Affiliations 

  • 1 Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
  • 2 Division of Clinical Immunology and Transfusion Sciences, School of Allied Health Sciences, University of Phayao, Phayao, Thailand
  • 3 Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand
  • 4 Thailand Center of Excellence in Physics, Commission on Higher Education, Bangkok, Thailand
  • 5 Viral Infections and Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1, Lyon Cedex 07, France
  • 6 Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu, Taiwan
Asian Pac J Allergy Immunol, 2018 06;36(2):126-135.
PMID: 28802032 DOI: 10.12932/AP-280217-0037

Abstract

BACKGROUND: AnkGAG1D4 is an artificial ankyrin repeat protein which recognizes the capsid protein (CA) of the human immunodeficiency virus type 1 (HIV-1) and exhibits the intracellular antiviral activity on the viral assembly process. Improving the binding affinity of AnkGAG1D4 would potentially enhance the AnkGAG1D4-mediated antiviral activity.

OBJECTIVE: To augment the affinity of AnkGAG1D4 scaffold towards its CA target, through computational predictions and experimental designs.

METHOD: Three dimensional structure of the binary complex formed by AnkGAG1D4 docked to the CA was used as a model for van der Waals (vdW) binding energy calculation. The results generated a simple guideline to select the amino acids for modifications. Following the predictions, modified AnkGAG1D4 proteins were produced and further evaluated for their CA-binding activity, using ELISA-modified method and bio-layer interferometry (BLI).

RESULTS: Tyrosine at position 56 (Y56) in AnkGAG1D4 was experimentally identified as the most critical residue for CA binding. Rational substitutions of this residue diminished the binding affinity. However, vdW calculation preconized to substitute serine for tyrosine at position 45. Remarkably, the affinity for the viral CA was significantly enhanced in AnkGAG1D4-S45Y mutant, with no alteration of the target specificity.

CONCLUSIONS: The S-to-Y mutation at position 45, based on the prediction of interacting amino acids and on vdW binding energy calculation, resulted in a significant enhancement of the affinity of AnkGAG1D4 ankyrin for its CA target. AnkGAG1D4-S45Y mutant represented the starting point for further construction of variants with even higher affinity towards the viral CA, and higher therapeutic potential in the future.

* Title and MeSH Headings from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.