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

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Presentation on theme: "Undergraduate Exercises with Trp Cage Paula Evans, Chet Fornari, Jeff Hansen, Jennifer Inlow, Larry Merkle."— Presentation transcript:

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

2 Background GLP1R Glucagon-like peptide 1 receptor GLP-1 natural peptide ligand of GLP1R 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 and decelerates gastric emptying.

3 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 ligand- receptor 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.

4 We can examine and analyze each question/hypothesis in terms of the BioQuest philosophy:

5 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 PyMol Data Sets PDB files (Trp Cage, Exendin, GLP-1, receptors)

6 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

7 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 DeeP View, MaTras (this study) and from NCBI’s Vast.

8 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 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

9 Multiple Sequence Alignment (MSA) of primary sequences of Trp cage, exendin, and GLP-1 reveal conserved residues MSA generated using CLUSTALW (default parameters) at Trp_cage NLYIQWLKDGGPSSGRPPPS Exendin --EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKG-----R----. :* **.*

10 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 3D structure comparison. Nucleic Acids Res. (2003) 31: ) Data Sets PDB files of 3 ligands

11 Overlay of 3-dimensional structures of GLP-1 (blue), exendin (red), and Trp cage (green) Figure generated using Matras (use PDB files 1l2y-, 1d0rA, 1jrjA) region of structural overlap

12 Structural Alignment of 1l2y- (trpCage in blue) with 1d0rA (GLP-1 in red) Figure generated using Matras (use PDB files 1l2y-, 1d0rA)

13 TrpCage with Exendin TrpCage with GLP-1 GLP-1 with Exendin Other pair-wise views generated by the MaTras program:

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

15 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, InterPro Biology Workbench (tree building algorithms) Data Sets primary sequences of ligands and receptors

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

17 Ectodomain (extracellular portion) of Methuselah, a homodimeric receptor related to GLP1R Figure generated using PyMol Potential ligand-binding site highlited in space-fill.


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