Characterization of bZIP protein binding specificities using a BACTH assay system
Poster Number
7
Introduction/Abstract
Basic leucine zipper domain (bZIP) transcription factors are proteins that dimerize to bind the promoter region of genes and control their expression. Interacting as classical helical coiled coils, the bZIP transcription factors exhibit the well-known pseudo- heptad repeat motif where every seventh residue is a leucine. Determination of the bZIP packing structure using the Knob-Socket (KS) model is a novel method to map the coiled-coil protein-protein interactions. The KS analysis and mapping has identified the residue sequences that may play a role in coiled-coil specificity.
Purpose
In this work I investigated bZIP protein interactions, and the amino acids within these coiled-coil proteins that dictate dimer specificity. Through KS mapping analysis, residue sequences that may play a role in coiled-coil specificity were identified, and their importance was analyzed experimentally through site-directed mutagenesis and BACTH studies.
Method
To confirm the KS analysis of bZIP specificity, a bacterial adenylate cyclase two hybrid (BACTH) system coupled with a beta galactosidase assay was used to test dimerization of coiled-coils. A BACTH system uses a protein interaction-mediated reconstitution of adenylate cyclase activity in E. coli. The catalytic domain of adenylate cyclase is composed of two fragments, T25 and T18. When separated, the enzyme is rendered nonfunctional. To take advantage of this, fusion proteins consisting of one of the adenylate cyclase fragments and a bZIP protein are made. If the bZIP proteins interact during co-expression, the T25 and T18 fragments will also interact and return functionality to the adenylate cyclase enzyme, as measured by cAMP production.
Results
T25 and T18 plasmids containing designed bZIP sequences based on the KS analysis of coiled-coil specificity were constructed and co-expressed in BTH101 (cya-) cells. Interactions between the bZIP proteins were monitored through selective media and quantified by measuring b-galactosidase levels, which are positively regulated by cAMP levels. The results support that the KS identified residues are a factor in determining bZIP coiled-coil specificity.
Significance
bZIP proteins are implicated in many cellular processes and have been known to be involved in the development of cancer. Identifying and characterizing the interactions that determine the dimerization specificity between bZIP proteins is a crucial factor in better understanding disease formation and proliferation, as well as developing drugs or therapeutics to combat these diseases.
Location
DeRosa University Center
Format
Poster Presentation
Poster Session
Afternoon
Characterization of bZIP protein binding specificities using a BACTH assay system
DeRosa University Center
Basic leucine zipper domain (bZIP) transcription factors are proteins that dimerize to bind the promoter region of genes and control their expression. Interacting as classical helical coiled coils, the bZIP transcription factors exhibit the well-known pseudo- heptad repeat motif where every seventh residue is a leucine. Determination of the bZIP packing structure using the Knob-Socket (KS) model is a novel method to map the coiled-coil protein-protein interactions. The KS analysis and mapping has identified the residue sequences that may play a role in coiled-coil specificity.
