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Updated: Jul 15, 2026
11:42
Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes
Published on: November 1, 2012
Crystal structure of the Sec4p.Sec2p complex in the nucleotide exchanging intermediate state
Yusuke Sato1, Shuya Fukai, Ryuichiro Ishitani
1Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan.
Summary
The Sec2p guanine nucleotide exchange factor (GEF) structure bound to Sec4p reveals how it triggers GDP release. This interaction deforms Sec4p, facilitating nucleotide exchange for vesicular transport regulation.
Area of Science:
- Molecular Biology
- Structural Biology
- Cell Biology
Background:
- Vesicular transport is crucial for exocytosis, a key cellular process.
- Rab GTPases, like yeast Sec4p, regulate vesicular transport.
- Guanine nucleotide exchange factors (GEFs), such as Sec2p, activate Rab GTPases.
Purpose of the Study:
- To elucidate the structural mechanism of Sec4p activation by the Sec2p GEF domain.
- To understand how Sec2p facilitates GDP release from Sec4p.
Main Methods:
- X-ray crystallography
- Protein complex structure determination
- Analysis of protein-protein interactions and conformational changes.
Main Results:
- Determined the crystal structure of the Sec2p GEF domain complexed with nucleotide-free Sec4p at 2.7 A resolution.
- Identified a hydrophobic platform formed by Sec2p residues that interacts with Sec4p's switch I and interswitch regions.
- Observed significant conformational changes in Sec4p, including deformation of switch I and II regions, creating a flat interface with Sec2p.
- Found a phosphate ion bound to Sec4p's P-loop, suggesting an intermediate state in nucleotide exchange.
Conclusions:
- The Sec2p-Sec4p complex structure reveals a mechanism for GDP release through induced conformational changes in Sec4p.
- The observed phosphate ion may indicate a transient state during the nucleotide exchange reaction.
- This structural insight aids in understanding the regulation of vesicular transport by Rab GTPase-GEF interactions.
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