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Assignment 5 - Homology modeling

How could the search for ultimate truth have revealed so hideous and visceral-looking an object?

Max Perutz (on his first glimpse of the Hemoglobin structure)

   

Where is the hidden beauty in structure, and where, the "ultimate truth"? In the previous assignments we have studied sequence conservation in APSES family domains and we have discovered homologues in all fungal species. This is an ancient protein family that had already duplicated to several paralogues at the time the cenancestor of all fungi lived, more than 600,000,000 years ago, in the Vendian period of the Proterozoic era of Precambrian times.

In order to understand how specific residues in the sequence contribute to the putative function of the protein, and why and how they are conserved throughout evolution, we would need to study an explicit molecular model of an APSES domain protein, bound to its cognate DNA sequence. Explanations of a protein's observed properties and functions can't rely on the general fact that it binds DNA, we need to consider details in terms of specific residues and their spatial arrangement. In particular, it would be interesting to correlate the conservation patterns of key residues with their potential to make specific DNA binding interactions. Unfortunately, no APSES domain structures in complex with bound DNA has been solved up to now, and the experimental evidence we have considered in Assignment 2 (Taylor et al., 2000) is not sufficient to unambiguously define the details of how a DNA double helix might be bound. Moreover, at least two distinct modes of DNA binding are known for proteins of the winged-helix superfamily, of which the APSES domain is a member.

In this assignment you will (1) construct a molecular model of the APSES domain from the Mbp1 orthologue in your assigned species, (2) identify similar structures of distantly related domains for which protein-DNA complexes are known, (3) assemble a hypothetical complex structure and(4) discuss whether the available evidence allows you to distinguish between different modes of ligand binding,

For the following, please remember the following terminology:

Target

The protein that you are planning to model.

Template

The protein whose structure you are using as a guide to build the model.

Model

The structure that results from the modeling process. It has the Target sequence and is similar to the Template structure.

 

A brief overview article on the construction and use of homology models is linked to the resource section at the bottom of this page. That section also contains links to other sites and resources you might find useful or interesting.

Preparation, submission and due date

Read carefully.

Be sure you have understood all parts of the assignment and cover all questions in your answers! Sadly, we see too many assignments which, arduously effected, nevertheless intimate nescience of elementary tenets of molecular biology. If the sentence above did not trigger an urge to open a dictionary, you are trying to guess, rather than confirm possibly important information.

Review the guidelines for preparation and submission of BCH441 assignments.

The due date for the assignment is Monday, December 5. at 12:00 noon.

   

 

Your documentation for the procedures you follow in this assignment will be worth 2 marks - 1 mark for generating the model and 1 mark for the selection/superposition and visualization of protein/DNA complexes.

 

(1) Preparation

(1.1) Template choice and template sequence

The SWISS-MODEL server provides several different options for constructing homology models. The easiest is probably the Automated Mode that requires only a target sequence as input, in this mode the program will automatically choose suitable templates and create an input alignment. I disagree however that that is the best way to use such a service: the reason is that template choice and alignment both may be significantly influenced by biochemical reasoning, and an automated algorithm cannot make the necessary decisions. Should you use a structure of reduced resolution that however has a ligand bound? Should you move an indel from an active site to a loop region even though the sequence similarity score might be less? Questions like that may yield answers that are counter to the best choices an automated algorithm could make. Therefore we will use the Alignment Mode of Swiss-Model in this assignment, choose our own template and upload our own alignment.

Template choice is the first step. Often more than one related structure can be found in the PDB. We have touched on principles of selecting template structures in the lecture and I have posted a short summary of template choice principles on this Wiki. One can either search the PDB itself through its Advanced Search interface; for example one can search for sequence similarity with a BLAST search, or search for structural similarity by accessing structures according to their CATH or SCOP classification. But one can always also use the BLAST interface at the NCBI, since the sequences contained in PDB files are accessible as a database subsection on the BLAST menu.

n assignment 2 you have already searched for structures of APSES domains in the PDB. If you need to repeat this:

• Use the NCBI BLAST interface to identify all PDB files that are clearly homologous to your target APSES domain.
  • In Assignment 2, you have defined the extent of the APSES domain in yeast Mbp1. In Assignment 3, you have aligned reference APSES domains with those you found in your species (and you hopefully saved that JALVIEW project for easy re-access). In assignment 4 you have confirmed by phylogenetic analysis which of these APSES domain sequences is the closest related orthologue to yeast Mbp1. From this data you can define the APSES domain sequence of that protein in your species. This sequence is the best candidate for having a conserved function similar to yeast Mbp1. This sequence is the target for the homology modeling procedure.
  • To find suitable PDB strctures to model a target sequence, use that sequence as input for a BLAST search, and select Protein Data Bank proteins(pdb) as the Database you search in. Hits that are homologues are all suitable templates in principle, but some are more suitable than others. Consider how the coordinate sets differ and which features would make each more or less suitable for creating a homology model; comment briefly on

• sequence similarity to your target
• size of expected model (= length of alignment)
• presence or absence of ligands
• experimental method and quality of the data set

Then choose the template you consider the most suitable and note why you have decided to use this template.

It is not straightforward at all how to number sequence in such a project. The "natural" numbering starts with the start-codon of the full length protein and goes sequentially from there. However, this does not map exactly to other numbering schemes we have encountered. As you know the first residue of the APSES domain (as defined by CDD) is not Residue 1 of the Mbp1 protein. The first residue of the 1MB1 FASTA file (one of the related PDB strcuctures) is the first residue of Mbp1 protein, but the last five residues are an artifical His tag. Is H125 of 1MB1 thus equivalent to R125 in MBP1_SACCE? The N-terminus of the Mbp1 crystal structure is disordered. The first residue in the structure is ASN 3, therefore N is the first residue in a FASTA sequence derived from the cordinate section of the PDB file (the ATOM records; whereas the SEQRES records start with MET ... and so on. You need to remember: a sequence number is not absolute, but assigned in a particular context.

The homology model will be based on an alignment of target and template. Thus we have to define the target sequence. As discussed in class, PDB files have an explicit and an implied sequence and these do not necessarily have to be the same. To compare the implied and the explicit sequence for the template, you need to extract sequence information from coordinates. One way to do this is via the Web interface for WhatIf, a crystallography and molecular modeling package that offers many useful tools for coordinate manipulation tasks.

• Navigate to the Administration sub-menu of the WhatIf Web server. Follow the link to Make sequence file from PDB file. Enter the PDB-ID of your template into the form field and Send the request to the server. The server accesses the PDB file and extracts sequence information directly from the ATOM   records of the file. The results will be returned in PIR format. Copy the results, edit them to FASTA format and save them in a text-only file. Make sure you create a valid FASTA formatted file! Use this implied sequence to check if and how it differs from the sequence ...

• ... listed in the SEQRES records of the coordinate file;
• ... given in the FASTA sequence for the template, which is provided by the PDB;
• ... stored in the protein database of the NCBI.

and record your results.

• Establish how the sequence numbers in the coordinate section of your template(*) correspond to your target sequence numbering.

(*) These residue numbers are important, since they are referenced e.g. by VMD when you visualize the structure. The easiest way to list them is via the Sequence Viewer extension of VMD..

Don't do this for every residue individually but define ranges. Look at the correspondence of the first and last residue of target and template sequence and take indels into account. Establishing sequence correspondence precisely is crucially important! For example, when a publication refers to a residue by its sequence number, you have to be able to relate that number to the residue numbers of the model as well as your target sequence..

   

(1.2) The input alignment

 

The sequence alignment between target and template is the single most important factor that determines the quality of your model. No comparative modeling process will repair an incorrect alignment; it is useful to consider a homology model rather like a three-dimensional map of a sequence alignment rather than a structure in its own right. In a homology modeling project, typically the largest amount of time should be spent on preparing the best possible alignment. Even though automated servers like the SwissModel server will align sequences and select template structures for you, it would be unwise to use these only because they are convenient. You should take advantage of the much more sophisticated alignment methods available. Analysis of wrong models can't be expected to produce right results.

The best possible alignment is usually constructed from a multiple sequence alignment that includes at least the target and template sequence and other related sequences as well. The additional sequences are an important aid in identifying the correct placement of insertions and deletions. Your alignment should have been carefully reviewed by you and wherever required, manually adjusted to move insertions or deletions between target and template out of the secondary structure elements of the template structure.

In most of the Mbp1 orthologues, we do not observe indels in the APSES domain regions - (and for the ones in which we do see indels, we might suspect that these are actually gene-model errors). Evolutionary pressure on the APSES domains has selected against indels in the more than 600 million years these sequences have evolved independently in their respective species. To obtain an alignment between the template sequence and the target sequence from your species, proceed as follows.

• Load your Jalview project with aligned APSES domain sequences or recreate it from the Mbp1 orthologue in your species and the APSES domains from the Reference APSES domain page that I prepared for Assignment 4. Include the sequence of your template protein and re-align.
• Delete all sequence you no longer need, i.e. keep only the APSES domains of the target (from your species) and the template (from the PDB) and choose Edit → Remove empty columns. This is your input alignment.
• Choose File→Output to textbox→FASTA to obtain the aligned sequences. They should both have exactly the same length, i.e. N- or C- termini have to be padded by hyphens if the original sequences had different length. Save the sequences in a text-file.

 
 

(2) Homology model

   

(2.1) SwissModel

 

Access the Swissmodel server at http://swissmodel.expasy.org . Navigate to the Alignment Mode page.

 

• Paste your alignment for target and model into the form field. Refer to the Fallback Data file if you are not sure about the format. Make sure to select the correct option (FASTA) for the alignment input format on the form.

• Click submit alignment and on the returned page define your target and template sequence. For the template sequence define the PDB ID of the coordinate file. Enter the correct Chain-ID.

If you run into problems, compare your input to the fallback data. It has worked for me, it will work for you. In particular we have seen problems that arise from "special" characters in the FASTA header like the pipe "|" character that the NCBI uses to separate IDs - keep the header short and remove all non-alphanumeric characters to be safe.

• Click submit alignment and review the alignment on the returned page. Make sure it has been interpreted correctly by the server. The conserved residues have to be lined up and matching. Then click submit alignment again, to start the modeling process.

• The resulting page returns information about the resulting model. Save the model coordinates on your computer. Read the information on what is being returned by the server (click on the red questionmark icon). Paste the Anolea profile into your assignment.

Do not paste a screenshot of the result, but copy and paste the image from the Web-page! You do not need to submit the actual coordinate files with your assignment.

(3) Model analysis

   

(3.1) The PDB file

 

Open your model coordinates in a text-editor (make sure you view the PDB file in a fixed-width font) and consider the following questions:

• What is the residue number of the first residue in the model? What should it be, based on the alignment? If the putative DNA binding region was reported to be residues 50-74 in the Mbp1 protein, which residues of your model correspond to that region?

(3.2) First visualization

 

In assignment 2 you have already studied a Mbp1 structure and compared it with your organism's Mbp1 APSES domain, Since a homology model inherits its structural details from the template, the model should look very similar to the original structure but contain the sequence of the target.

 

• Save your model coordinates to your computer and visualize the structure in VMD. Make an informative (parallel, not cross-eyed!) stereo view that shows the general orientation of the helix-turn-helix motif and the "wing", and paste it into your assignment.

 
 

(4) The DNA ligand

   

(4.1) Finding a similar protein-DNA complex

 

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 add a plausible 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?

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.

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, MSDfold provides a convenient interface for structure searches.

However we have also read previously that the APSES domains are members of a much larger superfamily, the "winged helix" DNA binding domains , of which hundreds of structures have 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. CATH does not provide information on complexes, but we can search the PDB with CATH codes in the following way:

• Access CATH domain 1.10.10.10.
• Navigate to the PDB home page and follow the link to Advanced Search
• In the options menu for "Choose a Query Type" select Structure Features → CATH classification. A window will open that allows you to navigate down through the CATH tree. The interface is awkward because it does not display the actual CATH codes along with the class names, but you can view the class names on the CATH page linked above. Click on the triangle icons before "Mainly Alpha"→"Orthogonal Bundle"→"ARC repressor mutant, subunit A" then click on the link to "winged helix repressor DNA binding domain". As of this writing, this subquery matches 368 structures.
• Click on the (+) button behind the subquery to add an additional query. Select the option "Structure Summary"→"Molecule / Chain type". In the option menus that pop up, select "Contains Protein → Yes", "Contains DNA → Yes""Contains RNA → Ignore". This selects files that contain Protein-DNA complexes.
• Check the box below this subquery to "Remove Similar Sequences at 90% identity" and click on "Evaluate Query". As of this writing, 83 complexes were returned.
• In the left-hand menu, under the Tabulate section, click on the "Collage" function to display icons of the structure files (Low resolution -80 pixels should be fine). This is a fast way to obtain an overview of the structures that have been returned. First of all you may notice that in fact not all of the structures are really different, despite selecting only to retrieve dissimilar sequences. This appears to be a deficiency of the algorithm. But you can also easily recognize how the recognition helix inserts into the major groove of most of the structures that were returned (at least those where the domain is not a very small part of a much larger complex). There is one exception: the structure 1DP7 shows how the human RFX1 protein binds DNA in a non-canonical way. This allows us to use structural superposition of your homology model and two of the winged-helix proteins to decide which mode of DNA binding seems to be more plausible for Mbp1 homologues.

 

• Follow the procedure outlined above, from a CATH entry page up to viewing a Collage (or alternatively a tabular view) of the retrieved coordinate files. You can be maximally concise in your documentation of following the procedure I have defined above, but do spend a bit of time to understand the key elements of the PDB's advanced search interface.

(4.2) 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, let's delete the second copy of the complex.

• Access the PDB and navigate to the 1DUX structure explorer page. Download the coordinates to your computer.
• Open the coordinate file in a text-editor 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 .
• 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
• 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.
• In the "'Stamp Alignment Options'" window, check the radio-button for Align the following ... Marked Structures and click on OK.
• In the Graphical Representations window, double-click on all "NewCartoon" representations for both molecules, to undisplay them.
• 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 the template's structure was determined in the absence of bound ligand.

 

• 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. Paste a copy of your image into your assignment. Remark briefly on which parts of the structure appear to superimpose best. Note whether this orientation of a B-DNA double-helix is a plausible model for DNA binding of your Mbp1 orthologue.

 
 

(4.2) 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).

The 1DP7 coordinate-file contains only one protein domain and only one B-DNA monomer in its asymmetric unit. This is a file for which we have to generate biological unit coordinates! Then, for simplicity we will delete the second protein monomer. As you know, there are at least two Web tools that make the so-called biological units available: the PDB itself, through the Biological Unit file that is accessible via the "Download Files" section on any Structure Explorer page, and the EBI through the PQS service. How the biological units are stored is subtly different for both cases and for our purpose this small difference is important. The PDB generates additional chains as copies of the original and delineates them with MODEL, ENDMDL records, just like in a multi-structure NMR file. The chain IDs and the atom numbers are the same as the original. The EBI's PQS service creates copies that have distinct atomnumbers and chain IDs. The difference is that the PDB file thus contains the same molecule in two different orientations, wheras the PQS file contains two independent molecules. This is an important difference when it comes to selecting residues, visualizing and superimposing structures. For VMD, the PQS way of doing things is the right way to go, since by default only the first MODEL will be displayed if several are available.

• Access the EBI PQS server, enter 1DP7 into the PDBidcode form field and click on Submit.
• On the results page, click on the link under 1dp7_0, which is the unique suggestion for a biological unit that the server has identified.
• On the PQS OUTPUT page that is retrieved, click on the 1dp7.mmol link, this will load the PDB formatted coordinate file.
• Save the coordinates as 1DP7_complex.pdb (or some other name that makes sense to you), open it in a text editor, delete the HETATM records from the end and the entire chain "B". Also make sure not to delete any of the TER records for chains "D", "P" or "A". Save the file.
• In the multiseq window, choose File→Import Data, Browse... to your 1DP7_complex file, select it and click on Open. Click OK to load the file.
• Mark all three protein chains by selecting the checkbox next to their name and again run the STAMP structural alignment.
• In the graphical representations window, double-click again on all cartoon representations that multiseq has generated to undisplay them, undisplay also the Tube representation of 1DUX, create a Tube representation for 1DP7, and select a Color by ColorID (a differenet color 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.

 

• Orient and scale your superimposed structures so that their structural similarity is apparent, the orientation is similar to the scene generated above and the 1DP7 "wing" can be clearly seen inserting into the DNA major groove. Paste a copy of your image into your assignment. Remark briefly on which parts of the structure appear to superimpose best. Note whether this orientation of a B-DNA double-helix is a plausible model for DNA binding of your Mbp1 orthologue.

 

(4.3) Interpretation

 

In your previous assignment, you have commented on conservation patterns in Mbp1 orthologues. You can refer back to your last results (easier to do), or you can import the APSES domain alignment for Mbp1 proteins and again color by conservation (easier to study) to briefly discuss the following question.

Analysis (2 marks)

• Considering the conservation patterns for Mbp1 orthologues, and assuming that all these orthologues bind DNA in a similar way, which model appears to be more plausible for protein-DNA interactions in APSES domains? Is it the canonical, or the non-canonical binding mode? Discuss briefly what you would expect to find and how this relates to your observations. Distinguish clearly between experimental evidence, computational inference and empirical hypothesis. You are of course welcome to paste detail views (stereo !) of particular sidechains, or surfaces etc. if this helps your arguments. Sometimes a picture is worth many words. But this is not a requirement, we are more interested in evidence-based reasoning than in the form of the presentation.

 
 

(5) Summary of Resources

 

Links and background reading

• Review (PDF, restricted) Manuel Peitsch on Homology Modeling
• Review (PDF, restricted) Aravind et al. Helix-turn-helix domains
• Review (PDF, restricted) Gajiwala & Burley, winged-Helix domains
• PDB file format (see the Coordinate Section if you are unsure about chain identifiers)
• Wikipedia on Structural Superposition (although the article is called "Structural Alignment")

Fallback Data page

Alignments

• APSES domains MUSCLE aligned

   

[End of assignment]

If you have any questions at all, don't hesitate to mail me at boris.steipe@utoronto.ca or post your question to the 2011 Course Mailing List .