X-ray crystallographic studies of three nucleic acid binding proteins.
Staker, Bart Lee
2000
Abstract
Three proteins, all of which bind nucleic acids, were crystallized and-their three-dimensional structures determined by X-ray crystallography. Hsp15 from <italic> Escherichia coli</italic> is a newly identified heat shock protein with homologs in many eubacteria. This highly basic protein binds to RNA in 50S ribosomal subunits. Diffraction data were phased by the multiple isomorphous replacement method. The structure, containing two 110-residue molecules in the asymmetric unit, was refined to 2.2 A resolution with <italic>R</italic><sub>work </sub> = 0.226 and <italic>R</italic><sub>free</sub> = 0.286. The protein folds into an alpha+beta domain followed by a solvent-exposed 22-residue alpha-helix. A distinctive L-shaped structural motif is highly conserved in at least eight different protein families which bind RNA, including the known structure of ribosomal protein S4. The motif contains positively-charged surface residues that define a likely RNA binding site. A similar fold, but with very low sequence homology, was detected in threonyl-tRNA synthetase. <italic>E. coli</italic> FtsJ is an rRNA methyltransferase whose expression is enhanced 20-fold during heat stress. The 1.5 A crystal structure (<italic>R</italic><sub>work</sub> = 0.198, <italic>R</italic><sub>free</sub> = 0.232) reveals a typical methyltransferase fold containing bound S-adenosyl-methionine. The molecular surface of FtsJ contains a likely nucleic acid binding groove of highly conserved, positively charged residues. This groove is structurally similar to the RNA binding groove of VP39, an m<super>7</super>G(5<super> '</super>)pppN-specific nucleoside-2<super>'</super>-O-methyltransferase. Murine monoclonal antibody 4B2 is an auto-antibody that binds to both single- and double-stranded DNA (ss/dsDNA). It was derived from MRL/mrl mice which mimic the human disease systemic lupus erythrematosus (SLE). The F(ab) fragment was purified from ascites fluid and crystallized. The 432-residue structure was determined by molecular replacement with the F(ab) from BV-0401 as the search model and refined to 2.5 A (<italic>R</italic><sub>work </sub> = 0.219, <italic>R</italic><sub>free</sub> = 0.279). The crystal structure reveals a putative binding cleft between the heavy and light chain complementary determinant regions (CDR). Positively-charged residues on the CDR loops suggest ionic interactions can be made with the sugar-phosphate backbone of ss/dsDNA and may be responsible for non-specific DNA interactions. This structure serves as a means for understanding the unique characteristics of cross-reactive anti-ss/dsDNA which are found in SLE and may lead to a better understanding of the pathogenic mechanisms of anti-DNA mediated glomerulonephritis in SLE.Subjects
Anti-dna Antibodies Crystallographic Heat Shock Proteins Methyltransferase Nucleic Acid Binding Proteins Studies Three X-ray Crystallography
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