Expression in Pichia pastoris and Backbone Dynamics of Dendroaspin and Erabutoxin b: Motion Variability within Three‐fingered Toxins

Abstract

Dendoaspin (DEN) and Erabutoxin b (EBTb) are three‐fingered toxins which possess a similar architecture with unrelated biological functions. They share a common structure consisting of a β‐sheet core that tightly cross‐linked by four conserved disulfide bonds. DEN inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin αIIbβ3 of platelets. In contrast, EBTb is a potent inhibitor of nicotinic acetylcholine receptor. To study their function and dynamics relationships, we expressed DEN and EBTb in Pichia pastoris (P. pastoris). The recombinant DEN expressed in P. pastoris inhibited platelet aggregation with a KI of 149 nM. The recombinant EBTb expressed in P. pastoris inhibited carbachol‐induced muscle contraction with a KI of 42 nM. They are the same potency as those of native proteins. NMR analysis showed that DEN and EBTb expressed in P. pastoris processes the same structures as those of native proteins. The results provide the first direct evidence that highly disulfide‐bonded three‐fingered toxins can be expressed in P. pastoris with the correct fold. Analysis of the dynamics properties of DEN and EBTb showed that they exhibited flexibility on the loops II and III and motions on multiple time scales. The RGD‐containing loop of DEN was the most flexible region and the apex of the loops II and III of EBTb exhibited flexible motions on the ps/ns timescale. Comparison of the dynamics properties of DEN and EBTb with other members of three‐fingered toxins suggests that their dynamics deviations may play an important role in interacting with different target proteins.