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J. Biol. Chem., Vol. 277, Issue 52, 50396-50402, December 27, 2002

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Processing of Escherichia coli Alkaline Phosphatase
SEQUENCE REQUIREMENTS AND POSSIBLE CONFORMATIONS OF THE 6 TO 4 REGION OF THE SIGNAL PEPTIDE^*

Andrey V. Kajava^§, Sergey N. Zolov^¶, Konstantin I. Pyatkov, Andrey E. Kalinin^¶**, and Marina A. Nesmeyanova^§¶

From the  Center for Molecular Modeling, CIT, National Institutes of Health, Bethesda, Maryland 20892, the ^¶ Laboratory of Protein Secretion in Bacteria, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia, and the  Institute of Cellular Biophysics, 142290 Pushchino, Russia

Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position 5 have a high incidence of Pro residues at positions 6 and 4, Val at position 3, and Ser at positions 4 and 2. This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position 5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions. Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic 5 Leu, Ile, or Tyr completely restores efficiency of the maturation. Introduction of Val at position 3 also partially suppresses the inhibition imposed by 5 Leu, while a Ser residue at position 4 or 2 does not restore processing. In addition, effective maturation of a mutant with Pro residues at positions from 6 throughout 4 proved that polyproline conformation of this region is permissive for processing. To understand the effects of the mutations, we modeled a peptide substrate into the active site of the signal peptidase using the known position of the -lactam inhibitor. The inhibitory effect of the 5 residue and its suppression by either Pro 6 or Pro 4 can be explained if we assume that Pro-containing 6 to 4 regions adopt a polyproline conformation whereas the region without Pro residues has a -conformation. These results permit us to specify sequence requirements at 6, 5, and 4 positions for efficient processing and to improve the prediction of yet unknown cleavage sites.

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