CompASM: an Amber-VMD alanine scanning mutagenesis plug-in


Alanine scanning mutagenesis (ASM) of protein–protein interfacial residues is a popular means to understand the structural and energetic characteristics of hot spots in protein complexes. In this work, we present a computational approach that allows performing such type of analysis based on the molecular mechanics/Poisson–Boltzmann surface area method. This computational approach has been used largely in the past and has proven to give reliable results in a wide range of complexes. However, the sequential preparation and manual submission of dozens of files has been often a major obstacle in using it. To overcome these limitations and turn this approach user-friendly, we have designed the plug-in CompASM (computational alanine scanning mutagenesis). This software has an easy-to-use graphical interface to prepare the input files, run the calculations, and analyze the final results. CompASM was built in TCL/TK programming language to be included in VMD as a plug-in. The CompASM package is distributed as an independent platform, with script code under the GNU Public License from

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  1. 1.

    Bordner AJ, Abagyan R (2005) Statistical analysis and prediction of protein–protein interfaces. Proteins 60(3):353–366. doi:10.1002/Prot.20433

    Article  CAS  Google Scholar 

  2. 2.

    Thorn KS, Bogan AA (2001) ASEdb: a database of alanine mutations and their effects on the free energy of binding in protein interactions. Bioinformatics 17(3):284–285

    Article  CAS  Google Scholar 

  3. 3.

    Moreira IS, Fernandes PA, Ramos MJ (2007) Computational alanine scanning mutagenesis—an improved methodological approach. J Comput Chem 28(3):644–654. doi:10.1002/jcc.20566

    Article  CAS  Google Scholar 

  4. 4.

    Ramos MJ, Moreira IS, Fernandes PA (2007) Hot spots—a review of the protein–protein interface determinant amino-acid residues. Proteins 68(4):803–812. doi:10.1002/Prot.21396

    Article  Google Scholar 

  5. 5.

    Moreira IS, Fernandes PA, Ramos MJ (2007) Hot spot occlusion from bulk water: a comprehensive study of the complex between the lysozyme HEL and the antibody FVD1.3. J Phys Chem B 111(10):2697–2706. doi:10.1021/Jp067096p

    Article  CAS  Google Scholar 

  6. 6.

    Kollman PA, Massova I, Reyes C, Kuhn B, Huo SH, Chong L, Lee M, Lee T, Duan Y, Wang W, Donini O, Cieplak P, Srinivasan J, Case DA, Cheatham TE (2000) Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acc Chem Res 33(12):889–897. doi:10.1021/Ar000033j

    Article  CAS  Google Scholar 

  7. 7.

    Fernandez-Recio J (2011) Prediction of protein binding sites and hot spots. Wires Comput Mol Sci 1(5):680–698. doi:10.1002/Wcms.45

    Article  CAS  Google Scholar 

  8. 8.

    Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, Spellmeyer DC, Fox T, Caldwell JW, Kollman PA (1996) A second generation force field for the simulation of proteins, nucleic acids, and organic molecules (vol 117, pg 5179, 1995). J Am Chem Soc 118(9):2309–2309

    Google Scholar 

  9. 9.

    Rocchia W, Sridharan S, Nicholls A, Alexov E, Chiabrera A, Honig B (2002) Rapid grid-based construction of the molecular surface and the use of induced surface charge to calculate reaction field energies: applications to the molecular systems and geometric objects. J Comput Chem 23(1):128–137

    Article  CAS  Google Scholar 

  10. 10.

    Rocchia W, Alexov E, Honig B (2001) Extending the applicability of the nonlinear Poisson–Boltzmann equation: multiple dielectric constants and multivalent ions. J Phys Chem B 105(28):6507–6514

    Article  CAS  Google Scholar 

  11. 11.

    Massova I, Kollman PA (1999) Computational alanine scanning to probe protein–protein interactions: a novel approach to evaluate binding free energies. J Am Chem Soc 121(36):8133–8143

    Article  CAS  Google Scholar 

  12. 12.

    Huo S, Massova I, Kollman PA (2002) Computational alanine scanning of the 1:1 human growth hormone–receptor complex. J Comput Chem 23(1):15–27

    Article  CAS  Google Scholar 

  13. 13.

    Case DA, Darden TA, Cheatham, Simmerling CL, Wang J, Duke RE, Luo R, Merz KM, Pearlman DA, Crowley M, Walker RC, Zhang W, Wang B, Hayik S, Roitberg A, Seabra G, Wong KF, Paesani F, Wu X, Brozell S, Tsui V, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Beroza P, Mathews DH, Schafmeister C, Ross WS, Kollman PA (2006) Amber 9

  14. 14.

    Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38

    Article  CAS  Google Scholar 

  15. 15.

    Bhat TN, Bentley GA, Boulot G, Greene MI, Tello D, Dallacqua W, Souchon H, Schwarz FP, Mariuzza RA, Poljak RJ (1994) Bound Water-molecules and conformational stabilization help mediate an antigen-antibody association. Proc Natl Acad Sci USA 91(3):1089–1093

    Article  CAS  Google Scholar 

  16. 16.

    Somers WS, Mosyak L, Zhang Y, Glasfeld E, Haney S, Stahl M, Seehra J (2000) The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography. EMBO J 19(13):3179–3191

    Article  Google Scholar 

  17. 17.

    Sauereriksson AE, Kleywegt GJ, Uhl M, Jones TA (1995) Crystal-structure of the C2 fragment of streptococcal protein-G in complex with the Fc domain of human-Igg. Structure 3(3):265–278

    Article  CAS  Google Scholar 

  18. 18.

    Ofran Y, Rost B (2007) ISIS: interaction sites identified from sequence. Bioinformatics 23(2):E13–E16. doi:10.1093/Bioinformatics/Btl303

    Article  CAS  Google Scholar 

  19. 19.

    Neuvirth H, Raz R, Schreiber G (2004) ProMate: a structure based prediction program to identify the location of protein–protein binding sites. J Mol Biol 338(1):181–199. doi:10.1016/J.Jmb.2004.02.040

    Article  CAS  Google Scholar 

  20. 20.

    Kortemme T, Baker D (2002) A simple physical model for binding energy hot spots in protein–protein complexes. Proc Natl Acad Sci USA 99(22):14116–14121

    Article  CAS  Google Scholar 

  21. 21.

    Darnell SJ, Page D, Mitchell JC (2007) An automated decision-tree approach to predicting protein interaction hot spots. Proteins 68(4):813–823. doi:10.1002/Prot.21474

    Article  CAS  Google Scholar 

  22. 22.

    Tuncbag N, Keskin O, Gursoy A (2010) HotPoint: hot spot prediction server for protein interfaces. Nucleic Acids Res 38:W402–W406. doi:10.1093/Nar/Gkq323

    Article  CAS  Google Scholar 

  23. 23.

    Moreira IS, Martins JM, Ramos RR, Fernandes PA, Ramos MJ (2012) Computational alanine scanning mutagenesis: a new method for hot-spot detection (Results to be published)

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The authors would like to thank the FCT (Fundação para a Ciência e Tecnologia) for financial support (Grants SFRH/BD/61324/2009 and PTDC/QUI-QUI/103118/2008).

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Correspondence to Maria João Ramos.

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Dedicated to Professor Marco Antonio Chaer Nascimento and published as part of the special collection of articles celebrating his 65th birthday.

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Ribeiro, J.V., Cerqueira, N.M.F.S.A., Moreira, I.S. et al. CompASM: an Amber-VMD alanine scanning mutagenesis plug-in. Theor Chem Acc 131, 1271 (2012).

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  • Protein–protein interactions
  • Amber
  • VMD
  • Software