Assignment #3

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Reminder

Please do not forget to write a short reflective statement about the most interesting thing you learned in that class/lab.
Do it immediately after class!  Else, you miss the purpose and it will become a hassle to write 10 of these items in raw. 

Exploring intein structures in chimera

Objectives:

 

Background information:

Inteins are molecular parasites that have their own life cycle. Once they are in gene in one member of the population, they spread by super-Mendelian inheritance. If as a consequence of sex of gene transfer, an infected and a non infected allele come together in one cell, the homing endonuclease activity of the intein makes a double-strand cut in the DNA of the non infected allele. During the repair of the break the intein is copied into the allele. The other activity of the intein is a self splicing activity: The DNA encoding the intein is translated and transcribed together with the host gene. At the protein level, the intein removes itself from the host protein (aka as extein).

Once an intein is fixed in a population, there is nothing left to do for the homing endonuclease. The endonuclease activity decays and may ultimately be deleted, leading to mini-inteins that only contain the self-splicing domain. However, specific inteins with functioning Homing Endonuclease have survived in some lineages for 100s of million of years.  For more discussion see here.

Most inteins are composed of two domains: one is responsible for protein splicing, and the other has endonuclease activity. A few inteins have lost the endonuclease domain completely and retain only the self-splicing domain and activity. The latter inteins are called mini-inteins .

The structures of several inteins have been determined through X-ray crystallography.  Today we will use the following: 

To do:

  1. Open 1VDE in chimera. This structure has two chains. Select chainA and save the selected residues only into their own pdb file (file > save pdb > fill out the form, check save selected residues only, save). Close the session.

  2. Reopen the saved chain A in chimera. Open Mycobacterium mini intein 1AM2. Depict the structures as ribbons and color them according to the secondary structure (if you press the shift key while executing an action, it should act on both chains; but there also is a check mark in the "Tools > Depiction > Color Secondary Structure".  However, it also might look nice to use slightly different colors. for the two chains.   Rotate the two structures until you can see the similarities between mini intein and the large intein. (Use the "favorites > model panel" to rotate one or the other structure). Which part of the structures appears to be similar?
    Your answer --->

    Align two structures using Tools > structure comparison > matchmaker. Does the alignment correspond to your expectation?
    Your answer --->

    Can you find which part of Saccharomyces cerevisiae intein corresponds to the endonuclease domain by comparison of the two structures?
    Using "tools > compare structure > match -align" create a pairwise alignment of the two inteins based on the match between the two structures (you need to have had run matchmaker first!). Color the putative self-splicing (i.e. the part that is present in 1VDE and in 1AM2) and endonuclease domains of 1VDE (no corresponding part in 1AM2) in two different colors (selecting consecutive residues works easily via the alignment window; if you press shift, you can add to the selection.)  Do you get the same result as above?
    Your answer --->
    Save your project.

    Find and select the N and C terminals (First a.a. and the last a.a.) in both structures. If you hover over the beginning or end, the name of the residues pops up in a little window. CTRL click selects the amino acid or atom, and shift control click adds to the selection. Under actions>atoms/bonds>show side-chains make the side chains of the first and last amino acid visible. Optional: hide the rest of the structure, if it distracts you. Rotate the structure of the sidechains of the first and last aa and decide which atoms are closest. Select these atoms (ctrl click and shift ctrl click), then go to tools>Structure analyses> distances and click create. Repeat this for a few atoms from the first and the last aa. How close are beginning and end (in Ångström and in nanometers)?
    Your answer --->
     

  3. Open Saccharomyces cerevisiae intein that is bound to its target DNA sequence.
    Does the DNA - Protein interaction in 1LWS agree with your previous assignment of the self-splicing domain? (see the saved structure from the previous exercise)
    Your answer --->

  4. Try to find a way to display the interactions between the amino acid side chains and the DNA helix.
    One way to do it is to select two DNA chains and select aa in the neighboring zone. To do this you could first select chain A, then invert the selection (you now should have selected Chain B and C). Then select zone 4 or 5 Angstrom. Make the side chains visible, and display either the side chains or the DNA as spheres. One way to look at individual interactions is to turn the molecule so that one looks down the DNA helix, and use the viewing controls only look at a cross section (or Slab) of the structure. If the bases of the DNA are displayed too cartoonish, you can change the display in Actions > Atoms/Bonds > nucleotide objects (select different option and click apply).
    Most of the interactions of aa side chains are with the major groove of the DNA. Do you find residues that interact with the minor groove? If yes, which aa are involved:
    Your answer --->

  5. The Lys 340 and Glu 366 are residues that are important for interaction with DNA. Select those residues (Tools> sequence>sequence allows to show the primary sequence, which is a good way to select a particular aa). Which base pairs interact with these amino acids? (if you hover over an atom, a pop-up window gives the base, the number of the base, and the chain (e.g., G 23.B is the 23 rd base in chain B, which is a Guanin). It might look nice to look at a cross section of the molecule perpendicular to the DNA.   
    Your answer --->


image

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Optional exercises:

Comparing divergent proteins with similar structures

GRASP ATP binding domain

Glutathione synthetase and D-Alanine D-Alanine Ligase were long ago (1990) recognized by Jim Knox (MCB faculty) to be so similar in structure that there could be no doubt about their common evolutionary origin. Later these and other enzymes were discovered to have a novel ATP binging site (the GRASP domain). These domains were identified using profile alignments (will be covered later in this course). A description of the GRASP domain family is at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249071/.

Save the following files to your computer and load them into chimera.

2DLN.pdb (D-Alanine D-Alanine Ligase - D-ala is an important part of the bacterial cell wall, more here

1GSA.pdb (glutathione synthetase from E. coli, glutathione is the biological equivalent of mercaptoethanol),

cpsBfrag.pdb, load cpsFfrag.pdb (Carbamoyl phosphate synthetase is an enzyme consisting of several domains. These are the front and the back of (1BXR)).

Based on your first impression, are these structures similar? homologous?

Your answer --->

Can you use Tools > Structure comparison > Matchmaker to align these structures?

Your answer --->

See here for an illustration of the structures in similar orientation.


Histones

If you have more time to spare and you are up for a challenge, take a look at the nucleosome (1AOI.pdb). Open it from within chimera. Save each of the 8 histones as a separate pdb file. Close the nucleosome file, open the 8 histone files and align them to one reference histone.

The nucleosome, one of the 8 histones is colored green. (2 copies of each paralog)

nucleosome

The eight histones aligned to one another.

Same as ribbon display

8 histones struct alignment

 

Below same as last figure, but histones are depicted side by side :

 

The proteasome core

Proteasomes are the recycling cans of the cell. Ubiquitin tagged protein are taken-up into the proteasome and broken down into aa. The proteasome (20S) core consists out of homologous alpha and beta subunits, arranged in 4 rings. The two central ones are beta subunits (2x7), the two rings at the ends are made from alpha subunits. Humans (and other eukaryotes) have 7 different genes encoding seven different alpha subunits, and 7 different genes encoding beta subunits, and each is present in duplicate in the 20S proteasome core. Altogether 28 subunits.

We are interested in creating a structure based sequence alignment of the different alpha and beta subunits. To do so, pick the bottom or top half of the proteasome (5LF7.pdb, or a similar high resolution structure from another organism), and save all the different subunits as separate pdb files (if you use 5LF7.pdb, the pdbs for the individual SU are here). First align all the alpha beta subunits separately, and then try to generate an alignment of the alpha with the beta subunits. Use matchmaker to align the structures, then use Tools > structure comarison > Match-Align to create a multiple sequence alignment.

 

human proteasome

The human 20S proteasome core (28 subunits, 14 paralogs)

proteasomeSU

Proteasome subunits (7 beta and 7 alpha subunits) from human hela cells (5LF7.pdb)

all proteasome SUs

All 14 subunits of the human proteasome core superimposed.

 

All 14 subunits of the human proteasome next to one another