Wu K C, Bryan J T, Morasso M I, Jang S I, Lee J H, Yang J M, Marekov L N, Parry D A, Steinert P M
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Biol Cell. 2000 Oct;11(10):3539-58. doi: 10.1091/mbc.11.10.3539.
Many alpha-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A(22) mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.
许多形成双股卷曲螺旋的α-螺旋蛋白拥有一个13个残基的触发基序，这似乎是卷曲螺旋稳定性所必需的。然而，按照目前的定义，中间丝（IF）蛋白链中不存在该基序，尽管如此，IF蛋白链仍能形成分段的双股卷曲螺旋。在本研究中，我们在IF链中寻找并鉴定了两个区域，它们对于形成卷曲螺旋分子所需的稳定性至关重要，因此可能起到触发基序的作用。我们利用角蛋白5/角蛋白14 IF系统进行了一系列点突变。将野生型和突变型链的组合在体外和体内组装，并使用尿素拆解试验检测双股（单分子）和双分子组装体的稳定性。我们的新数据表明，在2B杆状结构域片段的第100至113位残基之间存在一个区域，它对于分子稳定性和IF组装是绝对必需的。这个潜在的触发基序与共有序列略有不同，在第4位是Asp残基（而不是Glu），在第9位是Thr残基（而不是带电荷的残基），但在第6位绝对需要一个Glu残基。由于这13个残基高度保守，这个基序似乎有可能在所有IF链中发挥作用。同样，通过对两条链都进行突变的角蛋白IF进行测试，我们在1B杆状结构域片段的第79至91位残基之间鉴定出了第二个潜在的触发基序，它也可能在所有IF链中保守。然而，我们在1A杆状结构域片段中未能找到触发基序。此外，许多其他点突变对IF组装几乎没有可检测到的影响，除了2B杆状结构域片段中保守的Lys-23残基。交联和建模研究表明，当两个分子以A(22)模式排列时，Lys-23可能非常靠近Glu-106。因此，Glu-106残基在IF结构中可能具有双重作用：它可能参与触发基序的形成，为双股卷曲螺旋分子提供特殊稳定性，并且它可能参与IF结构双分子层级阶段的稳定。
