Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation.
Models of F-actin structure predict the importance of hydrophobic loop 262-274 at the interface of subdomains 3 and 4 to interstrand interactions in filaments. If this premise is correct, prevention of the loop conformational change--its swinging motion--should abort filament formation. To test this hypothesis, we used site-directed mutagenesis to create yeast actin triple mutant (LC)2CA (L180C/L269C/C374A). This mutation places two cysteine residues in positions potentially enabling the locking of loop 262-274 to the monomer surface via disulfide formation. Exposure of the purified mutant to oxidation catalysts resulted in an increased electrophoretic mobility of actin on SDS PAGE and a loss of two cysteines by DTNB titrations, consistent with disulfide formation. The polymerization of un-cross-linked mutant actin by MgCl2 was inhibited strongly but could be restored to wild type actin levels by phalloidin and improved greatly through copolymerization with the wild-type actin. Light scattering measurements revealed nonspecific aggregation of the cross-linked actin under the same conditions. Electron microscopy confirmed the absence of filaments and the presence of amorphous aggregates in the cross-linked actin samples. Reduction of the disulfide bond by DTT restored normal actin polymerization in the presence of MgCl2 and phalloidin. These observations provide strong experimental support for a critical role of the hydrophobic loop 262-274 in the polymerization of actin into filaments.
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Alexander
A
Shvetsov
Runa
R
Musib
Martin
M
Phillips
Peter A
PA
Rubenstein
Emil
E
Reisler
D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D000199:Actins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D000409:Alanine / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000235:genetics; D015636:Magnesium Chloride / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D010590:Phalloidine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure
D000199:Actins; D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D011108:Polymers; D029701:Saccharomyces cerevisiae Proteins; D015636:Magnesium Chloride; D010590:Phalloidine; D007930:Leucine; D003545:Cysteine; D000409:Alanine; D004229:Dithiothreitol