Structure-function study of the N-terminal domain of exocyst subunit Sec3

Area of Science:

• Cell Biology
• Molecular Biology
• Structural Biology

Background:

• The exocyst complex, an evolutionarily conserved octameric structure, is crucial for polarized exocytosis in eukaryotes.
• The Sec3 subunit guides exocytosis by interacting with phospholipids and small GTPases, acting as a spatial landmark.
• Understanding Sec3's structure and function is key to elucidating exocytosis mechanisms.

Purpose of the Study:

• To determine the ab initio structure of the N-terminal domain of Sec3 (Sec3N).
• To characterize the structural features of Sec3N, including its pleckstrin homology (PH) domain.
• To identify the molecular mechanisms underlying Sec3's role in phospholipid binding and spatial landmarking for exocytosis.

Main Methods:

• Ab initio structure determination of the Sec3N domain.
• Structural analysis to identify protein-protein and protein-lipid interaction sites.
• Site-directed mutagenesis to investigate the functional significance of identified residues in cellular localization and exocytosis.

Main Results:

• The structure of Sec3N reveals a novel subclass of pleckstrin homology (PH) domains and a new protein family.
• Sec3N possesses an alpha-helix and two beta-strands mediating dimerization via domain swapping.
• Key residues for phospholipid binding were identified; mutations disrupted plasma membrane localization and exocytosis in cells.

Conclusions:

• The Sec3N structure provides insights into the molecular basis of exocyst localization and function.
• The PH domain of Sec3 acts as a coincidence detector, integrating signals from phospholipids and the small GTPase Cdc42 at the plasma membrane.
• This study elucidates a novel structural and functional mechanism for spatial regulation of exocytosis.