Actin Cross-Linking and Inhibition of the Actomyosin Motor

• Published in: Biochemistry (Easton) 2002-01, Vol.41 (1), p.86-93
• Main Authors: Kim, Eldar, Bobkova, Elena, Hegyi, György, Muhlrad, Andras, Reisler, Emil
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Actomyosin - antagonists & inhibitors; Actomyosin - metabolism; Adenosine Triphosphatases - metabolism; Animals; Cell Movement - physiology; Cross-Linking Reagents - pharmacology; Cysteine - metabolism; Glutamine - metabolism; In Vitro Techniques; Lysine - metabolism; Models, Molecular; Muscle, Skeletal - metabolism; Myosin Subfragments - chemistry; Myosins - metabolism; Protein Conformation; Rabbits
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0113824

Intrastrand cross-linking of actin filaments by ANP, N-(4-azido-2-nitrophenyl) putrescine, between Gln-41 in subdomain 2 and Cys-374 at the C-terminus, was shown to inhibit force generation with myosin in the in vitro motility assays [Kim et al. (1998) Biochemistry 37, 17801−17809]. To clarify the immobilization of which of these two sites inhibits the actomyosin motor, the properties of actins with partially overlapping cross-linked sites were examined. pPDM (N,N‘-p-phenylenedimaleimide) and ABP [N-(4-azidobenzoyl) putrescine] were used to obtain actin filaments cross-linked (∼50%) between Cys-374 and Lys-191 (interstrand) and Gln-41 and Lys-113 (intrastrand), respectively. ANP, ABP, and pPDM cross-linked filaments showed similar inhibition of their sliding speeds and force generation with myosin (∼25%) in the in vitro motility assays. In analogy to ANP cross-linking of actin, pPDM and ABP cross-linkings did not change the strong S1 binding to actin and the V max and K m parameters of actomyosin ATPase. The similar effects of these three cross-linkings reveal the tight coupling between structural elements of the subdomain 2/subdomain 1 interface and show the importance of its dynamic flexibility to force generation with myosin. The possibility that actin cross-linkings inhibit rate-limiting steps in motion and force generation during myosin cross-bridge cycle was tested in stopped-flow experiments. Measurements of the rates of mantADP release from actoS1 and ATP-induced dissociation of actoS1 did not reveal any differences between un-cross-linked and ANP cross-linked actin in these complexes. These findings are discussed in terms of the uncoupling between force generation and other aspects of actomyosin interactions due to a constrained dynamic flexibility of the subdomain 2/subdomain 1 interface in cross-linked actin filaments.