Rebowski Grzegorz, Namgoong Suk, Boczkowska Malgorzata, Leavis Paul C, Navaza Jorge, Dominguez Roberto
Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
Boston Biomedical Research Institute, Watertown, MA 02472-2899, USA.
J Mol Biol. 2010 Oct 15;403(1):11-23. doi: 10.1016/j.jmb.2010.08.040. Epub 2010 Sep 8.
Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.
肌动蛋白丝成核因子以一种受调控的方式在细胞中启动聚合反应。这些分子的一个常见结构由串联的WASP同源2结构域（W结构域）组成，该结构域招募三到四个肌动蛋白亚基以形成聚合核。我们描述了一种由串联W结构域组装而成的肌动蛋白二聚体的低分辨率晶体结构，其中第一个W结构域与与其结合的肌动蛋白亚基的Cys374交联，而最后一个W结构域后面跟着胸腺素β4的C末端尖端正封端螺旋。虽然二聚体中肌动蛋白亚基的排列类似于肌动蛋白丝的长螺距螺旋，但也观察到了重要差异。这些差异是由W结构域与肌动蛋白丝中亚基间接触的空间位阻导致的。我们还确定了副溶血性弧菌VopL的第一个W结构域与肌动蛋白Cys374交联后的结构，并表明它与非交联的W-肌动蛋白结构几乎相同。这一结果验证了交联作为研究肌动蛋白成核复合物工具的用途，因为其天然的聚合倾向会干扰大多数结构方法。结合成核的生化分析，这些结构可能解释了为什么基于具有短W间连接子的串联W结构域的成核因子活性相对较弱，成核后不能与丝结合，并且不太可能影响丝的伸长。这些发现还可能解释了为什么与串联W结构域成核因子相关的Arp2/3复合物的成核促进因子在成核后会从分支连接处被排出。我们最终表明，简单地将胸腺素β4的C末端尖端正封端螺旋添加到串联W结构域上，可以将它们的活性从肌动蛋白丝成核转变为单体隔离。
