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

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 prefibril 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 ␤-sheet formation by the ␤-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 filmlike 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.

The C-terminus hot spot region helps in the fibril formation of bacteriophage-associated hyaluronate lyase (HylP2)

“…5a ). As reported, the increase in ThT fluorescence intensity for HylP2 fibrillation followed a sigmoidal pattern in which the nucleation phase (4 h) was followed by a log phase of elongation (6 h) and a plateau region of maturation 14 . The kinetics of fibril formation by N HylP C HylP2 (lacking the N-terminal domain desired for nucleation, but having the C-terminal domain with the hot spot region desired for the acceleration) was initiated by adding preformed HylP2 fibrils.…”

“…The changes in the quaternary structure of above proteins were monitored at a condition where the HylP2 was stabilized in a partially unfolded condition and formed fibrils 14 . The SEC profiles of HylP2, HylP, N HylP2 C HylP and N HylP C HylP2 at pH 5 show the elution volumes of ~8.2 ml, ~11.1 ml, ~13.1 ml and ~11.1 ml respectively ( Fig.…”

“…The mature fibrils generally lose their activity, though fibrillar structures in certain proteins fold as a functionally active fibril like in the case of nuclear protein controlling polyadenylation (PAPBN1), RNase A, phage hyaluronate lyase (HylP2) etc. 12 13 14 . These findings raise questions pertaining to fibrillar structure as to whether native-like structural domains undergo conformational modification or do they simply refold on fibril formation?…”

“…The Streptococcus pyogenes bacteriophage encodes hyaluronate lyases (HLs) termed as HylP, HylP1 and HylP2 14 17 18 19 20 21 . These bacteriophage HLs, despite showing a high degree of similarity to each other (homology-82%, identity-62%), differ with respect to the presence of a collagen-like motif, (Gly-X-Y) 10 present at the N-terminus of HylP 14 17 . The structure of phage HLs mainly consists of an N-terminal globular domain, followed by a triple-stranded β helix (TSβH) domain and a stretch of coiled coils with segmented α-helical nose at the C-terminus 19 20 .…”

“…It was therefore proposed that the N-terminus of HylP2 is essential for the nucleation as well as acceleration of fibril formation. The formation of functional fibril at pH 5, which is close to the optimum pH for enzyme activity is proposed to be advantageous during phage infection 14 .…”