Crystallization and crystallographic analysis of yeast Sec2p, a guanine nucleotide-exchange factor for the yeast Rab GTPase Sec4p.
Sec2p is a guanine nucleotide-exchange factor (GEF) for the yeast Rab GTPase Sec4p. Sec2p accelerates GDP release from Sec4p and promotes GDP-GTP exchange for Sec4p activation. In order to elucidate this nucleotide-exchange mechanism using X-ray crystallography, three constructs of native Sec2p (Sec2(1-160)p, Sec2(18-160)p and Sec2(31-160)p) and three constructs of selenomethionine-labelled Sec2p [Sec2(31-160)p, Sec2(31-160)p (M115L) and Sec2(31-160)p (M115L, K121M, T142M)] were crystallized. These six crystals diffracted to 8.8, 4.8, 2.6, 4.0, 3.3 and 3.0 A resolution, respectively. The data set from the SeMet-labelled Sec2(31-160)p (M115L, K121M, T142M) crystal was processed for SAD phasing; the crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 101.9, b = 176.6, c = 181.5 A.
Sato
Yusuke
Y
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.
Fukai
Shuya
S
Nureki
Osamu
O
D003460:Crystallization; D018360:Crystallography, X-Ray; D019204:GTP-Binding Proteins / Q000737:chemistry*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D020691:rab GTP-Binding Proteins / Q000737:chemistry*
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