Chicken GRIFIN: Structural characterization in crystals and in solution

Area of Science:

• Biochemistry
• Structural Biology
• Ophthalmology

Background:

• The physiological role of galectin-related inter-fiber protein (GRIFIN), abundant in vertebrate lenses, remains largely unknown.
• Interspecies variations in GRIFIN's lactose-binding capacity, particularly between fish/birds and mammals, lack clear functional significance.

Purpose of the Study:

• To elucidate the structural characteristics and stability of chicken GRIFIN (C-GRIFIN).
• To investigate the mechanism of lactose binding in C-GRIFIN and the impact of mammalian GRIFIN mutations on its function.

Main Methods:

• Ultracentrifugation and small-angle X-ray scattering to assess protein solution behavior.
• X-ray crystallography to determine the C-GRIFIN structure across a pH range.
• Hydrogen/deuterium exchange mass spectrometry to probe structural dynamics.
• Site-directed mutagenesis to study the effect of mammalian GRIFIN mutations.

Main Results:

• C-GRIFIN exists as a stable, compact homodimer in solution.
• The crystal structure revealed a canonical galectin-like lactose-binding site, with binding being enthalpically driven.
• A key mutation (Asn48Lys), characteristic of mammalian GRIFINs, abrogated lactose binding but preserved tissue-staining properties.

Conclusions:

• The study provides a detailed structural basis for understanding GRIFIN's structure-activity relationships.
• Findings offer insights into the functional divergence of GRIFIN across species, particularly concerning lactose binding.
• This work contributes to the comprehensive structural characterization of the chicken galectin network.