BINF 731

Assignment 2

Manal Othman

G00934163

1-Select a sequence of a human enzyme from one of the protein sequence databases that has a homolog in PDB with at least 30%, but not more than 70% of sequence identity. (Recommended length of the selected sequence is 100-250 residues).

·      From PDB search I wrote enzyme

·      I chose Homo sapiens

·      I chose Homo sapiens only

·      I chose from enzyme classification: Isomerases because it has only 35 enzyme

·      Then I chose the first one because it has 165 residues to meet the question requirement (100-250 residues) which is:

·       Multiconformer synchrotron model of CypA at 100 K: 4YUG Chain A

·      From PDB page of 4YUG I select sequence similarity tab

·      I selected 40% Sequence Similarity Cutoff.

·      Then I picked the first one which is:

Atomic resolution crystal structure of the ppiase domain of human cyclophilin g: 2WFI Chain A

I checked the sequence identity by JCE: pairwise alignment

·      So, the sequence ID: 51% < 70% to meet the question requirement.

2-Predict the three-dimensional structure for the selected sequence using at least two methods (e.g., one of the fold recognition algorithms and homology modeling with SwissModel).

A)- One of the fold recognition algorithms:

·      Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations by using advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants for protein sequence.

 

·      In this tool, users are guided through results by a simple interface at a level of detail they determine. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality.

 

·      I went to Phyre2. The human enzyme 4YUG:A was modeled using Phyre: is a threading-based fold recognition server. Unlike SwissModel, no templates are required for modeling, all that was required was to provide an e-mail address, job ID, and to paste the protein sequence into the "Amino Acid Sequence" box and submit the job. Results were e-mailed to me after about 2 minutes.

·      Here is the result for the modlling:

·      As the figure above, a list of protein structure models are obtained with their corresponding precision percentage. The first fold of the result obtained has an estimate of 100% confidence and 100% identity and the rest of the with less >65% of identity.

B)- Homology modeling with SwissModel:

·      SWISS-MODEL is a fully automated protein structure homology-modelling server. Homology modeling is currently the most accurate method to generate reliable three-dimensional protein structure models and is routinely used in many practical applications. is the most widely-used free web-based automated modeling facility today.

·      I went to SwissModel website

·      To identify template (2WFI:A)

·      Upload the 4YUG/A sequence.

·      Hit search of template.

·      All the results that appeared was from (100% to 70%) sequence id. So, I looked at the end of the page: It says: The full list of templates matching your target sequence includes the following 182 templates which are not in the list above. The full template list is available in text or html format.

·      I chose html format and look for the 2WFI/A

·      Hit build model (monomer): This is the result.

The details report of the result will be here.

 

To do the model with the template:

·      I copy this the template alignment sequence.

·      From top of page open the molding tab.

·      Select alignment mode.

·      Paste the template alignment sequence that I copied into Target-Template Alignment window.

·      Hit build model

The result:

3-Analyze the quality of your model using one of the structure validation or verification tools.

·      The quality of the model can be evaluated using any of the structure validation tools in SwissModel.

·      These tools can be found in under tools tap, which is "Structure Assessment".

·      I did my structure assessment with:

1-Anolea: Anolea atomic mean force potential

2-QMEAN6: Composite scoring function for model quality estimation

3-Procheck: Stereochemical quality check; min. Resolution: 2.5Å

Acopy of the result will be here

·      Anolea evaluates the model through identifying the areas of favorable energy environment to be (negative/green) and the (positive/red) represent the unfavorable energy environment.

·      Anolea structure assessment measure helps in building models with minimal energy.

·      The result shows that the overall quality of this model is very good, lots of green or low energy area, mostly from all the protein. There are some regions show residues residing in (red) unfavorable (high) energy environment.

·      The result from QMEAN score: 0.813

·      The result from Ramachandran plot in Procheck is: 77.1% core.

·      A detaild copy of the result will be here

 

4-Evaluate separately one of the biologically important parts of the structure (e.g., an active site or binding site).

·      To Evaluate the biologically important parts of my protein, I went to NCBI.

·      I select Sequence analysis.

·      From tools part, I select Conserved Domain Search Service (CD Search) that Identifies the conserved domains present in a protein sequence.

·      Submitting the 4YUG:A sequence, a graphical view of the results is shown as follows:

·      cyclophilin_ABH_like: Cyclophilin A, B and H-like cyclophilin-type peptidylprolyl cis-trans isomerase (PPIase) domain. This family represents the archetypal cystolic cyclophilin similar to human cyclophilins A, B and H. PPIase is an enzyme which accelerates protein folding by catalyzing the cistrans isomerization of the peptide bonds preceding proline residues.

 

·      From the first model that was done by Swissmodel, It shows that The 4YUG:A and its template 2WFI:A with a sequence identity 55.28% are not sharing the active site in residue W:121, which is Tryptophan (TRP).

5-Introduce residue substitution(s) at one or more positions of your original sequence, which would likely affect the structure and/or function of this protein.

·      A substitution mutation of any part of the protein sequence may likely result in changing of the function of the protein.

·      In 4YUG:A sequence, substituting the active side in residue TRP:121 with A (ALA) will effect the activity of this site.

·   I select the 121 residue because the substitution of W → A: 200-fold decrease of sensitivity to cyclopsporin A (CsA). cyclopsporin A is an immunosuppressive drug (Liu et al., 1991).

·      In  human W121 mutations to F121 or A121 yield cyclophilins with 75- and 200-fold decreased sensitivity to CsA, while kcat/Km values of rotamase activity in a tetrapeptide assay drop only 2- and 13-fold, respectively. This complementary gain and loss of CsA sensitivity to mutation to or from tryptophan validate the indole side chain as a major determinant in immunosuppressant drug recognition and the separation of PPIase catalytic efficiency from CsA affinity.

6-Model the structure of this mutant and compare it with the structure of the wild type model.

·      I upload the FASTA file for 4YUG:A from Pdb.

·      I made the mutation in the file, I substitute the residue 121: W to A (TRP to ALA).

·      I went to SwissModel website

·      Upload the mutant 4YUG/A sequence.

·      I fellow the same steps that I mentioned earlier in this repot to build model.

·      The template that I chose it here was the same as the one before which is: 2WFI:A

·      In addition, for comparison, the mutant model was submitted to the structure assessment tool in SwissModel.

·      By comparing the mutant model results against those for the wild type: The substitution mutation on residue 121:TRP made a slight increase of the favorable energy and make the anolea's plot more greenish.

·      The result from Ramachandran plot in Procheck remain the same value for the both model is: 77.1% core. Which is means that the change effect of the mutation from W to A was minimal in the structure side but huge in function side as I mentioned in the previous question.

·      A detaild copy of the mutant model will be here.

                                                       

Rererence:

·      Biochemistry   vol. 30 (9)       p. 2306-10Liu, J., Chen, C. M., & Walsh, C. T. (1991). Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue. Biochemistry, 30(9), 2306–10. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/2001362