Structure of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme

Area of Science:

• Biochemistry
• Structural Biology
• Cell Biology

Background:

• The Arg-Gly-Asp (RGD) motif is crucial for cell adhesion, mediating interactions with integrins.
• Understanding how RGD sequence conformation affects binding affinity is vital for biomaterial and therapeutic design.

Purpose of the Study:

• To investigate the impact of conformational constraint on the RGD sequence's cell adhesion activity.
• To elucidate the structural basis for enhanced RGD-mediated cell adhesion.

Main Methods:

• Construction and expression of a mutant human lysozyme (Cys-RGD4) with a constrained RGD sequence.
• Purification of Cys-RGD4 and assessment of its cell adhesion activity using baby hamster kidney cells.
• X-ray crystallography to determine the three-dimensional structure of Cys-RGD4 in complex with a lysozyme inhibitor.

Main Results:

• Cys-RGD4 exhibited higher cell adhesion activity compared to a non-constrained RGD lysozyme (RGD4).
• X-ray crystallography revealed a well-defined, rigid RGD region in Cys-RGD4 due to a novel disulfide bond.
• The RGD sequence adopted a type II' beta-turn conformation, stabilized by multiple hydrogen bonds.

Conclusions:

• Conformational rigidity of the RGD sequence, achieved through disulfide bonding, enhances integrin binding affinity.
• The type II' beta-turn structure of the RGD motif is essential for high-affinity cell adhesion.