Structural, Functional and Computational Studies of Proton-Coupled Eukaryotic Amino Acid Transporters

Cationic amino acid transporters (CATs) are membrane proteins tasked with the transport of positively charged amino acids across the cell membranes. Members of this family of transporters are involved in immune response, but our current understanding on how arginine and lysine - amino acids that play a role in immune signaling - are transported into the cell and total amounts is limited. A structural model of a CAT prokaryotic homolog (GkApcT) was recently solved and shown to be a proton-coupled transporter. However as inflammatory diseases are of increasing importance with lifespan elongation (e.g. rheumatoid arthritis) the need for therapeutic treatment of these conditions with agents of higher clinical effectiveness are needed. A mammalian eukaryotic structural model of CATs is required to inform structure-based medicinal chemistry efforts in anti-inflammatory drug design. To this end, the key goals of this project are solving a mammalian CAT structure and to functionally annotate members of the CAT family with functional biochemistry and simulation methods. Specifically deciphering whether CAT4 is proton-coupled like GkApcT and what amino acids it transports may suggest this orphan transporter's substrates. Additionally isoforms CAT2-A/B are known to have different affinities for arginine; however mechanistically the question remains of how this specifically is achieved with such high sequence similarity of the two transporters (e.g. is the difference kinetically (Vmax) or thermodynamically (KD) driven?). Liposomal reconstitution of the purified transporters will provide insight to these questions along with the employment of Molecular Dynamics (MD) simulations and free energy methods from computational chemistry to further interrogate dynamics.