Structural requirements for cooperativity in ileal bile acid-binding proteins

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• Ileal bile acid-binding proteins (I-BABP) are intracellular lipid-binding proteins crucial for bile acid homeostasis in enterocytes.
• I-BABP orthologues bind up to two ligands, but exhibit varying binding cooperativity due to sequence differences.

Purpose of the Study:

• To investigate the molecular basis of binding cooperativity in I-BABP.
• To engineer a noncooperative I-BABP into a cooperative mutant using a gain-of-function approach.

Main Methods:

• Solution structure determination of chicken I-BABP (cI-BABP) with glycochenodeoxycholate.
• Comparative structural analysis to identify key residues for cooperativity.
• Design and characterization of a double mutant (H99Q/A101S cI-BABP).
• Molecular dynamics simulations and essential dynamics analysis.

Main Results:

• The solution structure of cI-BABP revealed its capacity to bind two bile acid molecules.
• A double mutant (H99Q/A101S) was successfully engineered to exhibit cooperative binding.
• Molecular dynamics simulations identified specific residues mediating communication between binding sites.

Conclusions:

• Specific amino acid residues act as hot spots, mediating communication between I-BABP binding sites.
• The engineered cooperative binding mechanism in I-BABP aligns with an extended conformational selection model.
• This research provides insights into the molecular mechanisms governing bile acid transport and homeostasis.