Abstract
The structure of a recombinant pineapple cystatin (AcCYS) was determined by NMR with the RMSD of backbone and heavy atoms of twenty lowest energy structures of 0.56 and 1.11 Å, respectively. It reveals an unstructured N-terminal extension and a compact inhibitory domain comprising a four-stranded antiparallel β-sheet wrapped around a central α-helix. The three structural motifs (G45, Q89XVXG, and W120) putatively responsible for the interaction with papain-like proteases are located in one side of AcCYS. Significant chemical shift perturbations in two loop regions, residues 45 to 48 (GIYD) and residues 89 to 91 (QVV), of AcCYS strongly suggest their involvement in the binding to papain, consistent with studies on other members of the cystatin family. However, the highly conserved W120 appears not to be involved in the binding with papain as no chemical shift perturbation was observed. Chemical shift index analysis further indicates that the length of the α-helix is shortened upon association with papain. Collectively, our data suggest that AcCYS undergoes local secondary structural rearrangements when papain is brought into close contact. A molecular model of AcCYS/papain complex is proposed to illustrate the interaction between AcCYS and papain, indicating a complete blockade of the catalytic triad by AcCYS.
Highlights
Cystatins, the natural inhibitors of cysteine proteases are important regulatory proteins found in mammals, plants, and insects [1,2,3]
The a recombinant pineapple cystatin (AcCYS) gene encoding a cysteine protease inhibitor, cystatin was cloned from pineapple (Ananas comosus) stem [22]
The unique motif LARFAVxExN of phytocystatins was found in AcCYS
Summary
The natural inhibitors of cysteine proteases are important regulatory proteins found in mammals, plants, and insects [1,2,3]. They are involved in various biological and pathological processes, such as tumor invasion, inflammation, antigen processing, dystrophy, and metastasis [4]. Members of family 1, termed stefins are small intracellular proteins of about 100 residues with no disulfide bond. Cystatins of family 2 are extracellular proteins of about 120 residues with at least two internal disulfide bonds. Members of family 3, termed kininogens, are relatively large blood-circulated glycoproteins composed of several family 2-like cystatin domains
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