Undergraduate Exercises with Trp Cage Paula Evans Chet

Undergraduate Exercises with Trp Cage Paula Evans, Chet Fornari, Jeff Hansen, Jennifer Inlow, Larry Merkle

Background GLP 1 R Glucagon-like peptide 1 receptor GLP-1 natural peptide ligand of GLP 1 R Exendin peptide from gila monster venom Trp cage synthetic peptide similar in sequence and structure to Exendin GLP-1 increases glucose-dependent insulin secretion, decreases glucose-dependent glucagon secretion, and decelerates gastric emptying.

Questions and Hypotheses for Students to Explore 1. How do single-site mutations affect polypeptide structure? If we change specific amino acids, then detectable Structural 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 ligandreceptor interactions. 3. Which ligand residues interact with which receptor residues? The chemical properties of the amino acids where the structures of the three ligands overlap will be similar. 4. What are the phylogenetic relationships among these types of receptors? Ligands with similar structures will bind to common domains and motifs.

We can examine and analyze each question/hypothesis in terms of the Bio. Quest philosophy:

How do single-site mutations affect polypeptide structure? If we change specific amino acids, then detectable structural alterations will occur. Biological Principles chemical properties of amino acids mutations sequence/structure/function Analysis Tools CACHE Deep View and Py. Mol Data Sets PDB 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 changes that might result from mutation of a given amino acid residue, and compare these results with the results obtained from Dee. P View, Ma. Tras (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 ligandreceptor interactions. Biological Principles receptor-ligand interactions intermolecular forces of interaction Analysis Tools CLUSTALW, Bioquest Workbench CACHE, Chimera Data Sets primary sequences of Trp Cage, Exendin, GLP-1

Multiple Sequence Alignment (MSA) of primary sequences of Trp cage, exendin, and GLP-1 reveal conserved residues Trp_cage ----------NLYIQWLKDGGPSSGRPPPS Exendin --EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKG-----R---. : * **. * MSA generated using CLUSTALW (default parameters) at http: //www. ebi. ac. uk/clustalw/index. html

Which ligand residues interact with which receptor residues? The chemical properties of the amino acids where the structures of the three ligands overlap will be similar. Biological Principles chemical properties of amino acids receptor-ligand interactions Analysis Tools Protein Explorer Matras (Kawabata, T. MATRAS: A program for protein 3 D structure comparison. Nucleic Acids Res. (2003) 31: 3367 -9. ) Data Sets PDB files of 3 ligands

Overlay of 3 -dimensional structures of GLP-1 (blue), exendin (red), and Trp cage (green) region of structural overlap Figure generated using Matras http: //biunit. aist-nara. ac. jp/Matras/ (use PDB files 1 l 2 y-, 1 d 0 r. A, 1 jrj. A)

Structural Alignment of 1 l 2 y- (trp. Cage in blue) with 1 d 0 r. A (GLP-1 in red) Figure generated using Matras http: //biunit. aist-nara. ac. jp/Matras/ (use PDB files 1 l 2 y-, 1 d 0 r. A)

Other pair-wise views generated by the Ma. Tras program: Trp. Cage with GLP-1 Trp. Cage with Exendin GLP-1 with Exendin

Comparison of ligand residues in the region of structural overlap: conserved Lys and hydrophobic patch Exendin Trp cage gray: hydrophobic residues blue: positivelycharged residues green: Trp GLP-1

What are the phylogenetic relationships among these types of receptors? Ligands with similar structures will bind to common domains and motifs. Biological Principles receptor-ligand interactions structure/function Analysis Tools NCBI-CD, Smart, Pfam, Inter. Pro Biology Workbench (tree building algorithms) Data Sets primary sequences of ligands and receptors

Relation of Methuselah to family B GPCRs and other homologs from West, Anthony P. , Jr. et al. (2001) Proc. Natl. Acad. Sci. USA 98: 3744 -3749 Copyright © 2001 by the National Academy of Sciences

Ectodomain (extracellular portion) of Methuselah, a homodimeric receptor related to GLP 1 R Potential ligand-binding site highlited in space-fill. Figure generated using Py. Mol