| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 100 === Proteins with the three-fingered fold are widely distributed in various tissues where they exert a variety of functions. They comprise 60-80 amino acids with four or five disulfide bonds and are organized into three fingers connected by the double-stranded and triple-stranded antiparallel sheets. Despite the overall similarity in structure, these proteins in this family differ from each other in their biological activities. Dendroaspin (Den) is a three-fingered fold venom protein containing a PRGDMP motif from Dendroaspis jamesoni kaimose and rhodostomin (Rho) is a disintegrin from Calloselasma rhodostoma also with a PRGDMP motif. Although Den and Rho have different 3D structures, they are highly potent integrin inhibitors. To study their structure, function, and dynamics relationships, we expressed Den and Rho in Pichia pastoris (P. pastoris). The recombinant Den and Rho inhibited platelet aggregation with the KI values of 149.8 and 83.2 nM. Cell adhesion analysis showed that Den was 3.7 times less active than Rho when inhibiting the integrin αIIbβ3 and 2.5 times less active when inhibiting the integrin αvβ3. In contrast, Den and Rho were similarly active when inhibiting the integrin α5β1 with the IC50 values of 239.8 and 256.8 nM. NMR analysis showed that recombinant Den and Rho have different 3D conformations for their arginyl-glycyl-aspartic acid (RGD) motif. However, the comparison with Rho showed that the docking of Den into integrin αvβ3 resulted in a similar number of contacts. Analysis of the dynamic properties of the RGD loop in Den and Rho showed that they also had different dynamic properties. These results demonstrate that protein scaffolds affect the function, structure, and dynamics of their RGD motif, which suggests that the inhibitory potency and selectivity of RGD-containing proteins cannot be determined only by the sequence content of the RGD loop. To study how a similar fold can accommodate distinct functional topographies, we also expressed erabutoxin b (EBT b), a nicotinic acetylcholine receptor antagonist from Laticauda semifasciata, and muscarinic toxin 2 (MT2), a muscarinic receptor inhibitor from Dendroaspis angusticeps, in P. pastoris. Their yields were 10-15 mg/L and 5-10 mg/L, respectively. EBT b was shown to have muscle-type neuron toxicity. NMR analysis showed that EBT b and MT2 expressed in P. pastoris processes the same structures as those of native proteins. Analysis of the dynamics properties of DEN, EBT b, and MT2 showed that they exhibited difference in the flexibility on the loops I, II, and III and in the motions on multiple time scales. The RGD-containing loop of DEN, loop III, was the most flexible region. In contrast, EBTb and toxin exhibited rigidity on the loop I and flexibility on loops II and III. The loops II and III of MT2 were flexible; however, the loop III was rigid. Comparison of the dynamics properties of DEN, EBTb, and MT2 with other members of three-fingered toxins suggests that their dynamics deviations may play an important role in interacting with different target proteins. These results suggest that divergent functions of three-fingered toxins may be correlated with their backbone dynamics of loop regions.
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