Overlay of 3-dimensional structures of GLP-1 (blue), exendin (red), and Trp cage (green)

Figure generated using Matrashttp://biunit.aist-nara.ac.jp/Matras/(use PDB files 1l2y-, 1d0rA, 1jrjA)

region of structural overlap

Structural Alignment of 1l2y- (trpCage in blue) with 1d0rA (GLP-1 in red)

Figure generated using Matrashttp://biunit.aist-nara.ac.jp/Matras/(use PDB files 1l2y-, 1d0rA)

TrpCage withExendin

TrpCage with GLP-1

GLP-1 with Exendin

Other pair-wise views generated bythe MaTras program:

Comparison of ligand residues in the region of structuraloverlap: conserved Lys and hydrophobic patch

Trp cage

GLP-1

Exendin

gray: hydrophobic residues

blue: positively- charged residues

green: Trp

What are the phylogenetic relationships among thesetypes of receptors?

Ligands with similar structures will bind to commondomains and motifs.

Biological Principlesreceptor-ligand interactionsstructure/function

Analysis ToolsNCBI-CD, Smart, Pfam, InterProBiology Workbench (tree building algorithms)

Data Setsprimary sequences of ligands and receptors

Copyright ©2001 by the National Academy of Sciencesfrom West, Anthony P., Jr. et al. (2001) Proc. Natl. Acad. Sci. USA 98:3744-3749

Relation of Methuselah to family B GPCRs and other homologs

Ectodomain (extracellular portion) of Methuselah, a homodimeric receptor related to GLP1R

Figure generated using PyMol

Potential ligand-binding site highlited in space-fill.