Periplasmic chitooligosaccharide-binding protein requires a three-domain organization for substrate translocation

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Periplasmic solute-binding proteins (SBPs) mediate nutrient uptake and signal transduction in bacteria.
• Chitooligosaccharides ((GlcNAc)n) are important nutrients for Vibrios, with SBPs controlling their uptake.
• Existing models for SBP-mediated translocation involve a two-domain open/closed mechanism.

Purpose of the Study:

• To elucidate the translocation mechanism of chitooligosaccharides by SBPs in Vibrio cholerae.
• To propose a novel three-domain mechanism for (GlcNAc)n translocation.
• To investigate the structural and dynamic properties of the VcCBP protein.

Main Methods:

• X-ray crystallography to determine the structure of VcCBP in unliganded and liganded states.
• Molecular dynamics simulations to analyze protein domain movements and interface dynamics.
• Thermal unfolding experiments and Isothermal Titration Calorimetry (ITC) to assess binding affinities.

Main Results:

• VcCBP exhibits three distinct domains: Upper1, Upper2, and Lower.
• Molecular dynamics revealed independent domain motions and differential interface fluctuations upon ligand binding.
• Structural and binding data support a three-domain translocation mechanism where specific interfaces bind or release sugar molecules.

Conclusions:

• A novel three-domain mechanism for (GlcNAc)n translocation by VcCBP is proposed.
• This mechanism involves dynamic interactions between the three domains and their interfaces.
• The proposed mechanism may extend to other SBPs within the same protein cluster.