Abstract
P. falciparum Kelch 13 (Pfk13) is an essential protein that contains BTB and Kelch-repeat propeller domains (KRPD), which was predicted to bind substrate during ubiquitin-dependent degradation pathway. However, the function of Pfk13 and the structural alterations associated with artemisinin resistance mutations remain unknown. Herein, we screened two proteins, namely Pfk13-F446I and Pfk13-C580Y, which are closely associated with artemisinin, for structural prediction analysis. The 389 amino acids from 1011 nt to 2178 nt of KRPD were cloned into pFastBacTM1. The recombinant plasmids were heterologously expressed in Spodoptera frugiperda 9 cells (SF9) and a ~44 kDa protein band was yielded by SDS-PAGE and Western Blot. A total of five structure models were generated and predicted by AlphaFold for each protein. The models predicted that Pfk13-F446I would be located in the central protein cavity, proximal to mutations in cysteine residues primarily in β strands. Unlike Pfk13-F446I, the Pfk13-C580Y is located on the small channel that runs through the center of the K13 protein. Interestingly, the hydrogen bond between C580 and C533 in the wide type (WT) was not detected, suggesting that the hydrogen bond may be lost during the mutation. Besides, the Pfk13-F446I and Pfk13-C580Y mutation were found to add 11 and 9 hydrogen bonds variations that may lead to conformational change of the protein structure compared to WT, respectively. Future work should pay more attention to the binding characteristics of those mutations related with KPRD pockets and their binding substrates, which will further clarify the structure and function of Pfk13 and its mutant.