Elizabeth A. Komives
Protein-Protein Interactions and Catalysis
The long-term goal of the research in Dr. Komives's lab is to understand the parameters that determine the specificity of protein-protein interactions and the mechanisms by which these interactions contribute to catalytic efficiency. The relative importance of parameters such as hydrophobic effects, electrostatic interactions and dynamics will be defined by studying small peptide domains known to be involved in protein-protein interactions. These parameters will be explored by using a combination of molecular biological techniques, protein chemistry, and biophysical methods to probe the structure and function of the small polypeptide domains that resemble epidermal growth factor (called EGF-like domains) that are known to be involved in molecular recognition events.
EGF-like domains are small (typically ~40 amino acids) folded structures that contain six cysteines that form three disulfide bonds (see figure). Our lab is trying to understand the way in which protein-protein interactions are mediated by EGF-like domains. We use a combination of peptide synthesis and expression techniques to produce the EGF-like domains and we determine the structures of each domain by two-dimensional NMR methods. A combination of site-directed mutagenesis, surface plasmon resonance, titration calorimetry, NMR, and X-ray crystallography techniques are used to probe the protein-protein interactions mediated by each domain.
Our lab studies several proteins known to contain EGF-like domains. One project is to understand how the EGF-like domains of the anticoagulant protein, thrombomodulin, alter the function of thrombin. We have determined that two of the six EGF-like domains from thrombomodulin are necessary and sufficient for both the direct inhibition of fibrinogen clotting and the cofactor activity that turns thrombin into an anticoagulant. The structures of these two domains have been determined and are very interesting. One is rigid, and the other appears to be more flexible. The flexible EGF-like domain from thrombomodulin has a novel disulfide bonding pattern that has not been found in other EGF-like domains. Another project involves the EGF-like domains from the Drosophila development protein, Notch. This protein has 36 EGF-like domains and is involved in cell-cell interactions and differentiation. Two of the EGF-like domains from Notch have been shown to bind to Delta signalling cell-fate decisions. The binding is calcium-dependent, and we are currently doing structural and functional experiments to determine the role of the calcium in the protein-protein interaction.