SMAP-29 has two LPS-binding sites and a central hinge

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Antimicrobial peptides (AMPs) are crucial in innate immunity.
• SMAP-29, an AMP from sheep, exhibits antimicrobial properties.
• Understanding SMAP-29's interaction with bacterial membranes, particularly lipopolysaccharide (LPS), is key to its function.

Purpose of the Study:

• To elucidate the structure and membrane-binding properties of SMAP-29.
• To identify and characterize the lipopolysaccharide (LPS)-binding domains of SMAP-29.
• To investigate the mechanism and cooperativity of LPS binding by SMAP-29.

Main Methods:

• Circular dichroism (CD) spectroscopy to determine peptide structure in various environments.
• Nuclear Magnetic Resonance (NMR) spectroscopy to resolve the 3D structure of SMAP-29.
• Chromogenic Limulus assay to quantify LPS-binding affinity (EC50) of SMAP-29 and its fragments.

Main Results:

• SMAP-29 showed disordered structure in aqueous buffers but significant helicity in membrane-mimicking environments.
• NMR revealed a helical structure in residues 8-17 and a hydrophobic segment in residues 20-28.
• SMAP-29 possesses two LPS-binding domains, one at each terminus, with higher affinity in the N-terminal region.
• LPS binding to SMAP-29 exhibited positive cooperativity, primarily intramolecular, leading to high overall affinity (EC50 = 2.6 µM).
• Rabbit cathelicidin CAP-18[106-142] showed similar LPS-binding characteristics, suggesting a conserved mechanism.

Conclusions:

• SMAP-29 undergoes conformational changes to adopt helical structures in membrane-like environments.
• The cooperative binding of LPS by multiple domains significantly enhances SMAP-29's affinity for LPS.
• This cooperative binding mechanism is crucial for the high-affinity interaction of AMPs like SMAP-29 and CAP-18 with bacterial LPS.