Kannan N, Chander P, Ghosh P, Vishveshwara S, Chatterji D
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Protein Sci. 2001 Jan;10(1):46-54. doi: 10.1110/ps.26201.
The formation of alpha(2) dimer in Escherichia coli core RNA polymerase (RNAP) is thought to be the first step toward the assembly of the functional enzyme. A large number of evidences indicate that the alpha-subunit dimerizes through its N-terminal domain (NTD). The crystal structures of the alpha-subunit NTD and that of a homologous Thermus aquaticus core RNAP are known. To identify the stabilizing interactions in the dimer interface of the alpha-NTD of E. coli RNAP, we identified side-chain clusters by using the crystal structure coordinates of E. coli alpha-NTD. A graph spectral algorithm was used to identify side-chain clusters. This algorithm considers the global nonbonded side-chain interactions of the residues for the clustering procedure and is unique in identifying residues that make the largest number of interactions among the residues that form clusters in a very quantitative way. By using this algorithm, a nine-residue cluster consisting of polar and hydrophobic residues was identified in the subunit interface adjacent to the hydrophobic core. The residues forming the cluster are relatively rigid regions of the interface, as measured by the thermal factors of the residues. Most of the cluster residues in the E. coli enzyme were topologically and sequentially conserved in the T. aquaticus RNAP crystal structure. Residues 35F and 46I were predicted to be important in the stability of the alpha-dimer interface, with 35F forming the center of the cluster. The predictions were tested by isolating single-point mutants alpha-F35A and alpha-I46S on the dimer interface, which were found to disrupt dimerization. Thus, the identified cluster at the edge of the dimer interface seems to be a vital component in stabilizing the alpha-NTD.
在大肠杆菌核心RNA聚合酶（RNAP）中，α(2)二聚体的形成被认为是功能性酶组装的第一步。大量证据表明，α亚基通过其N端结构域（NTD）形成二聚体。已知α亚基NTD以及同源嗜热栖热菌核心RNAP的晶体结构。为了确定大肠杆菌RNAP的α-NTD二聚体界面中的稳定相互作用，我们利用大肠杆菌α-NTD的晶体结构坐标确定了侧链簇。使用一种图谱算法来识别侧链簇。该算法在聚类过程中考虑了残基的全局非键合侧链相互作用，并且在以非常定量的方式识别在形成簇的残基之间产生最多相互作用的残基方面是独特的。通过使用该算法，在与疏水核心相邻的亚基界面中鉴定出一个由极性和疏水残基组成的九个残基的簇。通过残基的热因子测量，形成该簇的残基是界面中相对刚性的区域。大肠杆菌酶中的大多数簇残基在嗜热栖热菌RNAP晶体结构中在拓扑和序列上是保守的。预测残基35F和46I在α二聚体界面的稳定性中很重要，其中35F形成簇的中心。通过分离二聚体界面上的单点突变体α-F35A和α-I46S对这些预测进行了测试，发现它们破坏了二聚化。因此，在二聚体界面边缘鉴定出的簇似乎是稳定α-NTD的关键成分。
