Freitag S, Le Trong I, Chilkoti A, Klumb L A, Stayton P S, Stenkamp R E
Department of Biological Structure, University of Washington, Seattle 98195-7420, USA.
J Mol Biol. 1998 May 29;279(1):211-21. doi: 10.1006/jmbi.1998.1735.
Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka approximately 10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.
先前的热力学和计算研究表明，芳香族相互作用在生成抗生物素蛋白-生物素结合的非凡结合自由能中起着重要的能量作用。我们在此报告了单一位点色氨酸突变体的晶体学特征，以研究这些芳香族相互作用改变的结构后果。四个色氨酸残基，即Trp79、Trp92、Trp108和Trp120，在疏水结合作用中发挥重要作用，它们与氢键网络和一个灵活的结合环共同导致紧密的配体结合（解离常数Ka约为10¹³ M⁻¹）。测定了无配体和生物素结合的突变体W79F、W108F、W120F和W120A在1.9至2.3 Å分辨率范围内的晶体结构。收集了这四个不同突变体的九个数据集，并将结构模型精修至R值范围为0.15至0.20。这里解决的主要问题是这些突变如何影响抗生物素蛋白的结合位点，特别是它们如何影响复合物中生物素的结合模式。在任何色氨酸突变体中，抗生物素蛋白的整体折叠都没有明显改变。除了一个例外，在未结合结构中仅观察到微小偏差。在未结合的W79F的一种晶体形式中，Phe29和Tyr43的侧链向突变位点有一个耦合位移，尽管在不同的晶体形式中未观察到这些位移。在结合结构中，突变体复合物中生物素在结合口袋中的取向没有明显改变。与野生型抗生物素蛋白-生物素复合物相比，在结合口袋中未观察到任何突变体有额外的晶体学水分子。因此，这些结构研究表明，热力学改变可归因于结合残基组成的局部改变，而不是结合位点结构的重排。
