Transcript and Trp cage

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 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.
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 BioQuest 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 PyMol
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
DeeP 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 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 3D 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 1l2y-, 1d0rA, 1jrjA)
Structural Alignment of 1l2y- (trpCage in blue) with 1d0rA (GLP-1 in red)
Figure generated using Matras
http://biunit.aist-nara.ac.jp/Matras/
(use PDB files 1l2y-, 1d0rA)
Other pair-wise views generated by
the MaTras program:
TrpCage with GLP-1
TrpCage 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, InterPro
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 GLP1R
Potential ligand-binding site highlited in space-fill.
Figure generated using PyMol