Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry
Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry
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DOI：
10.1021/bi035422a
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发表时间：
2004-03-09
期刊：
影响因子：
2.9
通讯作者：
Shock, A
中科院分区：
文献类型：
--
作者：
Crump, MP;Ceska, TA;Shock, A
淋巴细胞功能相关抗原-1(LFA-1)在细胞间黏附、淋巴细胞迁移和活化中起作用，是一个有吸引力的抗炎药物靶点。LFA-1的α-亚基与其他几种整合素一样，有一个N末端插入的结构域(1-结构域)，与200个氨基酸相似，在调节与LFA-1的配体结合方面发挥着核心作用。在LFA-1的I-结构域中发现了一个额外的区域，称为I-结构域变构位点(IDAS)，并被证明可以调节该蛋白的功能。经N-15-H-1HSQC(异核单量子相干)核磁共振(NMRS)滴定实验分析，IDAS被小分子LFA-1抑制剂占据。我们报道了一种新的芳硫基抑制剂，用等温滴定量热法(ITC)测定，它与I-结构域结合，K-d为18.3 nm。这个值与生化竞争分析(DELFIA)得出的IC50(10.9 NM)非常一致，DELFIA衡量了整个LFA-1与其配体ICAM-1结合的抑制程度。在建立了芳硫基抑制剂对分离的I-结构域的强烈亲和力后，我们使用了一系列技术来进一步表征结合，包括ITC、核磁共振和X射线结晶学。我们首先开发了一种有效的ITC结合试验，用于低溶解度抑制剂，避免了基于ELISA的分析的需要。此外，我们利用了一种基于核磁共振的快速分析方法来生成I-结构域抑制物模型。这是基于收集LFA-1结合形式的HCCH-TOCSY光谱，并鉴定在LFA-1抑制剂结合时产生化学位移变化的侧链甲基的子集。这个子集用于二维C-13-N-15和N-15过滤和编辑的二维核磁共振实验，以分别鉴定最小的对准内和配体-蛋白质NOE集(核Overhauser增强)。通过与X-射线晶体结构的比较，对核磁共振数据中的模型进行了评估，证实了该方法正确地预测了结合配体的基本特征。
LFA-1 (lymphocyte function-associated antigen-1) plays a role in intercellular adhesion and lymphocyte trafficking and activation and is an attractive anti-inflammatory drug target. The a-subunit of LFA-1, in common with several other integrins, has an N-terminally inserted domain (1-domain) of similar to200 amino acids that plays a central role in regulating ligand binding to LFA-1. An additional region, termed the I-domain allosteric site (IDAS), has been identified exclusively within the LFA-1 I-domain and shown to regulate the function of this protein. The IDAS is occupied by small molecule LFA-1 inhibitors when cocrystallized or analyzed by N-15-H-1 HSQC (heteronuclear single-quantum coherence) NMR (nuclear magnetic resonance) titration experiments. We report here a novel arylthio inhibitor that binds the I-domain with a K-d of 18.3 nM as determined by isothermal titration calorimetry (ITC). This value is in close agreement with the IC50 (10.9 nM) derived from a biochemical competition assay (DELFIA) that measures the level of inhibition of binding of whole LFA-1 to its ligand, ICAM-1. Having established the strong affinity of the arylthio inhibitor for the isolated I-domain, we have used a range of techniques to further characterize the binding, including ITC, NMR, and X-ray crystallography. We have first developed an effective ITC binding assay for use with low-solubility inhibitors that avoids the need for ELISA-based assays. In addition, we utilized a fast NMR-based assay for the generation of I-domain-inhibitor models. This is based around the collection of HCCH-TOCSY spectra of LFA-1 in the bound form and the identification of a subset of side chain methyl groups that give chemical shift changes upon binding of LFA-1 inhibitors. This subset was used in two-dimensional C-13-N-15 and N-15-filtered and -edited two-dimensional NMR experiments to identify a minimal set of intraligand and ligand-protein NOES, respectively (nuclear Overhauser enhancements). Models from the NMR data were assessed by comparison to an X-ray crystallographic structure of the complex, confirming that the method correctly predicted the essential features of the bound ligand.