School of Biological Sciences, Nanyang Technological University, Singapore.
PLoS One. 2010 Feb 10;5(2):e9146. doi: 10.1371/journal.pone.0009146.
Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.
METHODOLOGY/PRINCIPAL FINDINGS: In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547), NBD94(566-663) and NBD94(674-793), respectively. Using fluorescence correlation spectroscopy NBD94(444-547) has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547) in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663) and NBD94(674-793) enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments.
These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.
疟原虫的裂殖子入侵红细胞（RBC）涉及大量的受体配体相互作用。网织红细胞结合蛋白同源家族（RH）在红细胞识别和毒力方面起着重要作用。最近，已经表明 RH 除了受体结合之外，还可能作为 ATP/ADP 传感器发挥作用。一个 94 kDa 的区域命名为核苷酸结合域 94（NBD94）的约氏疟原虫 YM，代表 RH 的假定核苷酸结合区域，已被证明能够选择性地结合 ATP 和 ADP。ATP 或 ADP 的结合诱导 NBD94 的 C 端铰链区的核苷酸依赖性结构变化，并直接影响 RH 对 RBC 的结合能力。
方法/主要发现：为了找到能够结合核苷酸的最小结构单元及其偶联模块，即铰链区，已经生成了 NBD94 的三个截断结构域，分别命名为 NBD94(444-547)、NBD94(566-663)和 NBD94(674-793)。使用荧光相关光谱法，已经确定 NBD94(444-547)形成了最小的核苷酸结合片段，对 ATP 和 ADP 敏感，这被 4-氯-7-硝基苯并呋咱抑制。从小角度 X 射线散射数据计算 NBD94(444-547)在溶液中的形状，揭示了一个由两个球状结构域组成的拉长分子，通过约 73.1 A 长的螺旋段连接。由于构建物的高质量，形成了铰链区、NBD94(566-663)和 NBD94(674-793)，分别能够确定第一个晶体学和溶液结构。NBD94(566-663)的晶体结构由两个长度分别为 97.8 A 和 48.6 A 的螺旋组成，通过一个环连接。相比之下，溶液中 NBD94(674-793)的低分辨率结构代表了一种具有三个结构段的椅子形状。
这些结构首次揭示了核苷酸结合如何影响 RH 的整体结构，并证明了该区域作为新型药物靶点的潜力。
