Structural studies of the lysozyme coded by the pneumococcal phage Cp-1. Conformational changes induced by choline.
The CPL-1 lysozyme coded by the pneumococcal phage Cp-1 has been overproduced in Escherichia coli under the control of a modified lipoprotein lactose promoter. This result has provided the conditions to analyse the CPL-1 secondary structure by circular dichroism (CD). The CD spectra recorded in the far-ultraviolet region showed, at neutral pH, two minima at 210 nm and 230 nm and a shoulder at 217 nm, whereas two bands at 260 nm and 295 nm were observed in the near-ultraviolet region. It has been estimated, by using the CDPROT program, that the protein is composed of 19% alpha-helix, 32% beta-sheet, 28% beta-turn and 21% random coil. Minor changes in the CD spectra were detected either when the pH was varied over 6-10 or when the ionic strength was increased to 1 M NaCl. Choline, a well known modulator of the enzyme activity that is present in the pneumococcal cell wall, induced remarkable changes in the intensities of the bands at 210, 230 and 295 nm, with the appearance of an unusual positive band at 225 nm. The conformational change was reversible and correlated with the competitive inhibitory effect of choline on the lysozyme activity, supporting, by a new and direct experimental approach, the basic role of choline in the recognition of the cell wall substrate. The analyses of the secondary structure prediction and the CD data reported here are compatible with the two-domain structure of CPL-1 reinforce our hypothesis that the C-terminal region is directly involved in the binding of the enzyme to the pneumococcal teichoic and lipoteichoic acids.
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid. Spain.
J M
JM
Sanz
J L
JL
Garcia
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics*; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects; D002794:Choline / Q000494:pharmacology*; D002942:Circular Dichroism; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis* / Q000502:physiology; D010957:Plasmids; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis; D013296:Streptococcus pneumoniae / Q000187:drug effects
D011994:Recombinant Proteins; D004247:DNA; D009113:Muramidase; D002794:Choline