Araç Demet, Dulubova Irina, Pei Jimin, Huryeva Iryna, Grishin Nick V, Rizo Josep
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
J Mol Biol. 2005 Feb 18;346(2):589-601. doi: 10.1016/j.jmb.2004.12.004. Epub 2004 Dec 24.
Sec1/Mun18-like (SM) proteins and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play central roles in intracellular membrane fusion. Diverse modes of interaction between SM proteins and SNAREs from the syntaxin family have been described. However, the observation that the N-terminal domains of Sly1 and Vps45, the SM proteins involved in traffic at the endoplasmic reticulum, the Golgi, the trans-Golgi network and the endosomes, bind to similar N-terminal sequences of their cognate syntaxins suggested a unifying theme for SM protein/SNARE interactions in most internal membrane compartments. To further understand this mechanism of SM protein/SNARE coupling, we have elucidated the structure in solution of the isolated N-terminal domain of rat Sly1 (rSly1N) and analyzed its complex with an N-terminal peptide of rat syntaxin 5 by NMR spectroscopy. Comparison with the crystal structure of a complex between Sly1p and Sed5p, their yeast homologues, shows that syntaxin 5 binding requires a striking conformational change involving a two-residue shift in the register of the C-terminal beta-strand of rSly1N. This conformational change is likely to induce a significant alteration in the overall shape of full-length rSly1 and may be critical for its function. Sequence analyses indicate that this conformational change is conserved in the Sly1 family but not in other SM proteins, and that the four families represented by the four SM proteins found in yeast (Sec1p, Sly1p, Vps45p and Vps33p) diverged early in evolution. These results suggest that there are marked distinctions between the mechanisms of action of each of the four families of SM proteins, which may have arisen from different regulatory requirements of traffic in their corresponding membrane compartments.
Sec1/Mun18样（SM）蛋白和可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）在细胞内膜融合中起核心作用。已经描述了SM蛋白与来自 syntaxin家族的SNARE之间的多种相互作用模式。然而，在内质网、高尔基体、反式高尔基体网络和内体中参与运输的SM蛋白Sly1和Vps45的N端结构域与其同源syntaxin的相似N端序列结合这一观察结果，提示了大多数内膜区室中SM蛋白/SNARE相互作用的统一主题。为了进一步了解SM蛋白/SNARE偶联的这种机制，我们阐明了大鼠Sly1（rSly1N）分离的N端结构域在溶液中的结构，并通过核磁共振光谱分析了它与大鼠syntaxin 5的N端肽段形成的复合物。与它们的酵母同源物Sly1p和Sed5p之间复合物的晶体结构比较表明，syntaxin 5结合需要一种显著的构象变化，涉及rSly1N C端β链序列中两个残基的移位。这种构象变化可能会导致全长rSly1的整体形状发生重大改变，并且可能对其功能至关重要。序列分析表明，这种构象变化在Sly1家族中保守，但在其他SM蛋白中不保守，并且酵母中发现的四种SM蛋白（Sec1p、Sly1p、Vps45p和Vps33p）代表的四个家族在进化早期就已分化。这些结果表明，四种SM蛋白家族中每个家族的作用机制之间存在显著差异，这可能源于它们相应膜区室中运输的不同调节需求。
