The Sec1-Munc18 protein VPS33B forms a uniquely bidirectional complex with VPS16B.

Area of Science:

• Molecular Biology
• Cell Biology
• Structural Biology

Background:

• Vacuolar protein sorting proteins VPS33B and VPS16B are essential for cellular function, as indicated by severe genetic disorders caused by their loss-of-function variants.
• VPS33B belongs to the Sec1-Munc18 protein family, implicated in vesicular fusion, but the specific structure and function of the VPS33B-VPS16B complex remain largely unknown.

Purpose of the Study:

• To elucidate the molecular composition, structure, and stability of the human VPS33B-VPS16B complex.
• To investigate the impact of disease-causing variants on complex formation and function.
• To propose a structural model for the VPS33B-VPS16B complex and its role in membrane fusion.

Main Methods:

• Expression and purification of the human VPS33B-VPS16B complex in yeast.
• Biophysical techniques including circular dichroism (CD), size-exclusion chromatography-multiangle light scattering (SEC-MALS), and small-angle X-ray scattering (SAXS).
• Negative-stain electron microscopy (EM) and avidin tagging for structural and compositional analysis.

Main Results:

• The VPS33B-VPS16B complex is a high molecular weight assembly (∼315 kDa) with a predominantly α-helical structure.
• Quantitative analysis revealed a 2:3 stoichiometry of VPS33B to VPS16B, with a specific N-terminal region of VPS16B sufficient for complex formation.
• Structural studies (SAXS, EM) showed a distinct two-lobed shape, with each lobe containing a VPS33B molecule oriented oppositely, suggesting a potential for bidirectional function.

Conclusions:

• The study reveals the first potentially bidirectional Sec1-Munc18 protein complex, the VPS33B-VPS16B complex, with a unique two-lobed structure.
• The proposed model suggests the complex can interact with distinct SNARE complexes at opposing ends, facilitating membrane fusion.
• Understanding the structure and function of VPS33B-VPS16B provides insights into cellular trafficking and the pathogenesis of related genetic disorders.