Requirement for hydrophobic Phe residues in Pleurotus ostreatus proteinase A inhibitor 1 for stable inhibition.


Abstract

Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) has been shown to be unique among the various serine protease inhibitors in that its C-terminal region appears to be the reactive site responsible for its inhibitory action toward proteases. To investigate in more detail the mechanism of inhibition by POIA1, we have been studying its structural requirements for stable inhibition of proteases. In this study, we focused on hydrophobic Phe residues, which are generally located in the interior of protein molecules. A Phe-->Ala replacement at position 44 or 56 was introduced into a 'parent' mutant of POIA1 that had been converted into a strong and resistant inhibitor of subtilisin BPN' by replacement of its six C-terminal residues with those of the propeptide of subtilisin BPN' and the effects on inhibitory properties and structural stability were examined. Both of the mutated POIA1 molecules not only were found to exhibit decreased ability to bind to subtilisin BPN' (80-fold for the F44A mutant and 13-fold for the F56A mutant), but were also converted to temporary inhibitors that were degraded by the protease. The structural stability of the mutated POIA1 was also lowered, as shown by a 13 degrees C decrease in melting temperature for the F56A mutant. In particular, the F44A mutant was found to lose its tertiary structure, as judged from the circular dichroism spectrum, demonstrating that Phe44 is a strict requirement for structural formation by the POIA1 molecule. These results clearly indicate that stabilization of POIA1 by hydrophobic residues in its molecular interior is required for stable inhibition of the protease. This requirement for a stable tertiary structure is shared with other serine protease inhibitors, but other structural requirements seem to differ, in that strong binding with the protease is required for POIA1 whereas conformational rigidity around the reactive site is essential for many other protease inhibitors. Study holds ProTherm entries: 14838, 14839 Extra Details: hydrophobic residue; pleurotus ostreatus; protease inhibitor; protein,stability; temporary inhibition

Submission Details

ID: J2Zmg9oG

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Kojima S;Hisano Y,Protein Eng. (2002) Requirement for hydrophobic Phe residues in Pleurotus ostreatus proteinase A inhibitor 1 for stable inhibition. PMID:11983933
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1ITP 2002-02-13 Solution Structure of POIA1
1V5I 2005-03-08 1.5 Crystal structure of serine protease inhibitor POIA1 in complex with subtilisin BPN'

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.4 Serine proteinase inhibitor IA-1 Q7M4T5 PIA2_PLEOS
100.0 Serine proteinase inhibitor IA-1 Q7M4T6 PIA1_PLEOS