MSDsite help-solution:

You can use MSDsite service to mine for such a knowledge in the following suggested way:

I. MSDsite Loading:

Load MSD page by clicking here MSD and then to load the MSDsite click the link entitled MSDsite which you find under the links' list entitled "Services".

II. FOL binding:

We first want to see how the folate substrate (FOL) binds to DHFR:

Use the Ligand search fields from the form available on the MSDsite webpage and type FOL at the "Hetero:" box (see Fig.1) and then press the button .


Fig.1 MSDsite Input form (click image to enlarge)

MSDsite will generate a hit list of results (see Fig.2) by searching the MSD database for entries where FOL is bound. The list is by default sorted by Resolution value (refer to Resolution column) and shows the database entries representing enzymes that bind FOL (refer to ID column). Besides FOL, the main ligand, other ligands may be present that would represent cofactors, ions of interest to a particular investigation and/or compounds present in the crystallization solution (refer to Hetero list column).


Fig.2 MSDsite results page (click image to enlarge)

Remark. Other columns are present in the Results page, please feel free to explore.

Normally, to examine the binding of the ligand in question, we choose from the ID column the entry ID of interest and click on the provided link. For the sake of simplicity, we'll choose the 1rx7. Thus, click on the link 1rx7 to view summary of contacts (see Fig.3) (not detailed yet) per chain.


Fig.3 MSDsite contacts summary page (click image to enlarge)

For the purpose of following-up with the logic of this tutorial, in dealing with this result's page, we'll start from bottom upwards and focus only on the tutorial relevant sections. Jumps to different sections might also occur.

A. Table: Bound molecules

This table provides lots of useful links that are explained in the following:

1- Pattern/Active site column provide links to PROSITE patterns related to the main molecule under study (DHFR in this case). This particular pattern has been found to be conserved in almost all DHFRs from different species and is considered to constitute a signature specific to the DHFR family of enzymes. This can be useful to different functional and structural studies including Structure Modelling and Drug Design.

In order to see whether this PROSITE pattern makes contacts with the ligand and for more details on PROSITE patterns related to the protein sequence of the structure under investigation, click the link sequences at the top of the page.

The link will invoke a page entitled "Sequences 1rx7" that shows parts of the 1rx7 protein sequence where related PROSITE patterns are highlighted.

As seen above, amongst the listed patterns, the pattern PS00075 is the directly related one to DHFR, click the link to view and examine this DHFR signature (the pattern) in relation with FOL ligand. This uses the Astex/MSD-EBI viewer and you may like to click the link display the same as above using Rasmol viewer.

2- Hetero column provide the link "FOL" (see Fig.4) for detailed contacts of all DHFR residues with Folate. The link displays the 3-D structure of the environment; which is the ligand together with only those residues in contact with it.

The link display the same as above using Rasmol viewer.

The link gives contact statistics of the residues listed in the Environment column with any other Ligand found in the MSD database. These statistics would be useful in assessing the tendency of these residues in binding certain ligands.


Fig.4 MSDsite contacts details page (click image to enlarge)

3- Check-box() column can be ticked for the purpose of either:

1. Do search by the environment and this is done by first ticking the relevant check-box and then clicking on the button. This would allow you to find all MSD entries where any ligand is making contacts with the same residues of the FOL environment.

This can be useful in drug design attempts since similar/close binding pockets might show-up.
2. Use the multiple view function by first ticking the relevant check-box
   and then clicking the button .
   This will pop-up a separate page where your selection is listed and ready to add upon for multiple visualisation.

   Keep this page aside and follow on with it at section Ligands comparison(see further below).

4- Environment column shows the list of DHFR residues (their 3-letter codes, numbers and chain ID they belong to) making contacts with FOL. Colour shades are used to make it easy to identify the types of contacts made. Clicking on these links will invoke a page (Atomics bonds page) with the detailed contacts (see Fig.5) made between FOL and the residue in question. The atomic bond details include contact type, atoms involved and distances and other useful links.


Fig.5 MSDsite Atomic bonds per residue page (click image to enlarge)

B. Table: Bound molecule residues

The link FOL is for FOL chemical info which has been seen earlier.
The link is for doing ligand binding statistics. The page will show a bar-chart which represents a scan of FOL binding per residue-basis found in contact with in all of the MSD database.
These statistics would be useful in assessing the affinity of FOL to bind
proteins with binding pockets rich in certain types of residues.

III. MTX binding:

Load a new MSDsite page and type MTX at the "Hetero:" box from Ligand search fields form. From the results' hitlist choose the entry 1rg7 by clicking on it, and follow the section FOL binding's steps and explore the contacts details, perhaps by counting the number of bonds made and their types.

Select 1rg7 for multiple view and click the button . This will add the entry 1rg7 to the list of entries to be used in the multi-view function then
go to the section Ligands comparison.

IV. TOP binding:

Load a new MSDsite page and type TOP at the "Hetero:" box from Ligand search fields form. From the result hitlist choose the entry 1dyr by clicking on it, and follow the section FOL binding's steps and explore the contacts details, perhaps by counting the number of bonds made and their types.

Select 1dyr for multiple view and click the button . This will add the entry 1dyr to the list of entries to be used in the multi-view function then go to the section Ligands comparison.

V. Ligands comparison:

At this stage, see Fig.6, the page "Multi Entry View" should have three entries; 1rx7 (bound to FOL), 1rg7 (bound to MTX) and 1dyr (bound to TOP).

This page gives choices for multiple structural alignement for two or more selected entries:

First, tick the entries of your choice and select the "align by" option clicking on the relevant radio-button , then click either or to view and compare between the binding modes of ligands.


Fig.6 MSDsite Multi-view form (click image to enlarge)

VI. Ligand Binding Induced Structural Changes:

In order to examine the evidence of ligand binding induced changes in the structure of protein (DHFR in this case), we need to use an e.coli DHFR with no substrate/inhibitor ligand bound to it such as the structure represented by the entry 1ra9 and compare it with the above seen structure.

The comparison in this case involves doing structural alignment of whole structure and then visually inspect them preferably while they are backbone-displayed only.

For this task we need to use another MSD service, MSDfold that is the Secondary Structure Matching tool (SSM). Click in here SSM to load the service's web-page.

While on MSDfold main page (see Fig.7), Click the button to load the SSM Submission Form.


Fig.7 SSM Submission form (click image to enlarge)

The SSM allows for either pairwise or multiple structure alignement of protein entries (see Fig.8). The goal from studies like these may vary and thus necessitates different approaches for using the 3d-structural comparisons.


Fig.8 SSM Input form (click image to enlarge)

For the purpose of this tutorial we are going to use the pairwise option. Since we have a structure of e.coli DHFR with no substrate/inhibitor bound to it and on the other hand we have two structures of the same source DHFR binding FOL (a substrate) and MTX (an inhibitor), we can use SSM to discover what structural changes have happened to DHFR after binding FOL or MTX as seen below:

1. Structural effects of FOL on DHFR:

   On the SSM submitting form, using the "Query" column of the form and type 1ra9 at "PDB code" field and use the same field at the "Target" column to type 1rx7. Leave all SSM default options as they are and click the button .

   On the results page, you find the main results table click the link 1 which represents the matching structure 1rx7. The result pages will provide you with lots of useful and detailed data. One of the ways to visually observe the structural alignment is to click on the button see Fig.9.

   
   Fig.9 FOL structural effects when binding with e.coli DHFR (click image to enlarge)

   Main changes occur in the loop region which includes residues 16-18 when DHFR binds FOL, making this loop close on the FOL binding cleft.

2. Structural effects of MTX on DHFR:

   Follow the steps as seen above but with 1rg7 typed at the "Target" column. See Fig.10. which shows the MTX structural effect.

   
   Fig.10 MTX structural effects when binding e.coli with DHFR (click image to enlarge)

   MTX causes wider effect on the DHFR's loop region which includes residues 16-23 compared to when DHFR binds FOL, making this loop close dramatically on the MTX binding cleft.

   For more visualisation of the bigger effect of the inhibitor MTX when binding DHFR compared to subsrate FOL, use SSM to align 1rx7 and 1rg7. The dramatic effect of MTX observed in Fig.11 (look at the light gray loop and MTX in red) would count for its tight binding to DHFR thereby causing the inhibiting effect.

   
   Fig.11 Comparison of MTX and FOL structural effects when binding e.coli DHFR. The light gray loop pulled in more by MTX in red (click image to enlarge)