title: Crystal structure of the Sec4p.Sec2p complex in the nucleotide exchanging intermediate state.
abstract: Vesicular transport during exocytosis is regulated by Rab GTPase (Sec4p in yeast), which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. Here, we report the crystal structure of the Sec2p GEF domain in a complex with the nucleotide-free Sec4p at 2.7 A resolution. Upon complex formation, the Sec2p helices approach each other, flipping the side chain of Phe-109 toward Leu-104 and Leu-108 of Sec2p. These three residues provide a hydrophobic platform to attract the side chains of Phe-49, Ile-53, and Ile-55 in the switch I region as well as Phe-57 and Trp-74 in the interswitch region of Sec4p. Consequently, the switch I and II regions are largely deformed, to create a flat hydrophobic interface that snugly fits the surface of the Sec2p coiled coil. These drastic conformational changes disrupt the interactions between switch I and the bound guanine nucleotide, which facilitates the GDP release. Unlike the recently reported 3.3 A structure of the Sec4p.Sec2p complex, our structure contains a phosphate ion bound to the P-loop, which may represent an intermediate state of the nucleotide exchange reaction.
authors.affiliation: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Sato
authors.forename: Shuya
authors.initials: S
authors.lastname: Fukai
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Ishitani
authors.forename: Osamu
authors.initials: O
authors.lastname: Nureki
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D019281:Dimerization; D019204:GTP-Binding Proteins / Q000737:chemistry*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D020691:rab GTP-Binding Proteins / Q000737:chemistry*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C064456:SEC2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins