Self-assembly of Bacteriophage-associated Hyaluronate Lyase (HYLP2) into an Enzymatically Active Fibrillar Film

Parul Mishra,Vinod Bhakuni

doi:10.1074/jbc.m806730200

Abstract

The in vitro assembly of a soluble protein into its mature fibrillar form is usually accompanied by loss of its functional activity. Our study is the first demonstration of a natural enzyme (HylP2) retaining its enzymatic activity on conversion from pre-fibril to mature fibril and supports the contention that minor conformational changes in the native folded form of a protein can lead to the formation of a functional fibril. Hyaluronate lyase (HylP2) is a natural enzyme of bacteriophage 10403 of Streptococcus pyogenes. At pH 5.0, the enzyme undergoes partial unfolding localized in its N-terminal domain while the C-terminal domain maintains its folded trimeric conformation. This structural variant of HylP2 retains about 70% enzymatic activity with hyaluronan. It further self-assembles into a fibrillar film in vitro through solvent-exposed nonpolar surfaces and intermolecular beta-sheet formation by the beta-strands in the protein. Interestingly, the mature fibrillar film of HylP2 also retains about 60 and 20% enzymatic activity for hyaluronic acid and chondroitin sulfate, respectively. The possession of broad substrate specificity by the fibrillar form of HylP2 indicates that fluctuations in pH, which do not lead to loss of functionality of HylP2, might assist in bacterial pathogenesis. The formation of fibrillar film-like structure has been observed for the first time among the hyaluronidase enzymes. After acquiring this film-like structure in bacteriophage, HylP2 still retains its enzymatic activity, which establishes that these fibrils are a genuinely acquired protein fold/structure.

Highlights

It is widely recognized that fibril formation in globular proteins generally requires the presence of a partially unfolded state, referred to as the “amyloidogenic intermediate” [5,6,7,8]
S. pyogenes Hyaluronate Lyase Forms Active Fibrillar Film the bacterium itself has been reported to possess amyloid-like M proteins on its surface, which help in spreading bacterial infection by facilitating the attachment of this Grampositive bacterium to host soft tissues [21]
Limited proteolysis revealed that the domain organization is conserved in HylP2 even after the structural transition because of change in pH, and that the N-terminal domain affects the rate of fibril formation

Summary

Introduction

It is widely recognized that fibril formation in globular proteins generally requires the presence of a partially unfolded state, referred to as the “amyloidogenic intermediate” [5,6,7,8]. In addition to being present in the microorganisms, there is a naturally occurring functional amyloid in mammals (Pmel17) that functions as a template for the polymerization of melanin molecules in the melanosome [18], and strengthens the view that fibrillar structures could rather be a selected protein fold in the evolution of functional protein structures. For the first time, highlights the transformation of a functional, natively folded and nonfibrillar natural enzyme (HylP2) into fibril- like structures that appear as a thin membrane This form of protein is different in morphology from the disease-causing fibrils known to date, and the presence of enzymatic activity in such structures provides indications toward their plausible role in the biology of the organism. These structures can be considered to assist in phage access into the bacterial cells even under stress conditions (altered pH)

Methods

Results

Conclusion