ABangle: characterising the VH-VL orientation in antibodies

Area of Science:

• Structural Biology
• Immunology
• Computational Biology

Background:

• Antibody binding sites are formed by variable heavy (VH) and variable light (VL) domains within the antigen-binding fragment (Fab).
• Understanding VH-VL orientation is key for antibody specificity, affinity, modeling, docking, and engineering.
• Current methods using relative measures (e.g., RMSD) or a single absolute angle are insufficient to fully describe orientation.

Purpose of the Study:

• To develop a comprehensive method for fully characterizing VH-VL orientation in an absolute and consistent manner.
• To create a computational tool (ABangle) for automated calculation and comparison of VH-VL orientations.
• To analyze the influence of different residues on VH-VL orientation and compare flexibility in bound versus unbound antibody forms.

Main Methods:

• Developed a novel method using five angles (HL, HC1, LC1, HC2, LC2) and a distance (dc) to define VH-VL orientation.
• Created the ABangle computational tool for automatic calculation and comparison of VH-VL orientation parameters.
• Analyzed existing antibody structures and compared VH-VL orientation in bound and unbound states for different antigen specificities.

Main Results:

• The five-angle and distance method fully characterizes VH-VL orientation, resolving ambiguities from single-angle approaches.
• ABangle enables consistent comparison of VH-VL orientations across diverse antibody structures.
• Identified specific positions and residues influencing VH-VL orientation angles.
• Antibodies targeting protein antigens exhibit greater flexibility in their unbound form compared to those targeting hapten antigens.

Conclusions:

• The new method and ABangle tool provide a complete and absolute description of VH-VL orientation, advancing antibody structural analysis.
• Understanding residue-level influences on orientation aids in antibody engineering and design.
• Differential flexibility in unbound states has implications for antibody binding mechanisms and therapeutic applications.