Dissecting the roles of toxin bivalency membrane affinity and stoichiometry in dktx activation of trpv1

Abstract

Recent advances in structural biology show that lipids interact intimately with a wide range of membrane proteins, including ion channels, transporters, and membrane enzymes, implying that protein-lipid interfaces play important roles in protein function. A detailed understanding of the functional roles of these protein-lipid interfaces is required to fully understand the function of both integral and peripheral membrane proteins. A recently obtained structure of the rat TRPV1 channel complexed to its potent agonist, the double knot toxin (DkTx), composed of two inhibitory-cystine-knot (K1 knot and K2 knot), in lipid nanodiscs provides a detailed view of the DkTxand#8722;lipidand#8722;TRPV1 tripartite complex. This recent structural work provides one of the best available structural snapshots of protein-protein complexes in a native membrane-like milieu. DkTx exhibits an extremely slow wash-off of the toxin after channel activation. This is an intriguing observation given that the TRPV1-DkTx complex structures do not reveal any electrostatic, cation-pi, or pi stacking interactions between the toxin and channel residues, which commonly underpin tight complex formation between proteins. My Ph.D. focuses on elucidating the role of DkTx-membrane interaction in defining the characteristic slow wash-off of DkTx. Detailed electrophysiological studies performed in our laboratory have identified DkTx variants possessing remarkably reduced apparent TRPV1 affinity, all of which map to a toxin-membrane interface identified in the TRPV1-DkTx structure alluded to above. My first approach is to employ tryptophan fluorescence-based membrane partitioning experiments on toxin variants. The results of these experiments reveal a strong correlation between fast wash-off kinetics and mol. fraction partition coefficient forms the basis of the quottoxin relayquot mechanism. The single knots K1 and K2 of DkTx can individually activate TRPV1, albeit with much poorer potency and considerably faster wash-off rates than wild-type DkTx. Slow wash-off of DkTx