Alexey LadokhinAlexey Ladokhin
Associate Professor, Biochemistry and Molecular BIology
University of Kansas Medical Center

Conformational switching in diphtheria toxin translocation (T) domain: From protein folding to targeted drug delivery (2017-18)

The project seeks to understand the mechanism of bacterial toxin insertion into and translocation across membranes, a fundamental and unanswered question in cell biology. The mechanism, once understood, underlies another aim to develop a toxin-based molecular platform for targeting diseased acidic tissues produced by various maladies, including cancer.

The conversion of a protein structure from a water-soluble to membrane-inserted form is a key step in several cellular processes, including cellular entry of bacterial toxins, colicins, and viruses, as well as membrane-mediated regulation of apoptosis by the Bcl-2 family of proteins. Our long-term goal is to elucidate the molecular mechanism of protonation-triggered conformational switching that results in membrane insertion and to apply our findings to biomedical challenges, such as targeted drug delivery and regulation of apoptosis.

The first step toward this goal is deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the diphtheria toxin T-domain. This system is not only a paradigm for cell entry of toxins, but also has potential for targeted-delivery of anti-cancer therapies. Our goal is to use site-selective mutagenesis to alter the active range of conformational switching in the T-domain from current pH<6 (suitable for endosomal entry) to pH~6.5 (suitable for cancer targeting). To achieve this protein engineering task, first we will fill the current knowledge gap by establishing the mechanism of conformational switching through protonation of key titratable residues. We will examine effects of their replacement on conformational dynamics by using various tools of fluorescence spectroscopy, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics computer simulations and high-resolution structural techniques. We will also examine the feasibility of a T-domain-based system for delivering molecular cargo to cancer cells under acidic conditions.