Menéndez M, Gasset M, Laynez J, López-Zumel C, Usobiaga P, Töpfer-Petersen E, Calvete J J
Instituto de Química-Física Rocasolano CSIC, Madrid, Spain.
Eur J Biochem. 1995 Dec 15;234(3):887-96. doi: 10.1111/j.1432-1033.1995.887_a.x.
The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel beta-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5 degrees C and 78.6 degrees C, respectively. The calorimetric enthalpy changes (delta Hcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that beta-structure and turns are the major structural element of both PSP-I/PSP-II (53% of beta-sheet, 21% of turns) and aSFP (44% of beta-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, alpha-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-beta-structure proteins, and would support a beta-barrel topology like that predicted for the CUB domain. Other beta-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.
CUB结构域是一种广泛存在的由110个氨基酸组成的模块，存在于功能多样、通常受发育调控的蛋白质中，据预测其反平行β桶拓扑结构与免疫球蛋白V结构域中的类似。精黏附素被认为是由单个CUB结构域架构构成的该蛋白家族的一个亚组。为了验证所提出的结构模型，我们使用差示扫描量热法、远紫外圆二色光谱法和傅里叶变换红外光谱法分析了精黏附素蛋白家族的两个成员，即猪精浆蛋白I/II（PSP-I/PSP-II）异二聚体和牛酸性精液蛋白（aSFP）同二聚体的结构组织。PSP-I/PSP-II和aSFP的热解折叠是不可逆的，且遵循一步过程，其转变温度（Tm）分别为60.5℃和78.6℃。PSP-I/PSP-II热变性的量热焓变（ΔHcat）为439kJ/mol，aSFP二聚体为660kJ/mol。对PSP-I/PSP-II量热曲线的分析表明，整个二聚体构成协同解折叠单元。傅里叶变换红外光谱法以及使用凸约束分析对圆二色光谱进行去卷积表明，β结构和转角是PSP-I/PSP-II（β折叠占53%，转角占21%）和aSFP（β折叠占44%，转角占36%）的主要结构元件，并且猪和牛的蛋白质几乎不含有α螺旋结构（如果有的话）。综上所述，我们的结果表明，猪和牛的精黏附素分子可能都是全β结构蛋白，并且支持类似为CUB结构域预测的β桶拓扑结构。其他β结构折叠，如许多碳水化合物结合蛋白结构域特有的希腊钥匙模体，也不能被排除。最后，使用相同的生物物理技术组合来表征PSP-I/PSP-II和aSFP热变性形式的残余二级结构，并强调这些形式的聚集倾向。
