Yarmola E G, Somasundaram T, Boring T A, Spector I, Bubb M R
Department of Medicine, University of Florida, Gainesville, Florida 32610, USA.
J Biol Chem. 2000 Sep 8;275(36):28120-7. doi: 10.1074/jbc.M004253200.
Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to 0.4 micrometer and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin beta(4) to actin is inhibited by latrunculin A. Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin beta(4), then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if, as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin beta(4) is a surprising result that suggests that significant allosteric changes affect the thymosin beta(4) binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P2(1)2(1)2(1) and diffraction to 3.10 A. A high resolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties.
拉特肌动蛋白A被广泛用作一种在活细胞中隔离单体肌动蛋白的试剂。我们推测拉特肌动蛋白A的其他活性可能对其生物学活性很重要。我们的数据与形成一种化学计量比为1:1、平衡解离常数为0.2至0.4微米的复合物一致，并且没有证据表明肌动蛋白 - 拉特肌动蛋白A复合物参与肌动蛋白丝的延长。肌动蛋白结合蛋白和拉特肌动蛋白A独立地与肌动蛋白结合，而胸腺素β4与肌动蛋白的结合受到拉特肌动蛋白A的抑制。讨论了这种对肌动蛋白结合蛋白的差异效应的潜在影响。从结构角度来看，如果拉特肌动蛋白A在一个空间上影响胸腺素β4结合的位点与肌动蛋白结合，那么拉特肌动蛋白A抑制肌动蛋白上核苷酸交换的观察结果意味着对核苷酸结合裂隙有别构效应。或者，如果如先前假设的那样，拉特肌动蛋白A结合在肌动蛋白的核苷酸裂隙中，那么它抑制胸腺素β4结合的能力是一个令人惊讶的结果，表明显著的别构变化影响胸腺素β4结合位点。我们表明拉特肌动蛋白A和肌动蛋白形成一种具有正交空间群P2(1)2(1)2(1)且衍射至3.10埃的晶体结构。具有优化结晶条件的高分辨率结构应该能提供关于这些显著别构性质的见解。
