Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase

Abstract

a b s t r a c tHere, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to b-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model.

Four residues of propeptide are essential for precursor folding of nattokinase

“…Escherichia coli BL21(DE3) was the host bacterial strain for the pET-26b(þ) vector expression system. The gene encoding the precursor NK (propeptide and mature protease) was inserted into the plasmid pET-26b(þ) as described previously [7].…”

“…From the propeptide-subtilisin complex structure [7], the propeptide domain is made up of four antiparallel b-strands (named b-strand I, II, III, and IV, containing residues Lys8-Lys15, Gly34-Phe41, Asn45-Leu51, and Ser64-Ala74, respectively) and two a-helices (formed by Ser23-Lys33 and Glu53-Lys60). In addition, b-strands are formed by conserved regions and two a-helices are formed by nonconserved segments.…”

“…The propeptide can function as a template that imparts structural information to the subtilisin domain during precursor folding in vivo and in vitro [7,8]. Therefore, point mutations within the propeptide can alter the structure and activity of subtilisin, even though the mutation is not a part of the subtilisin domain.…”

“…The 77-residue propeptide at the N-terminal end of subtilisin assists in the folding of its cognate subtilisin domain and is termed as an IMC. Two 3D models of NK [13] and the propeptide-NK complex [7] were previously constructed. Site-directed mutagenesis and molecular dynamic simulations were used to probe the importance of the hydrogen bonds around the active site of NK [14].…”

“…Site-directed mutagenesis and molecular dynamic simulations were used to probe the importance of the hydrogen bonds around the active site of NK [14]. In addition, our earlier study demonstrated that specific interactions between the propeptide and subtilisin domain are important for the precursor folding, and conserved regions of the propeptide may be involved in the interactions [7,15]. Thus, certain residues in the conserved regions of the propeptide play an essential role during the precursor folding, and it is likely that these substitution mutations lead to defects in the production of active subtilisin during the precursor folding.…”