Structural insights into the exchange domain of sec2p: expression, purification, crystallization, and preliminary x-ray diffraction data analysis.
Sec2p is an essential yeast gene and is part of the cell polarization process that leads to budding. The N-terminal domain of sec2p (Sec2pN)--the guanine-nucleotide exchange factor for sec4p--has been expressed in Escherichia coli, purified, and crystallized. Crystals belong to the space group P2(1) with unit cell dimensions 178.1 x 98.4 x 180.0 A, beta = 91.7( degrees ), and diffract synchrotron-generated X-rays to better than 3.6 A resolution. Pseudo-precession plots reveal a Laue symmetry of 2/m, corresponding to the aforementioned space group, and unusual weak diffraction in the approximately 5-7 A resolution range. The Matthews number calculations for a typical crystal density suggest a range of 28 to 64 molecules per asymmetric unit. Self-rotation and native Patterson calculations demonstrate a pure helical array of protein subunits. Based on the X-ray diffraction data analysis and amino-acid sequence alignments, the paper presents a hypothetical model of the exchange domain of sec2p as a pair of coiled-coil helices that binds to sec4p and facilitates nucleotide disassociation.
Gill
Harindarpal S
HS
Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. hs.gill@yale.edu
D000595:Amino Acid Sequence; D003460:Crystallization; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012399:Rotation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; C064456:SEC2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006153:Guanosine Diphosphate; D019204:GTP-Binding Proteins; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins