Bioinformatics & Molecular Modelling

-Data Analysis

For all questions illustrate your answers fully, describing what you did at every step and providing
input/output illustrating what input/output was obtained.

You need to include, embedded, within your submitted work all relevant output from online
servers, as appropriate, as well as a written dialogue to fully illustrate what work you carried out.
For all parts describe how you obtained your data by stating the bioinformatics portal used and
the search strategy.

Please quote accession numbers of all database files used.

Q1

Aspartic proteinase enzymes are a family of enzymes involved in a number of important biological
processes. In animals the enzyme renin has a hypertensive action through its role in the renin-
angiotensin system. The retroviral aspartic proteinases, such as the HIV proteinase, are essential for
maturation of the virus particle and inhibitors have a proven therapeutic record in the treatment of
AIDS. The lysosomal aspartic proteinase cathepsin D has been implicated in tumorigenesis and the
stomach enzyme pepsin, which plays a major physiological role in hydrolysis of acid-denatured
proteins, is responsible for much of the tissue damage in peptic ulcer disease. Since aspartic
proteinases also play major roles in amyloid disease, malaria and common fungal infections such as
candidiasis, inhibitors to these enzymes are much sought after as potential therapeutic agents.

 Locate within the Protein Data Bank the 3-D structure of an aspartic proteinase enzyme.
State what your chosen entry is, and download the coordinates for the structure to use with
Rasmol to investigate your chosen structure.

 Identify, using an appropriate bioinformatics program, the active site residues for your
chosen PDB entry. Show the output generated by the program used.

 Using the program rasmol or Swiss-PDB Viewer produce an image of the structure that you
think clearly illustrates the major structural features within the enzyme and clearly shows
the location of the active site residues. State the commands used within the selected
program to obtain your image.

 Discuss which types of bioinformatics tools that could be used to design inhibitors for
aspartic proteinase.

20 marks
Q2

Cytochrome P450s are a family of proteins involved in phase I drug metabolism reactions. They are
highly expressed in the liver, in the endoplasmic reticulum membrane. In this question you will
explore the use of protein-protein interaction databases to find out what other proteins P450s
interact with and whether the potential partnerships could have biological significance.

 Use the UniProt file for human cytochrome P450 2D6 as your starting point. Summarise the
key structural features of P450 2D6 including how it is able to bind to the ER membrane, and
structural features of the active site.

 Use a range of PPI databases to identify possible protein partners. Summarise your findings.

 From your searches select three proteins with different activities that interact with P450
2D6, describe the evidence for the interaction and discuss whether these interactions could
be relevant to P450’s ability to metabolise drugs. Wherever possible select proteins for
which there is experimental evidence for the interaction.

25 marks

Q3

Using the human sequence for the 509 amino acid protein Tyrosine-protein kinase Lck (Uniprot
sequence entry P06239) determine the domain present within this protein sequence, using the Pfam
domain database. State the domain and the amino acids within the domain.

Run homology modelling for this sequence using SWISS-MODEL to obtain a 3-dimensional structure
for this sequence.

DISCUSS, in detail, the results of the modelling that you obtain, including an in-depth discussion of
the models obtained, the templates used by the program, and the output generated.

Download the coordinates of what you consider to be the ‘best’ model obtained, as a protein
databank (*.pdb) file, and create an image of your modelled structure using rasmol or Swiss-
PdbViewer which clearly shows the main features of the model.

25 marks

Q4
In lecture 6 on RNA informatics you were shown an analysis of RNA structure across the 3' UTR of
interleukin 2 (IL-2). Many other cytokines also carry an AU-rich element sequence, including
interleukin 6 (IL-6). Micro-RNAs are known to target some cytokine mRNAs. In this question you will
explore the interaction between the mRNA of IL-6 and a micro-RNA miR-365. It has been shown that
miR-365 inhibits expression of IL-6 through this interaction.

a) Retrieve the sequence files for human IL-6 and five other species. Align the 3' UTRs of the
six mRNAs and identify on your output potential AU-rich sequence elements.

b) Retrieve the two files containing the sequence of miR-365 from the miRNA database
www.mirbase.org. Run the complete sequences of the RNAs on Mfold and show the
predicted structure of the RNAs. Calculate the folding energy per base and comment on your
findings.

c) Model the binding of the mature sequence of each miRNA (this is given in the miRBase file)
with the 3' UTR of IL-6 mRNA. Assume that the miRNAs will bind to complementary
sequences in the IL-6 mRNA, but not necessarily with complete complementarity. You will
have to use alignment software to map complementary regions. Describe the procedure you
followed, discuss the output with reference to a diagram of the alignment.

d) On the basis of your models predict which miRNA would be inhibitory and explain why.
Does the mechanism involve the AU-rich element of IL-6?

30 marks