Cloning, Zn2+ binding, and structural characterization of the guanine nucleotide exchange factor human Mss4.
The Sec4/Ypt1/Rab family of small GTP-binding proteins are involved in the regulation of intracellular vesicular transport. A rat gene, mss4, that encodes a guanine nucleotide exchange factor (GEF) for Sec4 was recently cloned by its ability to rescue defects in protein transport of a yeast temperature-sensitive (ts) mutant, sec4-8. We describe herein the cloning, bacterial expression, and biochemical characterization of human Mss4. As expected, both the cDNA and its encoded amino acid sequences are highly conserved between the human and rat mss4. Soluble and functional Mss4 protein was obtained by expressing the gene as a glutathione-S-transferase fusion protein in Escherichia coli. Subsequent biochemical analysis revealed that Mss4 binds 1 equiv of Zn2+, and zinc is essential for the stability of the protein. Utilizing multidimensional heteronuclear NMR techniques, we assigned most of the 1H, 15N, and 13C resonances of this 14-kDa protein. Its secondary structure was also deduced from slowly exchanging amide protons, characteristic NOEs, and 3JNH-C alpha H coupling constants. The protein contains a central seven-stranded antiparallel beta sheet, flanked by two small beta sheets. Many resonances pertaining to a loop region of the molecule cannot be identified, suggesting that it might be involved in local movements. These studies provide the first structural insights into a protein possessing GEF activity.
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Howard Hughes Medical Institute, Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; C489794:RABIF protein, human; C079550:Rabif protein, rat; D029701:Saccharomyces cerevisiae Proteins; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C489795:MSS4 protein, S cerevisiae; D015032:Zinc