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There are many different approaches and programs avialable to align protein structure. Some of these are web-based, others have active discussion groups one can ask for help, in case one is stuck.
ATPase subunits
Objective: Have at least a rough understanding of the content of a protein data bank file. Save individual subunits into distinct pdb files Align structures of divergent proteins Align structures of a catalytic subunit during the catalytic cycle.
Go to the protein databank, download the file 1bmf. This protein is the head group of the beef-heart mitochondrial ATP synthase.
Open the file 1bmf.pdb in a texteditor (notepad+ or wordpad). [You could also use the file that chimera saves in the download folder, if you use the fetch by ID command]. Scroll down the file, notice of the journal citation, and in line 294 ff, take a note of which chain (A,B,C,D,E,F, and G) corresponds to which subunits. Both the alpha and beta subunits have an ATP binding site. The abbreviations E(mpty), DP (diphosphate), and TP (triphosphate) refer to the occupation of the beta subunit. Which chains correspond to alphaE, alphaDP, alpha TP, betaE, beta DP, beta TP?
The ATP synthase (aka as proton pumping ATPase) consists of ring of proteolipids that are integrated into the membrane, a head group (which is the structure in 1bmf), and a stator that keep the non-rotating parts fixed. The head group known as F1 portion of 6 ATP binding subunits (3 alpha and 3 beta subunits). The beta subunits bind and hydrolyze ATP, if the enzyme works as a proton pumping ATPase. These catalytic subunits rotate the central gamma subunit. In the intact enzyme, the gamma subunit is linked to the proteolipids, which than rotate relative to the stator. When they pass the stator the proteolipids (proteins that behave like a lipid, but they do NOT contain a lipid) they undergo a motion that moves a glutamate or aspartate residue into a different environment, where is picks up or dissociated a proton.
Why is the ATPsynthase important? Make a guess as to how much ATP are synthesized in the human body per day.
How is ATP synthesis coupled to the electron transport chain?
The beta and alpha subunit evolved from a very ancient gene duplication (this duplication had already occurred in the common ancestor of Bacteria, Archaea, and the eukaryotic nucleocytoplasm); this duplication had already occurred over 3.5 billion years ago. This means that the two subunit types (alpha and beta) evolved as separate subunits for over 7 billion years.
1) Open chimera and open the 1bmf file (File > fetch by ID 1bmf) Look at the structure in the first two preset modes (the surface may take some time to compute). Note the central gamma subunit (consisting mainly of alpha helices). [aside: in the related structure of a transcription termination factor, which unwinds a newly synthesized mRNA from the DNA template, the six ATP binding subunits have a similar arrangement and the place of the gamma subunit is taken by the RNA DNA duplex]. Also color the Ribbon by secondary structure (Tools > depiction > secondary structure). Select all non-standard residues (select > residue > ...) and show them as ball (Actions > Surface >show). Can you determine which chain, via (Select > chain), does not have an ATP or ATP analog bound?
F1 ATPase in ribon display. Gamma SU in purple or in blue.
Select each of the chains in turn and save is as a pdb (Select > chain …, then File >Save pdb – in the window, enter a name (use the names of the subunits), select an appropriate directory, and place a check mark into save selected atoms only (!!!).
Close your session
2) Open alphaTP and betaTP.
Open the model panel and inactivate one of the structures and turn/move the other so that you can move them side by side. Choose a display that helps you to recognize structural similarity – use different preset displays as a starting point. If you found a good-looking display, save the session (careful, if you want to safe many times along the way, give the session new names, else it overwrites the previously saved session)
Example of what your image might look like. The beta subunit is in slightly more orangey colors. Hopefully you are impressed by the degree of structural conservation over >7 billion years of evolution.
Does the similar arrangement of secondary structure elements relative to the ATP analog convince you that the two structures evolved from the same ancestral protein?
To generate an automated alignment, use Tools > Structure comparison > Matchmaker. In the matchmaker window, select one of the structures as reference, the other as structure to match. Place check marks to "include secondary structure scores", "compute secondary structure alignments", and "Show pairwise alignments". The choice of substitution matrix usually does not make a big difference. The numbers in the BLOSUM series reflect % identity of the sequences from which the scoring matrix was calculated, i.e., in this case the appropriate choice would be 30.
Both of the subunits bind ATP via the Rossman fold. A conserved part of this ATP binding site are the so-called Walker motifs. The Walker A motif is G-x-x-x-x-G-K-[TS]. Can you locate this sequence in the structure-based alignment? Are the Walker A motifs aligned between the two structures? What amino acid follows the GK[T/S] in the beta subunit? With what part of the ATP molecule do the Lysin and Threonine sidechains interact?
Visualize the catalytic cycle of the beta subunits: Load the three betas subunits as separate structures. Apply a good looking coloring scheme. Save session as … (in case something goes wrong, you can recover from the saved session) To align the subunits, Tools > Structure comparison > Matchmaker Select betaDP as reference, and align the other two structures (control click adds to the selection). Use the "model panel" to click on/off the visibility of the different chains. Where during the catalytic cycle (betaE > betaDP > betaTP > beta E does the structure change most? How would you describe this movement?
To create an animation that morphs one structure into another, load two or three of the beta subunits (E, DP, TP). Align the structures using matchmaker (see above). Then select tools> Structure Comparisons > Morph conformations. In the morph conformations window, click on add, and in the new window that pops up select the structures between which you want to morph. E.g., betaDP betaTP betaDP (this moves back and forth between the two structures with the nucleotide bound), or betaE, betaDP betaTP betaE (moves through the catalytic cycle). Click create to generate the frames between the structures.
The animation is saved as a new model. To see only the animation, unselect the display of the other structures in the model panel. To see the ATP, select betaTP, select the ANP, invert the selection of the selected model, and in actions, hide the display of the protein.
To save the animation as a movie (mpeg, mov or mp4), select Tools > Utilities > Movie Recorder. Select a format (mov with png had better colors on my screen), and click record. This samples frames, but does not yet record a movie. Once you have sampled enough frames, click stop, then select an output format, then click "make movie". (all in the movie recorder window). An mp4 versio of the movie is here.
Challenge: Make a movie using all 7 subunits in 1bmf. To do so, create 3 copies of 1bmf.pdb, and give each of them a distinctive name (e.g., 1bmf1.pdb, 1bmf2.pdb, 1bmf3.pdb). Open a new session, and load the 4 files. We need to choose one of the bmfs as reference structure (e.g., 1bmf2.pdb, the program cannot use the same pdb file as target and reference). Then go to matchmaker, and select the radio button in front of "specific chains in reference structure with specific chains in match structure". In the reference structure window select or highlight (control click adds to the selection) the three beta subunits in the reference bmf molecule (e.g., 1bmf2.pdb). We will align the other two pdb files to the reference. Think about the direction of the catalytic cycle you want to morph. In the first match, each betaX subunit needs to be matched to the one representing the previous step of the catalytic cycle. In the second match using the same reference, the betas need to be aligned to the ones representing the next step in the catalytic cycle in the third model. Aligning the structures using matchmaker works rather well; however, so far morphing between the three structures is not as envisioned. (It rotates the whole F1 … @#$%^&*(*&^%$#@, you will get extra credit, if you figure out a solution). A less than perfect solution is to save the three aligned structures each into a separate image (use png) and then use a gif maker (e.g. here) to animate the images.
Optional exercise:
Comparing other divergent proteins with similar structures
Glutathione synthetase and D-Alanine D-Alanine Ligase were long aago (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 aligments (will be covered later in this course). A description of the GRASP domian 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, Flash animation here) [you may get a warning/error message... ignore it] ,
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?
Can you use Tools > Structure comparison > Matchmaker to align these stuctures?
See here for an illustration of the structures in similar orientation.
Choose one of the following options, then click the button.
Send email to your instructor (and yourself) upon submit (If you created a nice image, please send it per email or via a link (UConn Dropbox)Send email to yourself only upon submit (as a backup) Show summary upon submit but do not send email to anyone.