Solid-state NMR spectroscopic analysis for structure determination of a zinc-bound catalytic amyloid fibril.

Abstract

Zinc ions are commonly involved in enzyme catalysis and protein structure stabilization, but their coordination geometry of zinc-protein complex is rarely determined. Here, in this chapter, we introduce a systematic solid-state NMR approach to determine the oligomeric assembly and Zn2+ coordination geometry of a de novo designed amyloid fibrils that catalyze zinc dependent ester hydrolysis. NMR chemical shifts and intermolecular contacts confirm that the peptide forms parallel-in-register β-sheets, with the two forms of Zn2+ bound histidines in each peptide. The amphiphilic parallel β-sheets assemble into stacked bilayers that are stabilized by hydrophobic side chains between β-sheets. The conformations of the histidine side chains, determined by 13C-15N distance measurements, reveal how histidines protrude from the β-sheet. 1H-15N correlation spectra show that the single-Zn2+ coordinated histidine associated with dynamic water. The resulting structure provides insight into how metal ions contribute to stabilizing the protein structure and driving its catalytic reactivity.