Effect of intramolecular cross-linking between glutamine-41 and lysine-50 on actin structure and function.
Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linking of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inhibits actomyosin motility and force generation (Kim et al., 1998; Biochemistry 37, 17,801-17,809). To shed light on this effect, additional modifications of the Gln-41 site on actin were carried out. Both intact G-actin and G-actin cleaved by subtilisin between Met-47 and Gly-48 in the DNase 1 binding loop of subdomain 2 were treated with bacterial transglutaminase. According to the results of Edman degradation, transglutaminase introduced an intramolecular zero-length cross-linking between Gln-41 and Lys-50 in both intact and subtilisin cleaved actins. This cross-linking perturbs G-actin structure as shown by the inhibition of subtilisin and tryptic cleavage in subdomain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases while the subtilisin cleavage dramatically decreases the thermostability of F-actin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The activation of S1 ATPase by actin and the sliding speeds of actin filaments in the in vitro motility assays were essentially unchanged by the cross-linking. Thus, although intramolecular cross-linking between Gln-41 and Lys-50 perturbs the structure of the actin monomer, it has only a small effect on actin polymerization and its interaction with myosin. These results suggest that the new cross-linking does not alter the intermonomer interface in F-actin and that changes in actomyosin motility reported for the Gln-41-Cys-374 intrastrand cross-linked actin are not due to decreased flexibility of loop 38-52 but to constrains introduced into the F-actin structure and/or to perturbations at the actin's C-terminus.
Eli-Berchoer
L
L
Departlment of Oral Biology, Institute of Dental Sciences, Hebrew University Hadassah School of Dental Medicine, Jerusalem, Israel.
Hegyi
G
G
Patthy
A
A
Reisler
E
E
Muhlrad
A
A
D000199:Actins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009218:Myosins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D011817:Rabbits; D013381:Subtilisins / Q000187:drug effects / Q000378:metabolism; D011503:Transglutaminases / Q000494:pharmacology
D000199:Actins; D003432:Cross-Linking Reagents; D011108:Polymers; D005973:Glutamine; D000255:Adenosine Triphosphate; D011503:Transglutaminases; D003850:Deoxyribonuclease I; D013381:Subtilisins; D009218:Myosins; D008239:Lysine