Mapping of the actomyosin interfaces.

Abstract

Recombinant DNA methods were used to obtain souble, r ts of the heavy chain of myosn subfragMent 1 (S-i). These ants Numerous studies using proteolytic fiagmentation, crosslinking experiments, or peptide competition have indicated that different myosin subfragment 1 heavy-chain fragment (S1-HC)areas are involved in interactions with actin: (i) the N-terminal 23kD)a fragment of S1-HC (1) and the 23/50-kDajunction (2), (i) the central 50-kDa domain of S1-HC (3, 4), (iii) the C-terminal fragment of S1-HC-i.e., the 50/20-kDa junction (5-7). This C-terminal area was postulated to be composed of three actin binding subsites (8). Crosslinking experiments with ATP analogs have shown that some residues (i.e., ref. 9) are close to the ATP binding site. Spatial proximity between the 23-and 20-kDa S1-HC segments delineating the ATPase site (7,(10)(11)(12) has also been demonstrated. All ofthese analyses required the use of adequate proteolytic enzymes, accessibility of corresponding protease-sensitive regions within the S1-HC, separation ofS1-HC by dena ion/renaturtion procedures, and also correct folding of the isolated polypeptides (3, 13). In addition, the protease-sensitive regions were virtually destroyed and were therefore inoperative. Through recombinant technology it is now possible to produce native and soluble myosin heavy-chain (MHC) fragments of any desired length, even those containing the two main intact protease-sensitive regions along the entire S1-HC. This enables reexamination of the MHC regions that may interact with actin and ATP. A fine delineation of the actomyosin interface(s), together with the three-dimensional structures of actin (14) and myosin (15-17), should help us understand the dynamic interactions between the two main motor molecules. The chimeric MHC fiagments are also of interest for understanding the molecular bases of functional variations between myosin isoforms (18) and the functional impact of (3-MHC missense mutations that cause familial hypertrophic cardiomyopathy (19). In this study, we designed different S1-HC from human cardiac 3-MHC and expressed these in Escherichia coli as soluble maltose binding protein (MBP) fusion polypeptides. After purification, their respective interactions with actin and ATP were analyzed in vitro. While this work was still in progress, the three-dimensional crystalline structure of the myosin head (15) and determination of the three-dimensional atomic models of F-actin decorated with the myosin head were reported (16,17). The present results are discussed in light ofthe newly proposed three-dimensional structure of the myosin head.MATERIALS AND METHODS ConstMcon of I-MHC Expession Comes. NM, N. M, Ni, and Ml constructs leading to the expression in TB1 E. coli cells of recombinant #8S1-HC of 100, 69, 73, 58, and 83 kDa, respectively, were derived from the ptrcf3HC13 plasmid (20), which contains a Nco I/HindIII. MHC cDNA fragment of 1585 bp encoding residues 1-524. NM was constructed by subcloning the Nco I/HindIU fragment into the pIH902 vec...

The cardiac myosin heavy chain Arg-403→Gln mutation that causes hypertrophic cardiomyopathy does not affect the actin- or ATP-binding capacities of two size-limited recombinant myosin heavy chain fragments

“…The recombinant /3-MHC fragments used in this study were previously shown to have functional capacities, they are represented under the trypsin S-1 map by rectangles with their respective names and positions according to the ,.-MHC numbering scheme [37]. Fragment NM binds ATP, and fragment M interacts with F-actin [20].…”

“…These different experimental approaches clearly demonstrated that the 403 mutation did not modify the capacities of the studied MHC fragments to bind ATP. Note that neither recombinant NM [20] nor NM403 hydrolysed ATP. Figure 6 Comparative F-actin binding between normal and 403 mutated recombinant fiS-1 heavy chain fragments (a) SDS/PAGE analysis of co-sedimentation assays carried out with 2 ,uM of each /SS-1 fragment and increasing amounts of F-actin (0.25, 0.5, 1, 2 and 4 ,M).…”

“…These studies demonstrated that the 403 mutation influences the rate of transition within the acto-myosin cross-bridge cycle and, more unexpectedly, thick filament formation. We recently used recombinant DNA methods to obtain soluble, undenatured tailored contiguous fl-MHC head fragments and determine their in vitro ATP-and F-actin-binding capacities [20]. Residue 403 is located in the middle of one such active actin-binding fragment (residues 249-524) and within the MHC fragment (residue 1-524) capable of interacting with ATP [20].…”

“…To examine the functional effects of the Arg-403 to Gln change in the ,-MHC, we compared the capacities of two different recombinant MHC fragments focused around normal or mutated residue 403 to interact with actin and ATP [20]. The actinbinding S-1-HC fragments, spanning from residues 249 to 524, centrally located in the myosin head [15], were referred to as M or M403, normal and mutated forms respectively.…”

“…The long Nterminal ATP-binding S-1 fragments, spanning residues 1 to 524, were referred to as NM or NM403, normal and mutated forms respectively ( Figures I and 4). These MHC fragments were easily obtained as soluble and undenatured proteins by recombinant DNA techniques using a 1585 bp cDNA fragment coding for the normal MHC fragment NM (and M) [20]. The cDNA fragment was mutagenized to produce mutated MHC fragments (see the 20 25…”