Characterization of an actin-myosin head interface in the 40–113 region of actin using specific antibodies as probes

Abstract

Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the 'multisite interface model', where the two actin sites could modulate each other during S-1 interaction.

The importance of C‐terminal amino acid residues of actin to the inhibition of actomyosin ATPase activity by caldesmon and troponin I

“…As a result of extensive studies it was established that, at various stages of ATP hydrolysis, three sequences of actin monomer are involved in the interaction with myosin subfragment 1. Two of them are situated in the N-terminal third ofactin [23][24][25][26][27], while the third, responsible for the interaction with skeletal muscle myosin light chain 1, is located in the C-terminal region [28]. Removal of three amino acid residues from the C-terminus by tryptic digestion of actin decreased its binding affinity to myosin as reflected by the fact that at low actin to myosin ratios truncated actin was less efficient in the activation of myosin Mg2+-ATPase activity than intact actin.…”

Two identical hydrophobic clusters are present on the same actin monomer: interaction between one myosin subfragment‐1 and two actin monomers

“…A conformational change in the 96 103 segment was induced on the actin monomer by the binding with anti-(18-28) antibodies [17], thus increasing exposure of residues in this segment (contact 1). This indicated a dynamic relationship essential in the motility process [40].…”

“…A multisite interface model has been suggested [17]. This model obviously did not take into account the location and simultaneous accessibility of myosin-binding sites on adjacent actin monomers nor the two real discontinuous ATP-dependent binding sites on the interface [19] and hydrophobic cluster organization.…”

“…Chemical cross-linking [12], NMR [13,14] and immunochemical studies [15,16,[17][18][19][20] have pinpointed actin segments in subdomain-1, residues 1 28, 96-103, 112-125, 338 348 and 360-372 which are able to bind with the myosin head.…”

“…Using .,uccessively EDC and DMS, we previously cross-linked two lnonomers to 20-kDa and 50-kDa skeletal S1 fragments, re-: Corresponding author. Fax: (33) (67) 60 94 78.spectively [10], and in vitro studies showed that subfragment-I alone can produce the sliding movement of the actin filament [111.Chemical cross-linking [12], NMR [13,14] and immunochemical studies [15,16,[17][18][19][20] have pinpointed actin segments in subdomain-1, residues 1 28, 96-103, 112-125, 338 348 and 360-372 which are able to bind with the myosin head.Furthermore, the (27 kDa-50 kDa-20 kDa) trypsin split myosin subfragment-1, which could no longer be activated by actin, did not bind to the two sites located in the 96-125 region, but it still interacted with the 338-348 and 360--372 segments [191. The presence of hydrophobic interactions in the actin myosin complex have already been suggested [21,22].…”