Tubulin secondary structure analysis, limited proteolysis sites, and homology to FtsZ
Tubulin secondary structure analysis, limited proteolysis sites, and homology to FtsZ
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DOI：
10.1021/bi961357b
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发表时间：
1996-11-12
期刊：
影响因子：
2.9
通讯作者：
Andreu, JM
中科院分区：
文献类型：
--
作者：
dePereda, JM;Leynadier, D;Andreu, JM
6种不同方法分析的α-β-微管蛋白二聚体的远紫外圆二色谱表明，其平均含量约为33%的螺旋、21%的β折叠和45%的其他二级结构。傅里叶变换红外光谱的解卷积表明：24%的片状结构，37%(最大)螺旋结构，38%(最小)其他结构。用多重序列方法PHD[REST，B.，&Sander，C.(1993a)J.Mel]对75个α-微管蛋白、106个β-微管蛋白、14个γ-微管蛋白序列和12个细菌细胞分裂蛋白FtsZ序列进行了分离比对，预测了它们的二级结构。比奥尔。232、584-599]。预测的二级结构平均为33%的a螺旋，24%的β折叠和43%的环状。这些预测已经与杂二聚体和紫杉醇诱导的微管表面的12种蛋白水解酶的有限降解位置进行了比较[de Pereda，J.M.，&Andreu，J.M.(1996年)生物化学，35,14184-14202]。在24个实验确定的划痕位置中，有18个位于预测的环或处于二级结构元素的极端位置。蛋白水解区A(包括α-微管蛋白中可乙酰化的Lys40和可能的Lys60，以及β-微管蛋白中的Gly93)和蛋白水解区B(在两条链上延伸到167到183个残基之间)在微管中可达。位于残基278和295之间的蛋白水解区C部分封闭在微管中。CL-微管蛋白切割位点Arg339-Ser340在蛋白水解区D的预测或螺旋后形成环状，该位点受到紫杉醇微管的保护，但出现了一个新的胰蛋白酶位点，可能位于同一螺旋的N端。D区还含有β-微管蛋白Cys354，它可以在微管中获得。蛋白水解区E包括每个微管蛋白链的C-末端高变环(10-20个残基)。它们遵循两个更大的预测螺旋区(在β-微管蛋白中残基为372-395和405-432)，这也是α-和β-微管蛋白序列中较长的保守部分。通过结合其他生化信息，提出了一组P-微管蛋白折叠的表面约束和距离约束。FtsZ序列与微管蛋白序列只有10%-18%相同。然而，预测的二级结构显示两个明显相似的区域(85-87和51-78%)，位于微管蛋白位置95-175和305-350，分别对应于FtsZ 65-135和255-300。第一个区域位于微管蛋白水解区A和B的两侧，它由一个预测的loop1-helix-loop2-sheet-loop3-helix-loop4-sheet折叠组成，其中包含基序(KR)GXXXXG(Loop1)和微管蛋白-FtsZ签名G盒基序(Sag)GGTG(SAT)G(Loop3)。一个简单的工作模型设想loop1和loop3在核苷酸结合部位一起，而环2和4在蛋白质的表面，这与微管蛋白的蛋白质分解和抗原可及性结果一致。该模型与微管蛋白和FtsZ突变体的研究相一致。有人认为，该区域是微管蛋白和FtsZ共同的结构和进化核，不同于典型的GTP酶。
The far-ultraviolet circular dichroism spectrum of the alpha beta-tubulin dimer analyzed by six different methods indicates an average content of approximately 33% ct helix, 21% beta sheet, and 45% other secondary structure. Deconvolution of Fourier transform infrared spectra indicates 24% sheet, 37% (maximum) helix, and 38% (minimum) other structure. Separate alignments of 75 alpha-tubulin, 106 beta-tubulin, and 14 gamma-tubulin sequences and 12 sequences of the bacterial cell division protein FtsZ have been employed to predict their secondary structures with the multiple-sequence method PHD [Rest, B., & Sander, C. (1993a) J. Mel. Biol. 232, 584-599]. The predicted secondary structures average of 33% a helix, 24% beta sheet, and 43% loop for the alpha beta dimer. The predictions have been compared with sites of limited proteolysis by 12 proteases at the surfaces of the heterodimer and taxol-induced microtubules [de Pereda, J. M., & Andreu, J. M. (1996) Biochemistry, 35, 14184-14202]. From 24 experimentally determined nicking sites, 18 are at predicted loops or at the extremes of secondary structure elements. Proteolysis zone A (including acetylable Lys40 and probably Lys60 in alpha-tubulin and Gly93 in beta-tubulin) and proteolysis zone B (extending between residues 167 and 183 in both chains) are accessible in microtubules. Proteolysis zone C, between residues 278 and 295, becomes partially occluded in microtubules. The cl-tubulin nicking site Arg339-Ser340 is at a loop following a predicted or helix in proteolysis zone D. This site is protected in taxol microtubules; however, a new tryptic site appears which is probably located at the N-terminal end of the same helix. Zone D also contains beta-tubulin Cys354, which is accessible in microtubules. Proteolysis zone E includes the C-terminal hypervariable loops (10-20 residues) of each tubulin chain. These follow the two larger predicted helical zones (residues 372-395 and 405-432 in beta-tubulin), which also are the longer conserved part of the alpha- and beta-tubulin sequences. Through combination of this with other biochemical information, a set of surface and distance constraints is proposed for the folding of P-tubulin. The FtsZ sequences are only 10-18% identical to the tubulin sequences. However, the predicted secondary structures show two clearly similar (85-87 and 51-78%) regions, at tubulin positions 95-175 and 305-350, corresponding to FtsZ 65-135 and 255-300, respectively. The first region is flanked by tubulin proteolysis zones A and B. It consists of a predicted loop1-helix-loop2-sheet-loop3-helix-loop4-sheet fold, which contains the motif (KR)GXXXXG (loop1), and the tubulin-FtsZ signature G-box motif (SAG)GGTG(SAT)G (loop3). A simple working model envisages loop1 and loop3 together at the nucleotide binding site, while loops 2 and 4 are at the surface of the protein, in agreement with proteolytic and antigenic accessibility results in tubulin. The model is compatible with studies of tubulin and FtsZ mutants. It is proposed that this region constitutes a common structural and evolutionary nucleus of tubulins and FtsZ which is different from typical GTPases.