Dynamic properties of actin. Structural changes induced by beryllium fluoride.
Beryllium fluoride (BeFx) has been widely used as a phosphate analogue in nucleotide-binding proteins. It was found to bind tightly to F- but not G-actin (Combeau C., and Carlier M. F. (1988) J. Biol. Chem. 263, 17429-17436) and to affect the three-dimensional structure of filaments by stabilizing the subdomain 2 region of the actin promoter (Orlova, A., and Egelman, E. H. (1992) J. Mol. Biol. 227, 1043-1053). In this work we examined the BeFx-induced structural and functional changes in G- and F-actin by using proteolysis, chemical modifications, ATPase, and in vitro motility assays. The results of proteolysis studies show that BeFx binds also to MgADP-G-actin and renders its subdomain 2 region more similar to that in MgATP-G-actin. This is manifested in enhanced subtilisin and decreased tryptic digestions in subdomain 2 of G-actin. BeFx had a strong effect on the proteolysis of MgADP-F-actin: both the tryptic and subtilisin digestions in subdomain 2 were completely inhibited. Significant protection against proteolysis in this region was observed even at 1:14 molar ratios of BeFx to actin indicating cooperative effects on the structure of the actin filament. A similar although milder effect of phosphate on the proteolysis of F-actin suggests that BeFx acts as a phosphate analogue in this system. BeFx also induces changes in the subdomain 1 region of F-actin. This is revealed via reduced rates of Cys-374 alkylation with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and an increased subtilisin cleavage near the C terminus of actin in the presence of BeFx. The BeFx-induced structural changes in actin have little effect on its interactions with myosin. BeFx inhibits only slightly the actin-activated ATPase activity of S1 by decreasing Vmax without affecting KM. Additionally, the binding of BeFx to actin does not change the sliding velocity of actin filaments in the in vitro motility assays. The BeFx-induced specific and distinct changes in G- and F-actin point to the dynamic nature of actin structure and the local differences between monomeric and polymeric forms of actin.
Muhlrad
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A
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Cheung
P
P
Phan
B C
BC
Miller
C
C
Reisler
E
E
D000199:Actins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001608:Beryllium / Q000494:pharmacology*; D005459:Fluorides / Q000494:pharmacology*; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation / Q000187:drug effects*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000199:Actins; D010446:Peptide Fragments; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D013381:Subtilisins; D014357:Trypsin; D000251:Adenosine Triphosphatases; D009218:Myosins; D001608:Beryllium; D005459:Fluorides