Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.
Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.
Way
M
M
Medical Research Council Laboratory of Molecular Biology, Cambridge, England.
Gooch
J
J
Pope
B
B
Weeds
A G
AG
D000199:Actins / Q000032:analysis / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000097:blood* / Q000235:genetics; D002872:Chromosome Deletion; D002918:Chymotrypsin / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors; D004926:Escherichia coli / Q000378:metabolism*; D018260:Gelsolin; D005786:Gene Expression Regulation; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000097:blood* / Q000235:genetics; D008854:Microscopy, Electron; D009154:Mutation; D014783:Viscosity
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D003851:Deoxyribonucleases; D002918:Chymotrypsin; D002118:Calcium