Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state

Abstract

The structure of myosin subfragment 1 (SI) in the weakly attached complex with actin was studied at three specific sites, at the 50-kDa/20-kDa and 27-kDa/SO-kDa junctions, and at the N-terminal region, using tryptic digestion as a structure-exploring tool. The structure of S1 at the vicinity of the 50-kDa/20-kDa junction is pH dependent in the weakly attached state because the tryptic cleavage at this site was fully protected by actin at pH 6.2, but the protection was only partial at pH 8.0. Since the actin protection is complete in rigor at both pH values, the results indicate that the structure of S1 at the 50-kDa/20-kDa junction differs in the two states at pH 8.0, but not at pH 6.2. Actin restores the ADP-suppressed tryptic cleavage after Lys213 at the 27-kDa/50-kDa junction in the strongly attached state, but not in the weakly attached state, which indicates structural difference between the two states at this site. ATP and ADP open a new site for tryptic cleavage in the N-terminal region of the S1 heavy chain between Arg23 and Ile24. Actin was found to suppress this cleavage in both weakly and strongly attached states, which shows that, in the vicinity of this site, the structure of S1 is similar in both states. The results indicate that the binding of S1 to actin induces localized changes in the S1 structure, and the extent of these changes is different in the various actin-S1 complexes.Myosin and actin are two cardinal proteins of muscle, whose interaction with each other, coupled with the hydrolysis of ATP, constitutes the molecular basis of contraction. The head segment of myosin, called S1, contains the distinct actinbinding and nucleotide-binding sites of myosin [I] and is sufficient, together with ATP and actin, to generate movement in vitro [2]. It is, therefore, obvious that its structural and functional characterization has great significance. The heavy chain of S1 is cleaved by limited tryptic digestion into three major fragments of 27 kDa, 50 kDa, and 20 kDa (aligned from the N-terminus in this order [3]). The fragments have become a valuable framework in which the nucleotide-binding and actin-binding sites and other specific functions can be assigned.It is generally assumed [4] that the contractile force originates from the hydrolysis of ATP taking place at the ATPbinding site of S1. Each intermediate in the ATP cycle induces specific conformational changes in S1, which define the actin-S1 relationship at each step. In the various actin-S1 complexes, the affinity of S1 for actin depends on the occupancy of the ATP-binding site. On the basis of affinity, three types of S1-actin complexes can be defined [5]: (a) rigor complex, noCorrespondence to A. Muhlrad,

Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N‐terminal region of the heavy chain

“…It seems more likely that a long-distance communication pathway exists between these sites. In favor of this suggestion are literature data showing that F-actin suppresses the N-terminal tryptic cleavage of S1 both in the strongly attached state [26] and in the weakly attached state [27]. The cleavage between Arg23 and Ile24 probably disrupts this communication pathway, thus preventing the global conformational changes in the myosin head induced by actin binding to loop 2.…”

“…Examination of the S1 structure has suggested that there are contacts between the essential light chain in the regulatory domain and some parts of the heavy chain in the motor domain [45]. Essential light-chain residues 103-115 form a helix and lie in close proximity to a helix-loop motif near the N-terminus of the heavy chain (residues [21][22][23][24][25][26][27][28][29][30][31]. This contact may serve as an additional communication pathway between the motor domain and the regulatory domain, and it may play a crucial role in the transmission of actin-induced conformational changes from loop 2 to the regulatory domain through the motor domain.…”

“…Nucleotide‐induced conformational changes in the myosin head probably expose this N‐terminal region to proteases. On the other hand, actin was found to suppress the nucleotide‐induced tryptic cleavage at the N‐terminal region of S1 in both strongly attached state (in the presence of ADP) [26] and weakly attached state (in the presence of ATP analogs) [27]. As the N‐terminal region is located spatially far from actin‐binding sites in the 3D structure of S1 [28], these effects of actin can be explained by long‐range conformational changes induced by the attachment of actin to its binding sites, primarily to loop 2 which is mainly responsible for the ‘weak’ binding of S1 to F‐actin.…”