Site-directed mutagenesis of the conserved aromatic residues in the 5 tandem modules starch binding domain of Lactobacillus amylovorus alpha-amylase

Five-residue-long deletions centered on A1a63, A1a75, and Glu118 of ribosomal protein L7/L12 gave low mutant yields (5% or less) when the mutant genes were cloned in phage M13mp18 and controlled by the L10 promotor. Deletions of Glu118–Lys120 or Lys120 (the COOH-terminus of L7/L12) gave higher mutant yields, up to 50% with L7/L12ΔLys120. L7/L12ΔLys120 was not preferentially found in the S100 and not preferentially removed by LiCl washing, but was preferentially extracted from 70S ribosomes in the presence of 28–35% ethanol in 0.25–0.5 M NH4Cl. It follows that ΔLys120 destabilizes the ribosome-binding domain of ribosomal protein L7/L12 in an ethanol-containing solvent, which raises the question whether Lys120 is part of the ribosome-binding domain of L7/L12 during some step of protein synthesis or whether it is essential to preserve the conformation of the physiological ribosome-binding domain under structurally stressful conditions.Key words: ribosome, ribosomal protein L7/L12, site-directed mutagenesis.

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Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2

Biochemical Journal ◽

10.1042/bj20020092 ◽

2002 ◽

Vol 366 (3) ◽

pp. 971-976 ◽

Cited By ~ 53

Author(s):

Lucimara CHIOATO ◽

Arthur H.C. de OLIVEIRA ◽

Roberto RULLER ◽

Juliana M. SÁ ◽

Richard J. WARD

Keyword(s):

Phospholipase A2 ◽

Lipid Membrane ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Phospholipid Bilayer ◽

Directed Mutagenesis ◽

Aromatic Residues ◽

Loop Region ◽

Terminal Loop ◽

Arginine Residues

Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca2+-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117—122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys122, these same substitutions did not significantly alter the Ca2+-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca2+-independent membrane-damaging activity but, with the exception of Lys122, had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115—119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A2, and support a model of a Ca2+-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.

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