Molecular Evolution - Mini Project #3

Protein phylogeny, Bayesian analysis, ancestral reconstruction - evolution of steroid receptors

In this miniproject you will use Bayesian methods to reconstruct the evolutionary history (and an ancestral sequence) for some steroid hormone receptors. Specifically, you will look at receptors for mineralocorticoids (MR) and for glucocorticoids (GR).

Modern tetrapods (amphibians, reptiles, birds, and mammals) have separate receptors for the steroid homormones cortisol (GR, which modulates metabolism, inflammation, and immunity) and aldosterone (MR, which modulates salt balance amongst other things). Hagfish and Lampreys ("primitive" jawless fish with cartilaginous skeletons) have only one receptor, which is activated by both aldosterone and cortisol. Sharks and such have two receptors, both of which are activated by both aldosterone and cortisol. Finally, bony fish and tetrapods have two receptors, one which is activated by aldosterone, and one which is activated by cortisol. What were the molecular steps which brought this about?

As always, report all steps of your approach along with your findings and conclusions in a mini report that you upload at CampusNet.

Protein phylogeny, Bayesian analysis, ancestral reconstruction

1. Use this table of sequences as the basis for constructing your data set. Specifically select all sequences ending in GR, GR1, GR2, and MR. Also include the hagfisfCR sequence and use this as outgroup. (Table taken from: Bridgham, Caroll, and Thornton, Science, 2006)

2. Align the sequences and prepare the alignment for use in MrBayes.

3. Read section 4.2 in the MrBayes manual to learn how to analyze protein sequences in MrBayes. Also check the help available from within MrBayes to learn what substitution models are available: Start MrBayes, issue the command help prset, and read the section titled aamodelpr (you will have to scroll back a bit).

4. Read sections 4.10, and 4.8.1 (in that order) in the MrBayes manual to learn how to reconstruct ancestral sequences.

5. Use MrBayes to construct a rooted phylogenetic tree for your protein sequences using your preferred amino acid substitution model, and requesting ancestral reconstruction for the internal node that corresponds to the ancestor of the entire ingroup.

6. Plot the phylogenetic tree you just constructed. Compare the structure of the clades containing the MR and HR sequences. What does this pattern tell you about the evolution of the present-day MR and GRs and how they relate to the Hagfish CR sequence?

7. Construct a fasta file with the most probable ancestral sequence (remember to include in report).

8. Looking at the set of present-day MR sequences, and the set of present-day GR sequences. Are there any sites that are conserved within one of these groups, but that differ between the groups? Make a list of these sites (and their amino acids). Now, compare these sites to the corresponding sites in your ancestral reconstruction.

9. See if any of your sequences have an associated X-ray structure in the PDB database. If not, find one or more closely related sequences that does have a structure. If possible, find a structure that includes a bound ligand molecule. (If you have never analyzed protein structures, then you can quickly find tools and info on this exercise page: Protein structures & PDB).

10. Figure out the relationship between positions in your alignment, and positions in the protein structure (if there are gaps in your alignment, then the position in the alignment and in the structure will be shifted).

11. Use PyMol (see link above) to make a plot of your protein structure, where all the sites identified above have been colored. Make sure to take into account that alignment positions and structure positions may have different numbering. Is it possible to make any general conclusions about the roles of one or more of these sites? (You can also check the PDB file itself for annotation about the role of individual sites).

12. Hand in at CampusNet.