Amyloid Forming Human Lysozyme Intermediates are Stabilized by Non-Native Amide-π Interactions.

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Human lysozyme variants can cause fatal hereditary systemic amyloidosis.
• Amyloid formation involves a transient intermediate state that is difficult to study structurally.

Purpose of the Study:

• To investigate the structure and properties of the transient intermediate state in an amyloid-forming human lysozyme variant (I59T).
• To gain insights into the mechanism of lysozyme amyloidosis for potential therapeutic targeting.

Main Methods:

• Utilized Chemical Exchange Saturation Transfer (CEST) and Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion Nuclear Magnetic Resonance (NMR) spectroscopy at low pH.
• Employed molecular dynamics (MD) simulations to model the folding pathway and free energy landscape.

Main Results:

• Identified and characterized a distinct intermediate state populated at 0.6% during thermal unfolding using 15N CEST and CPMG RD NMR.
• Observed unusual 1H chemical shifts in the intermediate state, confirmed by 1H CEST.
• MD simulations recapitulated the experimental findings, revealing a high-energy intermediate with a disordered beta-domain and C-helix stabilized by non-native interactions.

Conclusions:

• Provided the first direct structural information on the transient intermediate state of amyloid-forming human lysozyme.
• Elucidated the structural features stabilizing the intermediate, including non-native hydrogen bonding and amide-pi interactions.
• Offered critical insights into the molecular mechanisms underlying lysozyme amyloidosis, potentially guiding therapeutic strategies.