Kostyukova Alla S, Hitchcock-Degregori Sarah E, Greenfield Norma J
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
J Mol Biol. 2007 Sep 21;372(3):608-18. doi: 10.1016/j.jmb.2007.05.084. Epub 2007 Jun 2.
The tropomodulin (Tmod) family of proteins that cap the pointed, slow-growing end of actin filaments require tropomyosin (TM) for optimal function. Earlier studies identified two regions in Tmod1 that bind the N terminus of TM, though the ability of different isoforms to bind the two sites is controversial. We used model peptides to determine the affinity and define the specificity of the highly conserved N termini of three short, non-muscle TMs (alpha, gamma, delta-TM) for the two Tmod1 binding sites using circular dichroism spectroscopy, native gel electrophoresis, and chemical crosslinking. All TM peptides have high affinity for the second Tmod1 binding site (within residues 109-144; alpha-TM, 2.5 nM; gamma-TM, delta-TM, 40-90 nM), but differ >100-fold for the first site (residues 1-38; alpha-TM, 90 nM; undetectable at 10 microM, gamma-TM, delta-TM). Residue 14 (R in alpha; Q in gamma and delta) and, to a lesser extent, residue 4 (S in alpha; T in gamma and delta) are primarily responsible for the differences. The functional consequence of the sequence differences is reflected in more effective inhibition of actin filament elongation by full-length alpha-TMs than gamma-TM in the presence of Tmod1. The binding sites of the two Tmod1 peptides on a model TM peptide differ, as defined by comparing (15)N,(1)H HSQC spectra of a (15)N-labeled model TM peptide in both the absence and presence of Tmod1 peptide. The NMR and CD studies show that there is an increase in alpha-helix upon Tmod1-TM complex formation, indicating that intrinsically disordered regions of the two proteins become ordered upon binding. A model proposed for the binding of Tmod to actin and TM at the pointed end of the filament shows how the Tmod-TM accentuates the asymmetry of the pointed end and suggests how subtle differences among TM isoforms may modulate actin filament dynamics.
肌动蛋白丝末端尖锐、生长缓慢，能封闭该末端的原肌球蛋白调节蛋白（Tmod）家族蛋白的最佳功能需要原肌球蛋白（TM）。早期研究在Tmod1中确定了两个与TM的N端结合的区域，不过不同异构体与这两个位点结合的能力存在争议。我们使用模型肽，通过圆二色光谱、天然凝胶电泳和化学交联，来确定三种短的非肌肉TM（α、γ、δ-TM）高度保守的N端对Tmod1两个结合位点的亲和力并定义其特异性。所有TM肽对Tmod1的第二个结合位点（109 - 144位残基内；α-TM，2.5 nM；γ-TM、δ-TM，40 - 90 nM）具有高亲和力，但对第一个位点（1 - 38位残基；α-TM，90 nM；γ-TM、δ-TM在10 μM时未检测到）的亲和力相差100倍以上。14位残基（α中为R；γ和δ中为Q）以及在较小程度上4位残基（α中为S；γ和δ中为T）是造成这些差异的主要原因。序列差异的功能后果体现在，在存在Tmod1的情况下，全长α-TM比γ-TM对肌动蛋白丝伸长的抑制作用更有效。通过比较（15）N、（1）H HSQC光谱，在不存在和存在Tmod1肽的情况下，对（15）N标记的模型TM肽进行比较，确定了Tmod1的两个肽段在模型TM肽上的结合位点不同。核磁共振和圆二色性研究表明，Tmod1 - TM复合物形成后α-螺旋增加，这表明两种蛋白质的内在无序区域在结合时变得有序。提出的一个关于Tmod在丝的尖锐末端与肌动蛋白和TM结合的模型，展示了Tmod - TM如何加剧尖锐末端的不对称性，并暗示了TM异构体之间的细微差异可能如何调节肌动蛋白丝动力学。
