Journal of Computer-Aided Molecular Design

, Volume 19, Issue 6, pp 421–443

Design of Ligand Binding to an Engineered Protein Cavity Using Virtual Screening and Thermal Up-shift Evaluation

  • Claudia Machicado
  • Jon López-Llano
  • Santiago Cuesta-López
  • Marta Bueno
  • Javier Sancho
Article

DOI: 10.1007/s10822-005-7969-7

Cite this article as:
Machicado, C., López-Llano, J., Cuesta-López, S. et al. J Comput Aided Mol Des (2005) 19: 421. doi:10.1007/s10822-005-7969-7

Summary

Proteins could be used to carry and deliver small compounds. As a tool for designing ligand binding sites in protein cores, a three-step virtual screening method is presented that has been optimised using existing data on T4 lysozyme complexes and tested in a newly engineered cavity in flavodoxin. The method can pinpoint, in large databases, ligands of specific protein cavities. In the first step, physico-chemical filters are used to screen the library and discard a majority of compounds. In the second step, a flexible, fast docking procedure is used to score and select a smaller number of compounds as potential binders. In the third step, a finer method is used to dock promising molecules of the hit list into the protein cavity, and an optimised free energy function allows discarding the few false positives by calculating the affinity of the modelled complexes. To demonstrate the portability of the method, several cavities have been designed and engineered in the flavodoxin from Anabaena PCC 7119, and the W66F/L44A double mutant has been selected as a suitable host protein. The NCI database has then been screened for potential binders, and the binding to the engineered cavity of five promising compounds and three tentative non-binders has been experimentally tested by thermal up-shift assays and spectroscopic titrations. The five tentative binders (some apolar and some polar), unlike the three tentative non-binders, are shown to bind to the host mutant and, importantly, not to bind to the wild type protein. The three-step virtual screening method developed can thus be used to identify ligands of buried protein cavities. We anticipate that the method could also be used, in a reverse manner, to identify natural or engineerable protein cavities for the hosting of ligands of interest.

Key words

flavodoxin flexible docking ligand binding protein cavity protein stability reverse docking virtual screening 

Copyright information

© Springer 2005

Authors and Affiliations

  • Claudia Machicado
    • 1
    • 3
  • Jon López-Llano
    • 1
    • 3
  • Santiago Cuesta-López
    • 2
    • 3
  • Marta Bueno
    • 1
    • 3
  • Javier Sancho
    • 1
    • 3
  1. 1.Departamento de Bioquímica y Biología Molecular y CelularFacultad de Ciencias. Universidad de ZaragozaZaragozaSpain
  2. 2.Departamento de Física de la Materia CondensadaFacultad de Ciencias. Universidad de ZaragozaZaragozaSpain
  3. 3.Biocomputation and Complex Systems Physics Institute-BIFIUniversidad de ZaragozaZaragozaSpain

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