Stepwise construction of triple-helical heparin binding sites using peptide models

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Acetylcholinesterase (AChE) localization in neuromuscular junctions is crucial for synaptic function.
• The asymmetric form of AChE interacts with heparan sulfate proteoglycans via its collagen-like tail.
• Two specific heparin-binding consensus sequences (XBBXB) exist in the AChE collagen tail: N-terminal (GRKGR) and C-terminal (GKRGK).

Purpose of the Study:

• To investigate the basis for the higher heparin affinity of the C-terminal AChE binding site compared to the N-terminal site.
• To model the triple-helical binding sites of AChE using homologous peptide series.
• To understand the relationship between triple-helix stability and heparin binding affinity.

Main Methods:

• Construction of homologous peptide series based on (Gly-Xaa-Yaa)(8) to model AChE binding sites.
• Stepwise introduction of tripeptide units into a Gly-Pro-Hyp framework.
• Circular dichroism (CD) spectroscopy and calorimetry to assess peptide stability and heparin binding.
• Displacement assays to quantify peptide-heparin interaction strength.

Main Results:

• Peptide stability decreased with increasing incorporation of binding site triplets, consistent with independent contributions to stability.
• Heparin binding increased peptide stability, confirming interaction, with minimal enthalpy change suggesting electrostatic binding.
• Displacement assays showed an inverse correlation between heparin binding affinity and peptide thermal stability.
• C-terminal binding site models exhibited greater heparin affinity than N-terminal models only when surrounding residues were included.

Conclusions:

• The specific sequences and surrounding residues within the AChE collagen tail dictate heparin binding affinity.
• Heparin binding to AChE is primarily electrostatic and influences the stability of the collagenous structure.
• Contextual residues flanking the core binding motifs are critical for achieving higher affinity interactions, particularly for the C-terminal site.