A novel reverse two-hybrid method for the identification of missense mutations that disrupt protein–protein binding

Abstract

The reverse two-hybrid system is a powerful method to select mutations that disrupt the interaction between two proteins and therefore to identify the residues involved in this interaction. However, the usefulness of this technique has been limited by its relative complexity when compared to the classical two-hybrid system, since an additional selection step is required to eliminate the high background of uninformative truncation mutants. We have developed a new method that combines the classical and reverse two-hybrid systems to select loss-of-binding missense mutations in a single step. The strategy used to select against truncation mutants is based on the two-hybrid interaction between a C-terminal fusion peptide and the Tsg101 protein. We have applied this method to identify mutations in human glucokinase (GK) that disrupt glucokinase regulatory protein (GKRP) binding. Our results indicate that this method is very efficient and eliminates all the truncation mutants and false positives. The mutated residues identified in GK are involved in the GKRP binding interface or in stabilizing the super-open conformation of GK that binds GKRP. This technique offers an improvement over existing methods in terms of speed, efficiency and simplicity and can be used to study any detectable protein interaction in the two-hybrid system.

Coiled-coil-mediated dimerization of Atg16 is required for binding to the PROPPIN Atg21

“…This result indicates that our screen was not saturating, although some mutations in blade 2 were isolated repeatedly. It is likely due to the mutational bias associated with error-prone PCR methods, which has already been observed with the Taq polymerase in a previous work [ 48 ].…”

“…Previous studies have shown that Atg21 interacts with Atg16 and mediates the recruitment of the E3 complex Atg12-Atg5-Atg16 to the phagophore membrane [ 19 ]. In order to determine which residues in Atg21 are critical for binding Atg16, we performed a reverse double two-hybrid screen (RD2H) [ 48 ] to identify missense mutations in Atg21 that disrupt the interaction with Atg16. This method is based on generating random mutations in a fusion of Atg21 with the Gal4 transcriptional activation domain (GAD) and a PTAP motif-containing peptide at the N-terminal and C-terminal ends, respectively.…”

“…Plasmids used in this work are described in electronic supplementary material, table S2 and were constructed for the current study except pRS316-GFP-AUT7 [ 57 ] and pLexA-TSG101 [ 48 ]. Two-hybrid plasmids encoding Gal4 Binding Domain (GBD) or Gal4 Activation Domain (GAD) fusions to Atg5, Atg16, Atg21 and human WIPI2b were constructed by cloning the corresponding coding sequences in the BamH1 site of pGBKT7, pACT2 or pGAD424 (Clontech).…”

“…The aim of this work was to identify the amino acid residues in Atg21 and Atg16 that play a critical role in protein–protein interaction by using the recently developed reverse double two-hybrid method (RD2H) [ 48 ]. Overall, our findings are in agreement with the recently reported crystal structure of Atg21 bound to Atg16 CCD [ 19 ], but unexpectedly our results also indicate that CCD-mediated dimerization of Atg16 is required for its interaction with Atg21.…”

Deep mutational scanning of proteins in mammalian cells

“…As the sequence of a protein dictates its structure and function, mutagenesis can aid in understanding different functional properties such as stability, interactions, and enzymatic activity, as well as mechanisms underlying disease and evolution. Mutagenesis studies of proteins have, for example, aided in the identification of protein-protein interaction (PPI) interfaces 1 , 2 , 3 , 4 and contributed to the study of ion channel functioning. 5 Technologies collectively referred to as deep mutational scanning (DMS) have advanced this field by allowing the simultaneous and unbiased evaluation of large protein variant libraries, 6 reflected in the recent expansion of this research field.…”