I am working with the Acetylcholinesterase enzyme, PDB ID 1B41. A few ligands are already bound in three different locations of the protein. Active sites according to UniProt are 234-Acyl-ester intermediates in 365 and 478 charge relay systems. I need to screen more than 200 molecules insilico and am confused how to define its active site. Any suggestions?
Predicting the actual active of protein is quit complected. Every protein will have three to ten pockets region based on its complex structure. If the structure is model insilico then it will provide more number of active site region when combare to the NMR or Crystallography and those pocket regions are meaning less ( because some pockets region shown as it contain only one amino acids).
In such a case we need to be very careful in which method or tools we using to predict the active site regions. Based on this results we need to make sever article review on each residues of the active site to make clear data about the properties of each one. such as how those active site residues make inter-molecular hydrogen bond and how many donor and acceptor regions where presented and in which basics its showing interaction with our compound and its any biological properties for those residues had already pointed. I will recommend http://projects.biotec.tu-dresden.de/metapocket/ this online tools for active site prediction. Here you will get clear idea.
Only active site interaction never shown the proper result, you have to check your molecules with the C-terminal region of your enzymes.
Results may varies from different docking software based on its algorithms its used and different force field we apply. so better you try your results with at-least three different docking programmes then only you can able to choose the accurate interaction sites and properties. I will recommend Discovery studio, Glide and ICM pro. These are commercial and more effective for computational biology studies.
Anyway I am not expect in this field and if anyone like to give more information I will feel happy to boost my Knowledge. All the best for your research work.
Thanx a lot, I would surely try this server and takes ur suggestions :)
AChE is an esterase, and will have a trypsin like active site, i.e. SER, HIS and ASP (sometimes GLU) is highlighting the active site. Pinpoint where you find these three residues next to one another , this is where the active site is located. Most often the active site is located at some depth in a cleft in the protein surface. Use modeling to highlight the protein surface in the vicinity of the active site cleft. Identify which residues in the cleft surface that are likely to assist in substrate binding.
There are a lot of AChE available as protein sequences. You mention that you will have to screen more than 200. AChE's are of considerable interest in Neuro sciences, since they are involved in neuronal signal transmission.
Use a multialignment program to align all your AChE sequences in order to identify which residues that are conserved across the different AChE's. Check which of these you find in the active site and the active site cleft. Those are the residues you should focus your search on.
Please rememberIt may also be important for you to be aware that AChE's have many related proteins, e.g. other esterase's as well as triglyceride lipase's.
You can use free online tool of IIT Delhi for predicting Active sites:
http://www.scfbio-iitd.res.in/dock/ActiveSite.jsp
Also some othe online tools are:
PAR-3D, DoGSiteScorer.. etc....
Dear Anita Ma'm , I have used SCFBIO tools, I would try PAR-3D, or DoGsitescorer. Thank you so much.
Dear All, I have a paper on similar topic, they have used 29 amino acids gorge as binding site in their work, including three main charge relay system of AChE, can i quote that paper and use same binding site for my work. please go through the link Page No 3 left column , last paragraph under heading Result and discussion and subheading, Binding site analysis:
http://link.springer.com/article/10.1007/s11426-009-0281-y?null#page-3
Respected Steffen sir, thanks for such idea about multiple alignment program and conserved regions. Sir please suggest me appropriate alignment tool.
Dear Anuj Ranjan,
There are many such tools - one which I have used for such alignments is CLUSTALW
http://www.genome.jp/tools/clustalw/
I also have a couple of published papers actually addressing lipases and esterases, that you may find useful in your work:
Title: Identification of conserved residues in family of esterase and lipase sequences
Author(s): Drablos, F; Petersen, SB
Book Editor(s): Rubin, B; Dennis, EA
Source: LIPASES, PART A: BIOTECHNOLOGY Book Series: Methods in Enzymology Volume: 284 Pages: 28-61 Published: 1997
Times Cited: 17 (from Web of Science)
Title: Identification of conserved residues in family of esterase and lipase sequences
Author(s): Drablos, F; Petersen, SB
Book Editor(s): Rubin, B; Dennis, EA
Source: LIPASES, PART A: BIOTECHNOLOGY Book Series: Methods in Enzymology Volume: 284 Pages: 28-61 Published: 1997
Times Cited: 17 (from Web of Science)
Title: Identification of important motifs in protein sequences: Program MULTIM and its applications to lipase-related sequences
Author(s): Petersen, SB; Drablos, F; Petersen, MTN; et al.
Book Editor(s): Rubin, B; Dennis, EA
Source: LIPASES, PART A: BIOTECHNOLOGY Book Series: Methods in Enzymology Volume: 284 Pages: 61-85 Published: 1997
Times Cited: 3 (from Web of Science)
Title: Surface and electrostatics of cutinases
Author(s): Neves Petersen, MT; Martel, P; Petersen, EI; et al.
Book Editor(s): Rubin, B; Dennis, EA
Source: LIPASES, PART A: BIOTECHNOLOGY Book Series: Methods in Enzymology Volume: 284 Pages: 130-+ Published: 1997
Times Cited: 11 (from Web of Science)
Title: How do lipases and esterases work: the electrostatic contribution
Author(s): Petersen, MTN; Fojan, P; Petersen, SB
Source: JOURNAL OF BIOTECHNOLOGY Volume: 85 Issue: 2 Pages: 115-147 Published: FEB 13 2001
Times Cited: 38 (from Web of Science)
Title: What distinguishes an esterase from a lipase: A novel structural approach
Author(s): Fojan, P; Jonson, PH; Petersen, MTN; et al.
Source: BIOCHIMIE Volume: 82 Issue: 11 Pages: 1033-1041 DOI: 10.1016/S0300-9084(00)01188-3 Published: NOV 2000
Times Cited: 97 (from Web of Science)
Anuj Ranjan,
Great answers by Steffen Petersen. His papers on conservation of esterases and lipases are excellent.
I would disagree only slightly on the recommendation of CLUSTALW for multiple sequence alignment. CLUSTAL works well on sequences of higher percent sequence identity, but does not perform as well when sequences have lower identity.
As Steffen pointed out, AChE is in the esterase lipase superfamily. Members of this family can share as little as 10 - 11% sequence identity or even lower, as is common with a conserved fold. When you are comparing sequences of such low identity but strong structural conservation, you might want to try other sequence alignment tools. If you need or want to do a multiple sequence alignment, for global alignment of several low-identity sequences, try MAFFT or PROBCONS, for best local alignment or alignment of sequences with long internal gaps, try MAFFT or T-Coffee.
You can access all of these tools (MAFFT, PROBCONS, T-Coffee and CLUSTALW) from the Max Planck Institute's Bioinformatics Toolkit at:
http://toolkit.tuebingen.mpg.de/sections/alignment
You can also get a view of alignments based on structural similarity. For this purpose, you can use the VAST tool from the NCBI. VAST builds alignments based on structural similarities. Here is the page for the VAST alignment of other proteins from PDB compared to your protein:
http://www.ncbi.nlm.nih.gov/Structure/vast/vastsrv.cgi?sdid=48215
That page shows you a graphical view of aligned regions, but you can also get a table of features and percent identities by selecting "Table" view in the box at the top of the page.
You can view a multiple structural alignment of up to ten of these proteins to get a visual idea of the location of the active site (using Steffen's suggestions). You can also see other conserved regions of the aligned structures.
From the VAST page, you can also view sequence alignments using mFASTA, then use these sequences for alignment with another multiple sequence alignment tool.
Superimpose the structure on 1mx9 pdb , the catalytic triade of 1mx9 is already known (ser , his and glu ) the corresponding residues at your structure will be the active site residues
To put it in simple words, Acetylcholinesterase enzyme is well-studied enzyme. I would prefer to set the grid box centered over catalytic residues, preferentially serine. Decrease the grid spacing with 90x90x90 box. Set LGA runs to 200 and choose the largest cluster with min. average binding energy for your analysis.
This should work
In case you have many AAs in your active site.. How do you choose best one for docking???
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