Sharma Deepak, Balamurali M M, Chakraborty Kausik, Kumaran Sowmini, Jeganathan Sadasivam, Rashid Umar, Ingallinella Paolo, Varadarajan Raghavan
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Biochemistry. 2005 Dec 13;44(49):16192-202. doi: 10.1021/bi051120s.
CD4 is an important component of the immune system and is also the cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. Constructs consisting of all four extracellular domains of human CD4 as well as the first two domains (CD4D12) have previously been expressed and characterized. All of the gp120-binding residues are located within the first N-terminal domain (D1) of CD4. To date, it has not been possible to obtain domain D1 alone in a soluble and active form. Most residues in CD4 that interact with gp120 lie within the region 21-64 of domain D1 of CD4. On the basis of these observations and analysis of the crystal structure of CD4D12, a mutational strategy was designed to express CD4D1 and region 21-64 of CD4 (CD4PEP1) in Escherichia coli. K(D) values for the binding of CD4 analogues described above to gp120 were measured using a Biacore-based solution-phase competition binding assay. Measured K(D) values were 15 nM, 40 nM, and 26 microM for CD4D12, CD4D1, and CD4PEP1, respectively. All of the proteins interact with gp120 and are able to expose the 17b-binding epitope of gp120. Structural content was determined using CD and proteolysis. Both CD4D1 and CD4PEP1 were partially structured and showed an enhanced structure in the presence of the osmolyte sarcosine. The aggregation behavior of all of the proteins was characterized. While CD4D1 and CD4PEP1 did not aggregate, CD4D12 formed amyloid fibrils at neutral pH within a week at 278 K. These CD4 derivatives should be useful tools in HIV vaccine design and entry inhibition studies.
CD4是免疫系统的重要组成部分，也是HIV-1的细胞受体。CD4由一个胞质尾、一个跨膜区和四个细胞外结构域D1-D4组成。由人CD4的所有四个细胞外结构域以及前两个结构域（CD4D12）组成的构建体先前已被表达和表征。所有与gp120结合的残基都位于CD4的第一个N端结构域（D1）内。迄今为止，还无法以可溶且有活性的形式单独获得结构域D1。CD4中与gp120相互作用的大多数残基位于CD4结构域D1的21-64区域内。基于这些观察结果以及对CD4D12晶体结构的分析，设计了一种突变策略，以在大肠杆菌中表达CD4D1和CD4的21-64区域（CD4PEP1）。使用基于Biacore的溶液相竞争结合测定法测量上述CD4类似物与gp120结合的K(D)值。CD4D12、CD4D1和CD4PEP1的测量K(D)值分别为15 nM、40 nM和26 μM。所有这些蛋白质都与gp120相互作用，并能够暴露gp120的17b结合表位。使用圆二色光谱（CD）和蛋白水解确定结构内容。CD4D1和CD4PEP1均部分结构化，并且在渗透剂肌氨酸存在下显示出增强的结构。对所有蛋白质的聚集行为进行了表征。虽然CD4D1和CD4PEP1不聚集，但CD4D12在278 K下一周内在中性pH下形成淀粉样纤维。这些CD4衍生物应成为HIV疫苗设计和进入抑制研究中的有用工具。
