Sassoon J, Lilie H, Baumann U, Kohli J
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, Berne, 3012, Switzerland.
J Mol Biol. 2001 Jun 22;309(5):1101-15. doi: 10.1006/jmbi.2001.4723.
Cmb1, a novel HMG box protein from Schizosaccharomyces pombe, has been characterized biochemically using glutaraldehyde cross-linking, gel-filtration and analytical ultracentrifugation. It was identified as a monomeric, non-spherical protein, with a tendency to aggregate in solution. Limited proteolysis with trypsin and chymotrypsin showed that the C-terminal HMG box was a compact, proteolytically stable domain and the N-terminal region of Cmb1 was relatively unstructured and more easily digested. As Cmb1 was previously identified as a potential mismatch-binding protein, the binding constants and stoichiometry for both homoduplex and heteroduplex DNA were determined using an IASys resonant mirror biosensor. Cmb1 indeed demonstrated a tighter association with mismatched DNA, especially with the C/Delta-mismatch. Expression constructs of Cmb1 were made to study the sections of the protein involved in DNA binding. Constructs with the N-terminal region absent revealed that the C-terminal HMG box was the primary DNA-binding region. The presence of the N-terminal region did, however, facilitate tighter binding to both homoduplex and heteroduplex DNA. The amino acid residues isoleucine 14 and leucine 39 were located as putative intercalating residues using structure guided homology modelling. The model templates were derived from two distinct HMG:DNA complexes: HMG-D bound to homoduplex DNA and HMG 1 bound to cisplatin DNA. Binding studies using the Cmb1 HMG box with point mutations in these residues showed that isoleucine 14 was important for the binding of Cmb1 to homoduplex DNA, but affected binding to mismatches to a lesser extent. In contrast, leucine 39 appeared to have a more significant function in binding to mismatched DNA.
Cmb1是一种来自粟酒裂殖酵母的新型HMG盒蛋白，已通过戊二醛交联、凝胶过滤和分析超速离心对其进行了生化特性分析。它被鉴定为单体非球形蛋白，在溶液中有聚集倾向。用胰蛋白酶和糜蛋白酶进行的有限蛋白水解表明，C端HMG盒是一个紧密的、蛋白水解稳定的结构域，而Cmb1的N端区域相对无结构且更容易被消化。由于Cmb1先前被鉴定为潜在的错配结合蛋白，因此使用IASys共振镜生物传感器测定了同型双链和异型双链DNA的结合常数和化学计量。Cmb1确实与错配DNA表现出更强的结合，尤其是与C/Δ错配。构建了Cmb1的表达构建体以研究该蛋白中参与DNA结合的部分。缺失N端区域的构建体表明，C端HMG盒是主要的DNA结合区域。然而，N端区域的存在确实促进了与同型双链和异型双链DNA的更紧密结合。使用结构导向同源建模将异亮氨酸14和亮氨酸39定位为假定的嵌入残基。模型模板源自两种不同的HMG:DNA复合物：与同型双链DNA结合的HMG-D和与顺铂DNA结合的HMG 1。使用在这些残基处具有点突变的Cmb1 HMG盒进行的结合研究表明，异亮氨酸14对于Cmb1与同型双链DNA的结合很重要，但对与错配的结合影响较小。相比之下，亮氨酸39在与错配DNA的结合中似乎具有更重要的功能。
