Abstract
Protein S, the most abundant protein synthesized during development of the Gram-negative bacterium Myxococcus xanthus, assembles on the surface of the spores. It can be dissociated from the spores using divalent metal chelators and will reassemble on the spores in the presence of calcium. The amino acid sequence of protein S contains regions which have homology to the calcium-binding sites of calmodulin. Protein S was found to bind 2 mol of calcium/mol of protein with Kd values of 27 and 76 microM. Using oligonucleotide-directed site-specific mutagenesis, the gene coding for protein S was changed in each of two regions of homology to calmodulin (Ser40----Arg,Ser129----Arg), and a double mutant was also constructed. Each mutant gene was then transduced into the genome of a M. xanthus strain from which the wild-type genes had been deleted. All three mutants produced protein S normally during development. One of the mutants (Ser129----Arg) had normal amounts of protein S on its spores, whereas the other (Ser40----Arg) bound much less and the double mutant had virtually none. Analysis of the calcium binding affinities of the purified proteins showed that [Arg40]protein S and [Arg40, Arg129]protein S did not bind detectable quantities of calcium, whereas [Arg129]protein S bound less calcium than the wild-type protein and with a reduced affinity.
Highlights
From the $Department of Medicine, Divisionof Digestive Diseasesand the Department of Cell Biologyand Anatomy, Cornel1 University MedicalCollege, New York, New York 10021and the §Department of Biochemistry, University of Medicine & Dentistry of New Jersey, Robert Wood Johnson Medical Schoolat Rutgers, Piscataway, New Jersey 08854
In addition to the homology between these two genes, there is striking internalhomology in the proteinsequences; they can be divided into four homologous domains, with domains 1 and 3 as well as domains 2 and 4 having extensive homologies (8).This pattern of internal homology genewas transduced into the genome ofa M. is found in calmodulin and the y-crystallins,two classes xanthus strain from which the wild-type genes had of proteins that have been identified only in eukaryotes
It accumulates in thedevelop- mation found in calmodulin (14), but the proposed structure ing cells until the onsetof sporulation, after which most of it based on y-crystallindoes show possible calcium-bindingsites is found assembled on the surface of the myxospores and a involving the sequence from G l P to Ser4”and its equivalents smaller amount inside (3-5)
Summary
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 263, No 3, Issue of January 25, pp. 1199-1203,1988 Printed in U.S.A. The sequence of protein S contains regions which have ops gene is induced very late in development, after sporulahomology to the calcium-binding sites of calmodulin. Using oligonucleotide-directed site-specific mutagenesis, the gene coding for protein S was changed in each of two regions of homology to calmodulin(Ser4’+Arg, Ser12’+Arg), and a double mutanwtas alsoconstructed.Each mutant tion; and its product is founodnly inside the spores (4, 5). Protein Stherefore does not have the helix-loop-helix conforstage of mound formation (3) It accumulates in thedevelop- mation found in calmodulin (14), but the proposed structure ing cells until the onsetof sporulation, after which most of it based on y-crystallindoes show possible calcium-bindingsites is found assembled on the surface of the myxospores and a involving the sequence from G l P to Ser4”and its equivalents smaller amount inside (3-5). Protein S has beenpurified and crystallized (6),and a preliminary x-ray crystallographic study has been performed(7)
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