León Rafael, Murray Jill I, Cragg Gina, Farnell Benjamin, West Nathan R, Pace Tamara C S, Watson Peter H, Bohne Cornelia, Boulanger Martin J, Hof Fraser
Department of Chemistry, University of Victoria, P.O. Box 3065, Victoria, British Columbia V8W 3V6, Canada.
Biochemistry. 2009 Nov 10;48(44):10591-600. doi: 10.1021/bi901330g.
S100A7 (psoriasin) is a member of the S100 family of signaling proteins. It is implicated in and considered a therapeutic target for inflammation and cancer, yet no small molecule ligands for S100A7 have been identified. To begin the development of specific small molecule inhibitors of S100A7 function, we have used a series of surface binding fluorescent dyes to probe the surface hydrophobic sites. Two naphthalene-based dyes (2,6-ANS and 1,8-ANS) were found to bind S100A7 in a distinct cleft. We characterized the binding interaction by determining both the structure of S100A7 bound to 2,6-ANS and the structure of S100A7 bound to 1,8-ANS to 1.6 A. In both cases, two molecules of dye were docked such that the naphthalene groups were positioned in two symmetry-related grooves that are formed by the N-terminal helices of each monomer. We observed that Met12 acts as a gatekeeper to the binding cleft, adopting an "open" conformation for the more elongated 2,6-ANS while remaining in a "closed" conformation for the more compact 1,8-ANS. Steady-state fluorescence experiments revealed that S100A7 binds two copies of 2,6-ANS, each with a K(d) of 125 muM. Time-resolved fluorescence lifetime measurements indicated that the two molecules of 2,6-ANS bind in two independent binding sites with different fluorescence lifetimes, suggesting that the S100A7 homodimer is not perfectly symmetric in solution. Isothermal titration calorimetry studies demonstrate that S100A7 has a higher affinity for 2,6-ANS than 1,8-ANS. Yeast two-hybrid studies were also used to probe contributions of individual residues of an S100A7 triple mutant with respect to Jab1 binding. Mutation of Leu78, which forms part of the Met12 cleft occupied by 2,6-ANS, reduced the level of Jab1 binding, suggesting a potentially important role for the Met12 hydrophobic pocket in defining a Jab1 interface. Additional Y2H studies also delineate contributions of Gln88 and in particular Asp56 that shows the most significant abrogated binding to Jab1. Collectively, these data suggest a complex interaction between S100A7 and the much larger Jab1. These studies form the basis for the development of small molecule reporters and modifiers of S100A7 form and function.
S100A7（牛皮癣素）是信号蛋白S100家族的成员。它与炎症和癌症有关，被认为是炎症和癌症的治疗靶点，但尚未发现S100A7的小分子配体。为了开始开发S100A7功能的特异性小分子抑制剂，我们使用了一系列表面结合荧光染料来探测表面疏水位点。发现两种基于萘的染料（2,6-ANS和1,8-ANS）在一个独特的裂隙中与S100A7结合。我们通过确定与2,6-ANS结合的S100A7的结构以及与1,8-ANS结合至1.6埃的S100A7的结构来表征这种结合相互作用。在这两种情况下，两个染料分子对接，使得萘基团位于由每个单体的N端螺旋形成的两个对称相关的凹槽中。我们观察到Met12作为结合裂隙的守门人，对于更长的2,6-ANS采用“开放”构象，而对于更紧凑的1,8-ANS保持“封闭”构象。稳态荧光实验表明S100A7结合两个拷贝的2,6-ANS，每个的解离常数（K(d)）为125μM。时间分辨荧光寿命测量表明两个2,6-ANS分子在两个具有不同荧光寿命的独立结合位点结合，这表明S100A7同二聚体在溶液中并非完全对称。等温滴定量热法研究表明S100A7对2,6-ANS的亲和力高于1,8-ANS。酵母双杂交研究也用于探测S100A7三突变体的各个残基对Jab1结合的贡献。构成2,6-ANS占据的Met12裂隙一部分的Leu78突变降低了Jab1结合水平，表明Met12疏水口袋在定义Jab1界面中可能起重要作用。额外的酵母双杂交研究还描绘了Gln88特别是Asp56的贡献，Asp56显示出与Jab1的结合被最显著地消除。总体而言，这些数据表明S100A7与大得多的Jab1之间存在复杂的相互作用。这些研究为开发S100A7形式和功能的小分子报告物和调节剂奠定了基础。
