Bartegi A, Roustan C, Chavanieu A, Kassab R, Fattoum A
Centre de Recherches de Biochimie Macromoléculaire, CNRS ERS 155, Université Montpellier 1, France.
Eur J Biochem. 1997 Dec 1;250(2):484-91. doi: 10.1111/j.1432-1033.1997.0484a.x.
The atomic model of the F-actin-myosin subfragment 1 complex (acto-S-1) from skeletal muscle suggests that the transition of the complex from a weakly to a strongly binding state, generating mechanical force during the contractile cycle, may involve the attachment of the upper 50-kDa subdomain of myosin subfragment 1 (S-1) to the interface between subdomains 1 and 3 of actin. For the human cardiac myosin, this putative interaction would take place at the ordered loop including Arg403 of the beta-heavy chain sequence, a residue whose mutation into Gln is known to elicit a severe hypertrophic cardiomyopathy caused by a decrease of the rate of the actomyosin ATPase activity. Moreover, in several nonmuscle myosins the replacement of a Glu residue within the homolog loop by Ser or Thr also results in the reduction of the actomyosin ATPase rate that is alleviated by phosphorylation. As an approach to the characterization of the unknown interaction properties of F-actin with this particular S-1 loop region, we have synthesized four 17-residue peptides corresponding to the sequence Gly398-Gly414 of the human beta-cardiac myosin. Three peptides included Arg403 (GG17) or Gln403 (GG17Q) or Ser409 (GG17S) and the fourth peptide (GG17sc) was a scrambled version of the normal GG17 sequence. Using fluorescence polarization, cosedimentation analyses and photocross-linking, we show that the three former peptides, but not the scrambled sequence, directly associate in solution to F-actin, at a nearly physiological ionic strength, with almost identical affinities (Kd approximately 40 microM). The binding strength of the F-actin-GG17 peptide complex was increased fivefold (Kd = 8 microM) in the presence of subsaturating concentrations of added skeletal S-1 relative to actin, without apparent competition between the peptide and S-1. Each of the three actin-binding peptides inhibited the steady-state actin-activated MgATPase of skeletal S-1 by specifically decreasing about twofold the Vmax of the reaction without changing the actin affinity for the S-1-ATP intermediate. Cosedimentation assays indicated the binding of about 0.65 mol peptide/mol actin under conditions inducing 70% inhibition. Collectively, the data point to a specific and stoichiometric interaction of the peptides with F-actin that uncouples its binding to S-1 from ATP hydrolysis, probably by interfering with the proper attachment of the S-1 loop segment to the interdomain connection of actin.
骨骼肌中F-肌动蛋白-肌球蛋白亚片段1复合物（肌动蛋白-S-1）的原子模型表明，该复合物在收缩周期中从弱结合状态转变为强结合状态并产生机械力，可能涉及肌球蛋白亚片段1（S-1）的上部50 kDa亚结构域与肌动蛋白亚结构域1和3之间的界面结合。对于人类心肌肌球蛋白，这种假定的相互作用将发生在包括β重链序列中Arg403的有序环处，已知该残基突变为Gln会引发由肌动球蛋白ATP酶活性速率降低引起的严重肥厚性心肌病。此外，在几种非肌肉肌球蛋白中，同源环内的Glu残基被Ser或Thr取代也会导致肌动球蛋白ATP酶速率降低，而磷酸化可缓解这种降低。作为表征F-肌动蛋白与该特定S-1环区域未知相互作用特性的一种方法，我们合成了四种对应于人类β-心肌肌球蛋白序列Gly398-Gly414的17个残基的肽。三种肽包含Arg403（GG17）或Gln403（GG17Q）或Ser409（GG17S），第四种肽（GG17sc）是正常GG17序列的 scrambled 版本。使用荧光偏振、共沉降分析和光交联，我们表明前三种肽而非 scrambled 序列在接近生理离子强度下直接在溶液中与F-肌动蛋白结合，亲和力几乎相同（Kd约为40 microM）。在添加亚饱和浓度的骨骼肌S-1相对于肌动蛋白的情况下，F-肌动蛋白-GG17肽复合物的结合强度增加了五倍（Kd = 8 microM），肽和S-1之间没有明显竞争。三种肌动蛋白结合肽中的每一种都通过特异性降低反应的Vmax约两倍而不改变肌动蛋白对S-1-ATP中间体的亲和力，从而抑制骨骼肌S-1的稳态肌动蛋白激活的MgATP酶。共沉降分析表明在诱导70%抑制的条件下约0.65摩尔肽/摩尔肌动蛋白的结合。总体而言，数据表明肽与F-肌动蛋白之间存在特异性和化学计量的相互作用，该相互作用通过干扰S-1环段与肌动蛋白结构域间连接的正确附着，使肌动蛋白与S-1的结合与ATP水解解偶联。
