Hagan Nathan A, Fabris Daniele
University of Maryland Baltimore County, Department of Chemistry and Biochemistry, 1000 Hilltop Circle, Baltimore, MD 21228, USA.
J Mol Biol. 2007 Jan 12;365(2):396-410. doi: 10.1016/j.jmb.2006.09.081. Epub 2006 Oct 3.
The specific binding of HIV-1 nucleocapsid protein (NC) to the different forms assumed in vitro by the stemloop 1 (Lai variant) of the genome's packaging signal has been investigated using electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The simultaneous observation of protein-RNA and RNA-RNA interactions in solution has provided direct information about the role of NC in the two-step model of RNA dimerization and isomerization. In particular, two distinct binding sites have been identified on the monomeric stemloop structure, corresponding to the apical loop and stem-bulge motifs. These sites share similar binding affinities that are intermediate between those of stemloop 3 (SL3) and the putative stemloop 4 (SL4) of the packaging signal. Binding to the apical loop, which contains the dimerization initiation site (DIS), competes directly with the annealing of self-complementary sequences to form a metastable kissing-loop (KL) dimer. In contrast, binding to the stem-bulge affects indirectly the monomer-dimer equilibrium by promoting the rearrangement of KL into the more stable extended duplex (ED) conformer. This process is mediated by the duplex-melting activity of NC, which destabilizes the intramolecular base-pairs surrounding the KL stem-bulges and enables their exchange to form the inter-strand pairs that define the ED structure. In this conformer, high-affinity binding takes place at stem-bulge sites that are identical to those present in the monomeric and KL forms. In this case, however, the NC-induced "breathing" does not result in dissociation of the double-stranded structure because of the large number of intermolecular base-pairs. The different binding modes manifested by conformer-specific mutants have shown that NC can also provide low affinity interactions with the bulged-out adenine bases flanking the DIS region of the ED conformer, thus supporting the hypothesis that these exposed nucleotides may constitute "base-grips" for protein contacts during the late stages of the viral lifecycle.
利用电喷雾电离-傅里叶变换质谱（ESI-FTMS）研究了HIV-1核衣壳蛋白（NC）与基因组包装信号的茎环1（赖氏变体）在体外呈现的不同形式之间的特异性结合。对溶液中蛋白质-RNA和RNA-RNA相互作用的同时观察提供了关于NC在RNA二聚化和异构化两步模型中作用的直接信息。特别是，在单体茎环结构上确定了两个不同的结合位点，分别对应于顶端环和茎-凸起基序。这些位点具有相似的结合亲和力，介于包装信号的茎环3（SL3）和假定的茎环4（SL4）之间。与包含二聚化起始位点（DIS）的顶端环结合，直接与自互补序列的退火竞争，以形成亚稳的亲吻环（KL）二聚体。相反，与茎-凸起结合通过促进KL重排为更稳定的延伸双链体（ED）构象间接影响单体-二聚体平衡。这个过程由NC的双链解链活性介导，它使围绕KL茎-凸起的分子内碱基对不稳定，并使其交换形成定义ED结构的链间碱基对。在这种构象中，高亲和力结合发生在与单体和KL形式中相同的茎-凸起位点。然而，在这种情况下，由于大量分子间碱基对的存在，NC诱导的“呼吸”不会导致双链结构的解离。构象特异性突变体表现出的不同结合模式表明，NC还可以与ED构象DIS区域侧翼的凸起腺嘌呤碱基提供低亲和力相互作用，从而支持了这些暴露的核苷酸可能在病毒生命周期后期构成蛋白质接触的“碱基抓手”这一假设。
