Expression, purification, and characterization of anuran saxiphilins using thermofluor, fluorescence polarization, and isothermal titration calorimetry.

Anuran saxiphilins (Sxphs) are "toxin sponge" proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins. For complete details on the use and execution of this protocol, please refer to Chen et al. (2022).1.

Definition of a saxitoxin (STX) binding code enables discovery and characterization of the anuran saxiphilin family.

American bullfrog (Rana castesbeiana) saxiphilin (RcSxph) is a high-affinity "toxin sponge" protein thought to prevent intoxication by saxitoxin (STX), a lethal bis-guanidinium neurotoxin that causes paralytic shellfish poisoning (PSP) by blocking voltage-gated sodium channels (NaVs). How specific RcSxph interactions contribute to STX binding has not been defined and whether other organisms have similar proteins is unclear. Here, we use mutagenesis, ligand binding, and structural studies to define the energetic basis of Sxph:STX recognition. The resultant STX "recognition code" enabled engineering of RcSxph to improve its ability to rescue NaVs from STX and facilitated discovery of 10 new frog and toad Sxphs. Definition of the STX binding code and Sxph family expansion among diverse anurans separated by ∼140 My of evolution provides a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and for developing novel proteins to sense or neutralize STX and related PSP toxins.

Inward-facing conformation of a multidrug resistance MATE family transporter.

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism of MATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-sought-after inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combination with the outward-facing one supports an alternating access mechanism for the MATE family transporters.