Processing of Escherichia coli alkaline phosphatase: role of the primary structure of the signal peptide cleavage region

J Mol Biol. 1998 Apr 10;277(4):859-70. doi: 10.1006/jmbi.1997.1617.

Abstract

A wide range (69) of mutant Escherichia coli alkaline phosphatases with single amino acid substitutions at positions from -5 to +1 of the signal peptide were obtained for studying protein processing as a function of the primary structure of the cleavage region. Amber suppressor mutagenesis, used to create mutant proteins, included: (i) introduction of amber mutations into respective positions of the phoA gene; and (ii) expression of each mutant phoA allele in E. coli strains producing amber suppressor tRNAs specific to Ala, Cys, Gln, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser and Tyr. Most amino acid substitutions at positions -3 and -1 resulted in a complete block of protein processing. These data give new experimental support for the "-3, -1 rule". Only Ala, Gly and Ser at position -1 allowed protein processing, and Ala provided the highest rate of processing. The results revealed the more conservative nature of the amino acids at the -1 position of signal peptides of Gram-negative bacteria as compared with those of eukaryotic organisms. Position -3 was less regular, since not only Ala, Ser and Gly, but also Leu and Cys at this position, allowed the processing. Mutations at position -4 had an insignificant effect on the processing. Surprisingly, efficient processing was provided mainly by large amino acid residues at position -2 and by middle-sized residues at position -5, indicating that the processing rate is affected by the size of amino acid residues not only at positions -1 and -3. Conformation analysis of the cleavage site taken together with the mutation and statistical data suggests an extended beta-conformation of the -5 to -1 region in the signal peptidase binding pocket.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / chemistry*
  • Alkaline Phosphatase / genetics
  • Alkaline Phosphatase / metabolism*
  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites / genetics
  • DNA Primers / genetics
  • DNA, Bacterial / genetics
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Protein Processing, Post-Translational
  • Protein Sorting Signals / chemistry
  • Protein Sorting Signals / genetics
  • Protein Sorting Signals / metabolism
  • RNA, Bacterial / genetics
  • RNA, Transfer / genetics

Substances

  • DNA Primers
  • DNA, Bacterial
  • Protein Sorting Signals
  • RNA, Bacterial
  • RNA, Transfer
  • Alkaline Phosphatase