Random mutagenesis MAPPIT analysis identifies binding sites for Vif and Gag in both cytidine deaminase domains of Apobec3G

Isabel Uyttendaele; Delphine Lavens; Dominiek Catteeuw; Irma Lemmens; Celia Bovijn; Jan Tavernier; Frank Peelman

doi:10.1371/journal.pone.0044143

Random mutagenesis MAPPIT analysis identifies binding sites for Vif and Gag in both cytidine deaminase domains of Apobec3G

PLoS One. 2012;7(9):e44143. doi: 10.1371/journal.pone.0044143. Epub 2012 Sep 10.

Authors

Isabel Uyttendaele¹, Delphine Lavens, Dominiek Catteeuw, Irma Lemmens, Celia Bovijn, Jan Tavernier, Frank Peelman

Affiliation

¹ Department of Medical Protein Research, VIB, Ghent, Belgium.

Abstract

The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultaneously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

APOBEC-3G Deaminase
Binding Sites
Cytidine Deaminase / chemistry*
Cytidine Deaminase / metabolism
HEK293 Cells
HIV-1 / metabolism
Humans
Models, Molecular
Mutagenesis*
Mutant Proteins / metabolism
Mutation / genetics
Protein Binding
Protein Interaction Mapping
Protein Multimerization
Protein Structure, Tertiary
Two-Hybrid System Techniques*
gag Gene Products, Human Immunodeficiency Virus / metabolism*
vif Gene Products, Human Immunodeficiency Virus / metabolism*

Substances

Mutant Proteins
gag Gene Products, Human Immunodeficiency Virus
vif Gene Products, Human Immunodeficiency Virus
APOBEC-3G Deaminase
APOBEC3G protein, human
Cytidine Deaminase

Grants and funding

This work is supported by the Fund of Scientific Research (FWO, www.fwo.be) Grant (G.0747.10N), Interuniversitary Attraction Poles (P6:28) (http://www.belspo.be/belspo iap/index_en.stm) and a Ghent University Methusalem grant (http://www.ugent.be/en/research/topresearch methusalem_laureates.htm/). DL and IL are postdoctoral fellows of the Fund of Scientific Research (FWO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.