BIO Assignment Week 9

Assignment for Week 9
Protein Ligand Complex

Note! This assignment is currently active. All significant changes will be announced on the mailing list.

 
 

Concepts and activities (and reading, if applicable) for this assignment will be topics on next week's quiz.

 

Introduction

One of the really interesting questions we can discuss with reference to our model is how sequence variation might result in changed DNA recognition sites, and then lead to changed cognate DNA binding sequences. In order to address this, we would need to generate a plausible structural model for how DNA is bound to APSES domains.

Since there is currently no software available that would accurately model such a complex from first principles, we will base a model of a bound complex on homology modeling as well. This means we need to find a similar structure for which the position of bound DNA is known, then superimpose that structure with our model. This places the DNA molecule into the spatial context of the model we are studying. However, you may remember from the third assignment that the APSES domains in fungi seem to be a relatively small family. And there is no structure available of an APSES domain-DNA complex. How can we find a coordinate set of a structurally similar protein-DNA complex?

This assignment is based on the homology model you built. You will (1) identify similar structures of distantly related domains for which protein-DNA complexes are known, (2) assemble a hypothetical complex structure and (3) consider whether the available evidence allows you to distinguish between different modes of ligand binding,

Modeling a DNA ligand

 

 

Finding a similar protein-DNA complex

 

Remember that homologous sequences can have diverged to the point where their sequence similarity is no longer recognizable, however their structure may be quite well conserved. Thus if we could find similar structures in the PDB, these might provide us with some plausible hypotheses for how DNA is bound by APSES domains. We thus need a tool similar to BLAST, but not for the purpose of sequence alignment, but for structure alignment. A kind of BLAST for structures. Just like with sequence searches, we might not want to search with the entire protein, if we are interested in is a subdomain that binds to DNA. Attempting to match all structural elements in addition to the ones we are actually interested in is likely to make the search less specific - we would find false positives that are similar to some irrelevant part of our structure. However, defining too small of a subdomain would also lead to a loss of specificity: in the extreme it is easy to imagine that the search for e.g. a single helix would retrieve very many hits that would be quite meaningless.

At the NCBI, VAST is provided as a search tool for structural similarity search.

Task:

1. Navigate to the VAST search interface page.
2. Enter 1bm8 as the PDB ID to search for and click Go.
3. Study the result.

You will see that VAST finds a large number of partially similar structures, but it would be almost impossibly tedious to find structures of protein DNA complexes that are similar in the core of the interaction domain. It turns out that our search is not specific enough in two ways: we have structural elements in our PDB file that are unnecessary for the question at hand, and thus cause the program to find irrelevant matches. But, if we constrain ourselves to just a single helix and strand (i.e. the 50-74 subdomain that has been implicated in DNA binding, the search will become too non-specific. Also we have no good way to retrieve functional information from these hits: which ones are DNA-binding proteins, that bind DNA through residues of this subdomain and for which the structure of a complex has been solved? It seems we need to define our question more precisely.

Task:

1. Open VMD and load the 1BM8 structure or your homology model.
2. Display the backbone as a Trace (of CA atoms) and color by Index
3. In the sequence viewer, highlight residues 50 to 74.
4. In the representations window, find the yellow representation (with Color ID 4) that the sequence viewer has generated. Change the Drawing Method to NewCartoon.
5. Now (using stereo), study the topology of the region. Focus on the helix at the N-terminus of the highlighted subdomain, it is preceded by a turn and another helix. This first helix makes interactions with the beta hairpin at the C-terminal end of the subdomain and is thus important for the orientation of these elements. (This is what is referred to as a helix-turn-helix motif, or HtH motif, it is very common in DNA-binding proteins.)
6. Holding the shift key in the alignment viewer, extend your selection until you cover all of the first helix, and the residues that contact the beta hairpin. I think that the first residue of interest here is residue 33.
7. Again holding the shift key, extend the selection at the C-terminus to include the residues of the beta hairpin to where they contact the helix at the N-terminus. I think that the last residue of interest here is residue 79.
8. Study the topology and arrangement of this compact subdomain. It contains the DNA-binding elements and probably most of the interactions that establish its three-dimensional shape. This subdomain even has a name: it is a winged helix DNA binding motif, a member of a very large family of DNA-binding domains. I have linked a review by Gajiwala and Burley to the end of this page; note that their definition of a canonical winged helix motif is a bit larger than what we have here, with an additional helix at the N-terminus and a second "wing". )

Armed with this insight, we can attempt again to find meaningfully similar structures. At the EBI there are a number of very well designed structure analysis tools linked off the Structural Analysis page. As part of its MSD Services, PDBeFold provides a convenient interface for structure searches for our purpose

Task:

1. Navigate to the PDBeFold search interface page.
2. Enter 1bm8 for the PDB code and choose Select domain from the drop down menu. Select Secondary Structure elements 4 to 7 i.e. those elements that span the range you have previously defined..
3. Note that you can enter the lowest acceptable match % separately for query and target. This means: what percentage of secondary structure elements would need to be matched in either query or target to produce a hit. Keep that value at 80 for our query, since we would want to find structures with almost all of the elements of the winged helix motif. Set the match to 10 % for the target, since we are interested in such domains even if they happen to be small subdomains of large proteins.
4. Keep the Precision at normal. Precision and % query match could be relaxed if we wanted to find more structures.
5. Finally click on: Submit your query.
6. On the results page, click on the number next to the top hit that is not one of our familiar Mbp1 structures to get a detailed view of the result. Most likely this is 1no7:b, a herpesvirus capsid domain. Click on View Superposed. This will open a window with the structure coordinates superimposed in the Jmol molecular viewer. Control-click anywhere in the window area to open a menu of viewing options. Select Style → Stereographic → Wall-eyed viewing. Then study the superposition. You will note that the secondary structure elements match quite well, but clearly, we have not found a winged-helix domain. If you consider the secondary structure alignment in the results page, you will notice that there are a significant number of gaps between the elements.
7. Go back to the table of results and resort the table by number of gaps. This will bring a different protein to the top (again, excepting Mbp1 structures.) Explore its structure as well.

All in all this appears to be well engineered software! It gives you many options to access result details for further processing. I think this may be useful. But for our problem, we would have to search through too many structures because, once again, we can't tell which ones of the hits are DNA binding domains, especially domains for which the structure of a complex has been solved.

Stereo-view of a subdomain within the 1MB1 structure that includes residues 36 to 76. The color gradient ramps from blue (36) to green (76) and the "wing" is clearly seen as the green pair of beta-strands, extending to the right of the helix-turn-helix motif.

 

APSES domains represent one branch of the tree of helix-turn-helix (HTH) DNA binding modules. (A review on HTH proteins is linked from the resources section at the bottom of this page). Winged Helix domains typically bind their cognate DNA with a "recognition helix" which precedes the beta hairpin and binds into the major groove; additional stabilizing interactions are provided by the edge of a beta-strand binding into the minor groove. This is good news: once we have determined that the APSES domain is actually an example of a larger group of transcription factors, we can compare our model to a structure of a protein-DNA complex. Superfamilies of such structural domains are compiled in the CATH database. Unfortunately CATH itself does not provide information about whether the structures have been determined as complexes. But we can search the PDB with CATH codes and restrict the results to complexes. Essentially, this should give us a list of all winged helix domains for which the structure of complexes with DNA have been determined. This works as follows:

Task:

• For reference, access CATH domain superfamily 1.10.10.10; this is the CATH classification code we will l use to find protein-DNA complexes.

1. Navigate to the PDB home page and follow the link to Advanced Search
2. In the options menu for Choose a Query Type select Structure Features → CATH Classification Browser. A window will open that allows you to navigate down through the CATH tree. You can view the Class/Architecture/Topology names on the CATH page linked above. Click on the triangle icons (not the text) for Mainly Alpha → Orthogonal Bundle → ARC repressor mutant, subunit A then click on the link to winged helix repressor DNA binding domain. Or, just enter "winged helix" into the search field. This subquery should match more than 500 coordinate entries.
3. Click on the (+) button behind Add search criteria to add an additional query. Select the option Structure Features → Macromolecule type. In the option menus that pop up, select Contains Protein→Yes, Contains DNA→Yes, Contains RNA→Ignore, Contains DNA/RNA hybrid→Ignore. This selects files that contain Protein-DNA complexes.
4. Check the box below this subquery to Remove Similar Sequences at 90% identity and click on Submit Query. This query should retrieve more than 90 complexes.
5. Scroll down to the beginning of the list of PDB codes and locate the Reports menu. Under the heading View select Gallery. This is a fast way to obtain an overview of the structures that have been returned. Adjust the number of Results to see all 90 of the images and choose Options→Resize medium.
6. Finally we have a set of winged-helix domain/DNA complexes, for comparison. Scroll through the gallery and study how the protein binds DNA.

First of all you may notice that in fact not all of the structures are really different, despite having requested only to retrieve dissimilar sequences, and not all images show DNA. This appears to be a deficiency of the algorithm. But you can also easily recognize how in most of the the structures the recognition helix inserts into the major groove of B-DNA (eg. 1BC8, 1CF7) and the wing - if clearly visible at all in the image - appears to make accessory interactions with the DNA backbone.. There is one exception: the structure 1DP7 shows how the human RFX1 protein binds DNA in a non-canonical way, through the beta-strands of the "wing". This is interesting since it suggests there is more than one way for winged helix domains to bind to DNA. We can therefore use structural superposition of your homology model and two of the winged-helix proteins to decide whether the canonical or the non-canonical mode of DNA binding seems to be more plausible for Mbp1 orthologues.

 

Preparation and superposition of a canonical complex

 

The structure we shall use as a reference for the canonical binding mode is the Elk-1 transcription factor.

Stereo-view of the canonical DNA binding mode of the Winged Helix domain family. Shown here is the Elk-1 transcription factor - an ETS DNA binding domain - in complex with a high-affinity binding site (1DUX). Note how the "recognition helix" inserts into the major groove of the DNA molecule. The color gradient ramps from blue (34) to green (84). Note how the first helix of the "helix-turn-helix" architecture serves only to position the recognition helix and makes few interactions by itself.

The 1DUX coordinate-file contains two protein domains and two B-DNA dimers in one asymmetric unit. For simplicity, you should delete the second copy of the complex from the PDB file. (Remember that PDB files are simply text files that can be edited.)

Task:

1. Find the 1DUX structure in the image gallery and open the 1DUX structure explorer page in a separate window. Download the coordinates to your computer.
2. Open the coordinate file in a text-editor (TextEdit or Notepad - NOT MS-Word!) and delete the coordinates for chains D,E and F; you may also delete all HETATM records and the MASTER record. Save the file with a different name, e.g. 1DUX_monomer.pdb .
3. Open VMD and load your homology model. Turn off the axes, display the model as a Tube representation in stereo, and color it by Index. Then load your edited 1DUX file, display this coordinate set in a tube representation as well, and color it by ColorID in some color you like. It is important that you can distinguish easily which structure is which.
4. You could use the Extensions→Analysis→RMSD calculator interface to superimpose the two strutcures IF you would know which residues correspond to each other. Sometimes it is useful to do exactly that: define exact correspondences between residue pairs and superimpose according to these selected pairs. For our purpose it is much simpler to use the Multiseq tool (and the structures are simple and small enough that the STAMP algorithm for structural alignment can define corresponding residue pairs automatically). Open the multiseq extension window, select the check-boxes next to both protein structures, and open the Tools→Stamp Structural Alignment interface.
5. In the "'Stamp Alignment Options'" window, check the radio-button for Align the following ... Marked Structures and click on OK.
6. In the Graphical Representations window, double-click on all "NewCartoon" representations for both molecules, to undisplay them.
7. You should now see a superimposed tube model of your homology model and the 1DUX protein-DNA complex. You can explore it, display side-chains etc. and study some of the details of how a transcription factor recognizes and binds to its cognate DNA sequence. However, remember that your model's side-chain orientations have not been determined experimentally but inferred from the template, and that the template's structure was determined in the absence of bound DNA ligand.

1. Orient and scale your superimposed structures so that their structural similarity is apparent, and the recognition helix can be clearly seen inserting into the DNA major groove. You may want to keep a copy of the image for future reference. Consider which parts of the structure appear to superimpose best. Note whether it is plausible that your model could bind a B-DNA double-helix in this orientation.

 
 

Preparation and superposition of a non-canonical complex

The structure displaying a non-canonical complex between a winged-helix domain and its cognate DNA binding site is the human Regulatory Factor X.

Stereo-view of a non-canonical wHTH-DNA complex, discovered in with the stucture of human Regulatory Factor X (hRFX) binding its cognate X-box DNA sequence (1DP7). Note how the helix that coresponds to the recognition helix in the canonical domain lies across the minor groove whereas the beta-"wing" inserts into the major groove. The color gradient ramps from blue (18) to green (68).

Before we can work with this however, we have to fix an annoying problem. If you download and view the 1DP7 structure in VMD, you will notice that there is only a single strand of DNA! Where is the second strand of the double helix? It is not in the coordinate file, because it happens to be exactly equivalent to the frist starnd, rotated around a two-fold axis of symmetry in the crystal lattice. We need to download and work with the so-called Biological Assembly instead. But there is a problem related to the way the PDB stores replicates in biological assemblies. The PDB generates the additional chains as copies of the original and delineates them with MODEL and ENDMDL records, just like in a multi-structure NMR file. The chain IDs and the atom numbers are the same as the original. The PDB file thus contains the same molecule in two different orientations, not two independent molecules. This is an important difference regarding how such molecules are displayed by VMD. If you try to use the biological unit file of the PDB, VMD does not recognize that there is a second molecule present and displays only one chain. And that looks exactly like the one we have seen before. We have to edit the file, extract the second DNA molecule, change its chain ID and then append it to the original 1DP7 structure...

Task:

1. On the structure explorer page for 1DP7, select the option Download Files → PDB File.
2. Also select the option Download Files → Biological Assembly.
3. Uncompress the biological assembly file.
4. Open the file in a text editor.
5. Delete everything except the second DNA molecule. This comes after the MODEL 2 line and has chain ID D. Keep the TER and END lines. Save this with a new filename (e.g. 1DP7_DNAonly.pdb).
6. Also delete all HETATM records for HOH, PEG and EDO, as well as the entire second protein chain and the MASTER record. The resulting file should only contain the DNA chain and its copy and one protein chain. Save the file with a new name, eg. 1DP7_BDNA.PDB.
7. Use a similar procedure as BIO_Assignment_Week_8#R code: renumbering the model in the last assignment to change the chain ID.

PDBin <- "1DP7_DNAonly.pdb"
PDBout <- "1DP7_DNAnewChain.pdb"

pdb  <- read.pdb(PDBin)
pdb$atom[,"chain"] <- "E"
write.pdb(pdb=pdb,file=PDBout)

1. Use your text-editor to open both the 1DP7.pdb structure file and the 1DP7_DNAnewChain.pdb. Copy the DNA coordinates, paste them into the original file before the END line and save.
2. Open the edited coordinate file with VMD. You should see one protein chain and a B-DNA double helix. (Actually, the BDNA helix has a gap, because the R-library did not read the BRDU nucleotide as DNA). Switch to stereo viewing and spend some time to see how amazingly beautiful the complementarity between the protein and the DNA helix is (you might want to display protein and nucleic in separate representations and color the DNA chain by Position → Radial for clarity) ... in particular, appreciate how not all positively charged side chains contact the phosphate backbone, but some pnetrate into the helix and make detailed interactions with the nucleobases!
3. Then clear all molecules
4. In VMD, open Extensions→Analysis→MultiSeq. When you run MultiSeq for the first time, you will be asked for a directory in which to store metadata. You can use the default, or a directory of your choice; you may subsequently skip all steps that ask you to install "required" databases locally since we will not need them for this task.
5. Choose File→Import Data, browse to your directory and load one by one:

-Your model;

-The 1DUX complex;

-The 1DP7 complex.

1. Mark all three protein chains by selecting the checkbox next to their name and choose Tools→ STAMP structural alignment.
2. Align the Marked Structures, choose a scanscore of 2 and scanslide of 5. Also choose Slow scan. You may have to play around with the setting to get the molecules to superimpose: but the can be superimposed quite well - at lest the DNA-binding helices and the wings should line up.
3. In the graphical representations window, double-click on the cartoon representations that multiseq has generated to undisplay them, also undisplay the Tube representation of 1DUX. Then create a Tube representation for 1DP7, and select a Color by ColorID (a different color that you like). The resulting scene should look similar to the one you have created above, only with 1DP7 in place of 1DUX and colored differently.
4. Orient and scale your superimposed structures so that their structural similarity is apparent, and the differences in binding elements is clear. Perhaps visualizing a solvent accessible surface of the DNA will help understand the spatial requirements of the complex formation. You may want to keep a copy of the image for future reference. Note whether it is plausible that your model could bind a B-DNA double-helix in the "alternative" conformation.

 

Interpretation

Task:

1. Spend some time studying the complex.
2. You should clearly think about the following question: considering the position of the two DNA helices relative to the YFO structural model, which binding mode appears to be more plausible for protein-DNA interactions in the YFO Mbp1 APSES domains? Is it the canonical, or the non-canonical binding mode? Is there evidence that allows you to distinguish between the two modes?
3. Before you quit VMD, save the "state" of your session so you can reload it later. We will look at residue conservation once we have built phylogenetic trees. In the main VMD window, choose File→Save State....

That is all.

 

Links and resources

Gajiwala & Burley (2000) Winged helix proteins. Curr Opin Struct Biol 10:110-6. (pmid: 10679470)

[ PubMed ] [ DOI ] The winged helix proteins constitute a subfamily within the large ensemble of helix-turn-helix proteins. Since the discovery of the winged helix/fork head motif in 1993, a large number of topologically related proteins with diverse biological functions have been characterized by X-ray crystallography and solution NMR spectroscopy. Recently, a winged helix transcription factor (RFX1) was shown to bind DNA using unprecedented interactions between one of its eponymous wings and the major groove. This surprising observation suggests that the winged helix proteins can be subdivided into at least two classes with radically different modes of DNA recognition.

Aravind et al. (2005) The many faces of the helix-turn-helix domain: transcription regulation and beyond. FEMS Microbiol Rev 29:231-62. (pmid: 15808743)

[ PubMed ] [ DOI ] The helix-turn-helix (HTH) domain is a common denominator in basal and specific transcription factors from the three super-kingdoms of life. At its core, the domain comprises of an open tri-helical bundle, which typically binds DNA with the 3rd helix. Drawing on the wealth of data that has accumulated over two decades since the discovery of the domain, we present an overview of the natural history of the HTH domain from the viewpoint of structural analysis and comparative genomics. In structural terms, the HTH domains have developed several elaborations on the basic 3-helical core, such as the tetra-helical bundle, the winged-helix and the ribbon-helix-helix type configurations. In functional terms, the HTH domains are present in the most prevalent transcription factors of all prokaryotic genomes and some eukaryotic genomes. They have been recruited to a wide range of functions beyond transcription regulation, which include DNA repair and replication, RNA metabolism and protein-protein interactions in diverse signaling contexts. Beyond their basic role in mediating macromolecular interactions, the HTH domains have also been incorporated into the catalytic domains of diverse enzymes. We discuss the general domain architectural themes that have arisen amongst the HTH domains as a result of their recruitment to these diverse functions. We present a natural classification, higher-order relationships and phyletic pattern analysis of all the major families of HTH domains. This reconstruction suggests that there were at least 6-11 different HTH domains in the last universal common ancestor of all life forms, which covered much of the structural diversity and part of the functional versatility of the extant representatives of this domain. In prokaryotes the total number of HTH domains per genome shows a strong power-equation type scaling with the gene number per genome. However, the HTH domains in two-component signaling pathways show a linear scaling with gene number, in contrast to the non-linear scaling of HTH domains in single-component systems and sigma factors. These observations point to distinct evolutionary forces in the emergence of different signaling systems with HTH transcription factors. The archaea and bacteria share a number of ancient families of specific HTH transcription factors. However, they do not share any orthologous HTH proteins in the basal transcription apparatus. This differential relationship of their basal and specific transcriptional machinery poses an apparent conundrum regarding the origins of their transcription apparatus.

 

Footnotes and references

 

Ask, if things don't work for you!

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• Do consider how to ask your questions so that a meaningful answer is possible:
  • How to create a Minimal, Complete, and Verifiable example on stackoverflow and ...
  • How to make a great R reproducible example are required reading.