Carboxyl-Group Footprinting Maps the Dimerization Interface and Phosphorylation-induced Conformational Changes of a Membrane-associated Tyrosine Kinase

Abstract

Her4 is a transmembrane receptor tyrosine kinase belonging to the ErbB-EGFR family. It plays a vital role in the cardiovascular and nervous systems, and mutations in Her4 have been found in melanoma and lung cancer. The kinase domain of Her4 forms a dimer complex, called the asymmetric dimer, which results in kinase activation. Although a crystal structure of the Her4 asymmetric dimer is known, the dimer affinity and the effect of the subsequent phosphorylation steps on kinase domain conformation are unknown. We report here the use of carboxyl-group footprinting MS on a recombinant expressed, Her4 kinase-domain construct to address these questions. Carboxyl-group footprinting uses a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, in the presence of glycine ethyl ester, to modify accessible carboxyl groups on glutamate and aspartate residues. Comparisons of Her4 kinase-domain monomers versus dimers and of unphosphorylated versus phosphorylated dimers were made to map the dimerization interface and to determine phosphorylation induced-conformational changes. We detected 37 glutamate and aspartate residues that were modified, and we quantified their extents of modification by liquid chromatography MS. Five residues showed changes in carboxyl-group modification. Three of these residues are at the predicted dimer interface, as shown by the crystal structure, and the remaining two residues are on loops that likely have altered conformation in the kinase dimer. Incubating the Her4 kinase dimers with ATP resulted in dramatic increase in Tyr-850 phosphorylation, located on the activation loop, and this resulted in a conformational change in this loop, as evidenced by reduction in carboxyl-group modification. The kinase monomer-dimer equilibrium was measured using a titration format in which the extent of carboxyl-group footprinting was mathematically modeled to give the dimer association constant (1.5-6.8 ؋ 10 12 dm 2 /mol). This suggests that the kinase-domain makes a significant contribution to the overall dimerization affinity of the fulllength Her4 protein. Her4 (also called ErbB-4) is a transmembrane receptor tyrosine kinase belonging to the ErbB-Epidermal Growth Factor Receptor (EGFR) family; it plays a vital role in the cardiovascular system, nervous system, and other tissues (1, 2). Genetic knock-out of Her4 in mice results in embryonic lethality because of malformations of the heart and hindbrain (3). Cancer-associated mutations in Her4 were found in malignant melanoma and lung adenocarcinomas (4, 5).The structure of Her4 consists of an extracellular domain that mediates ligand binding, a transmembrane domain, a short intracellular juxtamembrane domain, a tyrosine kinase domain and a C-terminal-tail domain that contains multiple autophosphorylation sites (6). As with other receptor tyrosine kinases, ligand binding stimulates Her4 dimerization, which results in activation of its tyrosine-kinase domain and autophosphorylation of multiple Tyr residues (6-8). Biophysical and protein crysta...

Carboxyl Group Footprinting Mass Spectrometry and Molecular Dynamics Identify Key Interactions in the HER2-HER3 Receptor Tyrosine Kinase Interface

“…6). Analogous salt bridge interactions were previously observed in HER4 homodimers (23). Hydrophobic interactions also play a key role at the dimerization interface, and we will further explore those interactions in the future (46).…”

“…It can also be utilized to rapidly characterize several experimental conditions in parallel. We previously utilized this strategy to examine the dimer interface of the HER4/ErbB4 homodimer, determine its dimer association constant on a lipid membrane, and characterize the conformational changes resulting from activation loop tyrosine phosphorylation (23).…”

Mass Spectrometry-based Footprinting Reveals Structural Dynamics of Loop E of the Chlorophyll-binding Protein CP43 during Photosystem II Assembly in the Cyanobacterium Synechocystis 6803

“…PSII complexes from three genetically modified strains were prepared, and their D/E residues were subjected to protein footprinting by chemical modification with GEE (17,18). A schematic diagram of the experimental design and procedure is shown in Fig.…”

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

“…44 In this study, carboxyl group footprinting was used to characterize the conformational changes introduced in HER4 (a transmembrane receptor tyrosine kinase) during the dimerization and the phosphorylation process. For the purpose of our study, we derived solvent-accessibility areas from the known crystal structure of HER4 monomer (PDB ID: 3BCE), and plotted them against the relative extent of modification of the N-lobe of HER4 kinase domain as calculated in the previous work.…”

“…The carbodiimidelabeling method with GEE tagging has previously been used to probe the structure of a number of proteins, such as the mammalian polyamine transport system and the membrane-attached antenna protein (including mapping a protein-protein interface), and to study phosphorylation-induced conformation changes of a membrane associated kinase. [44][45][46][47][48][49] For this work, we evaluated the reproducibility of protein labeling kinetics at the specific side chain residues with this reagent using a mAb. Specifically, the mAb was exposed to carbodiimide in the presence of GEE label for varying amounts of time from 0 to 10 minutes in triplicate and at various concentrations, providing a detailed quantitative characterization of side chain reactivity.…”