Xu Yi, Keene Douglas R, Bujnicki Janusz M, Höök Magnus, Lukomski Slawomir
Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX 77030, USA.
J Biol Chem. 2002 Jul 26;277(30):27312-8. doi: 10.1074/jbc.M201163200. Epub 2002 Apr 25.
The collagens are a family of animal proteins containing segments of repeated Gly-Xaa-Yaa (GXY) motifs that form a characteristic triple-helical structure. Genes encoding proteins with repeated GXY motifs have also been reported in bacteria and phages; however, it is unclear whether these prokaryotic proteins can form a collagen-like triple-helical structure. Here we used two recently identified streptococcal proteins, Scl1 and Scl2, containing extended GXY sequence repeats as model proteins. First we observed that prior to heat denaturation recombinant Scl proteins migrated as homotrimers in gel electrophoresis with and without SDS. We next showed that the collagen-like domain of Scl is resistant to proteolysis by trypsin. We further showed that circular dichroism spectra of the Scl proteins contained features characteristic of collagen triple helices, including a positive maximum of ellipticity at 220 nm. Furthermore the triple helices of Scl1 and Scl2 showed a temperature-dependent unfolding with melting temperatures of 36.4 and 37.6 degrees C, respectively, which resembles those seen for collagens. We finally demonstrated by electron microscopy that the Scl proteins are organized into "lollipop-like" structures, similar to those seen in human proteins with collagenous domains. This implies that the repeated GXY tripeptide motif is a structural indicator of collagen-like triple helices in proteins from such phylogenetically distant sources as bacteria and humans.
胶原蛋白是一类动物蛋白，其包含重复的Gly-Xaa-Yaa（GXY）基序片段，这些片段形成特征性的三螺旋结构。在细菌和噬菌体中也报道了编码具有重复GXY基序的蛋白质的基因；然而，尚不清楚这些原核生物蛋白质是否能形成类似胶原蛋白的三螺旋结构。在此，我们使用最近鉴定出的两种链球菌蛋白Scl1和Scl2，它们含有延长的GXY序列重复，作为模型蛋白。首先，我们观察到在热变性之前，重组Scl蛋白在有无SDS的凝胶电泳中均以同三聚体形式迁移。接下来，我们表明Scl的类胶原结构域对胰蛋白酶的蛋白水解具有抗性。我们进一步表明，Scl蛋白的圆二色光谱包含胶原三螺旋的特征，包括在220nm处椭圆率的正最大值。此外，Scl1和Scl2的三螺旋显示出温度依赖性解折叠，解链温度分别为36.4和37.6摄氏度，这与胶原蛋白的情况相似。我们最终通过电子显微镜证明，Scl蛋白被组织成“棒棒糖样”结构，类似于在具有胶原结构域的人类蛋白中看到的结构。这意味着重复的GXY三肽基序是来自细菌和人类等系统发育距离遥远的来源的蛋白质中类似胶原三螺旋的结构指标。
