Katz B A
Arris Pharmaceutical Corporation, 385 Oyster Point Boulevard, South San Francisco, CA 94080, USA.
J Mol Biol. 1997 Dec 19;274(5):776-800. doi: 10.1006/jmbi.1997.1444.
The remarkable stability of the streptavidin tetramer towards subunit dissociation becomes even greater upon binding of biotin. At two equivalent extensive monomer-monomer interfaces, monomers tightly associate into dimers that in turn associate into the tetramer at a less extensive dimer-dimer interface. To probe the structural basis for the enhancement of the stability of streptavidin by biotin, the crystal structures of apostreptavidin and its complexes with biotin and other small molecule and cyclic peptide ligands were determined and compared at resolutions as high as 1.36 A over a range of pH values from as low as 1.39. At low pH dramatic changes occur in the conformation and intersubunit hydrogen bonds involving the loop comprising Asp61 to Ser69. The hydrogen-bonded salt bridge between Asp61 Odelta2 and His87 Ndelta1, observed at higher pH, is replaced with a strong hydrogen bond between Asp61 Odelta1 and Asn85 Odelta1. Through crystallography at multiple pH values, the pH where this conformational change occurs, and thus the pKa of Asp61, was determined in crystals of space group I222 and/or I4122 of apostreptavidin and complexes. A range in pKa values for Asp61 was observed in these structures, the lowest being 1.78+/-0.19 for I222 streptavidin-biotin in 2.9 M (NH4)2SO4. At low pH the decrease in pKa of Asp61 and preservation of the intersubunit Asp61 Odelta2-Ndelta1 His87 hydrogen-bonded salt bridge in streptavidin-biotin versus apostreptavidin or streptavidin-peptide complexes is associated with an ordering of the flexible flap comprising residues Ala46 to Glu51, that in turn orders the Arg84 side-chain of a neighboring loop through resulting hydrogen bonds. Ordering of Arg84 in close proximity to the strong intersubunit interface appears to stabilize the conformation associated with the Asp61 Odelta2-Ndelta1 His87 hydrogen-bonded salt bridge. Thus, in addition to the established role of biotin in tetramer stabilization by direct mediation of intersubunit interactions at the weak interface through contact with Trp120, biotin may enhance tetramer stability at the strong interface more indirectly by ordering loop residues.
抗生物素蛋白四聚体对亚基解离具有显著的稳定性，在结合生物素后这种稳定性会变得更强。在两个相当大的单体 - 单体界面处，单体紧密结合形成二聚体，二聚体再在一个不太广泛的二聚体 - 二聚体界面处结合形成四聚体。为了探究生物素增强抗生物素蛋白稳定性的结构基础，测定并比较了脱生物素抗生物素蛋白及其与生物素、其他小分子和环肽配体形成的复合物的晶体结构，分辨率高达(1.36)埃，(pH)值范围低至(1.39)。在低(pH)值下，涉及从Asp61到Ser69的环的构象和亚基间氢键会发生显著变化。在较高(pH)值下观察到的Asp61 Oδ2与His87 Nδ1之间的氢键盐桥，在低(pH)值时被Asp61 Oδ1与Asn85 Oδ1之间的强氢键所取代。通过在多个(pH)值下进行晶体学研究，确定了脱生物素抗生物素蛋白及其复合物在空间群I222和/或I4122晶体中发生这种构象变化的(pH)值，从而确定了Asp61的(pKa)值。在这些结构中观察到Asp61的(pKa)值存在一定范围，在(2.9 M (NH4)2SO4)中的I222抗生物素蛋白 - 生物素复合物中，最低为(1.78 ± 0.19)。在低(pH)值下，抗生物素蛋白 - 生物素复合物中Asp61的(pKa)值降低，以及亚基间Asp61 Oδ2 - Nδ1 His87氢键盐桥相对于脱生物素抗生物素蛋白或抗生物素蛋白 - 肽复合物得以保留，这与包含Ala46至Glu51残基的柔性侧翼的有序化有关，进而通过形成的氢键使相邻环的Arg84侧链有序化。Arg84在靠近强亚基间界面处的有序化似乎稳定了与Asp61 Oδ2 - Nδ1 His87氢键盐桥相关的构象。因此，除了生物素通过与Trp120接触在弱界面处直接介导亚基间相互作用来稳定四聚体这一既定作用外，生物素可能通过使环残基有序化更间接地增强强界面处的四聚体稳定性。
