Folding Pathway Mediated by an Intramolecular Chaperone: A FUNCTIONAL PEPTIDE CHAPERONE DESIGNED USING SEQUENCE DATABASES

Yukihiro Yabuta,Ezhilkani Subbian,Catherine Oiry,Ujwal Shinde

doi:10.1074/jbc.m212003200

Yukihiro Yabuta, Ezhilkani Subbian + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m212003200

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Abstract

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or "intramolecular chaperones" to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined alpha-beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT.subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.

Highlights

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or ‘‘intramolecular chaperones’’ to facilitate correct folding
Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity
The peptide sequence (ProD)1 was designed through a decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment

Summary

Introduction

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or ‘‘intramolecular chaperones’’ to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. Because propeptides perform functions different from the catalytic domains, they may be subjected to different mutational frequencies because of different functional constraints (14 –16) Given that they impart structural information to their catalytic domains [17,18,19], propeptides within one family could adopt similar structural scaffolds despite digressions in their polypeptide sequences. This makes propeptides attractive models for protein redesign and for understanding the relation between sequence and structure.

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