Scholz C, Mücke M, Rape M, Pecht A, Pahl A, Bang H, Schmid F X
Biochemisches Laboratorium, Universität Bayreuth, Bayreuth, D-95440, Germany.
J Mol Biol. 1998 Apr 3;277(3):723-32. doi: 10.1006/jmbi.1997.1604.
The trigger factor is associated with bacterial ribosomes and catalyzes proline-limited protein folding reactions. Its folding activity is very high and conserved in evolution, as shown for the homologous enzymes from Escherichia coli and Mycoplasma genitalium. The folding protein substrate (a variant of ribonuclease T1) binds with high affinity to the trigger factors, and permanently unfolded proteins are strong, competitive inhibitors. We used this inhibition to characterize the substrate binding sites of the trigger factors. Unfolded alpha-lactalbumin binds very tightly and inhibits the trigger factor from M. genitalium with a KI value of 50 nM. The binding of inhibitory proteins is independent of proline residues, as shown for unfolded tendamistat, which binds to the trigger factor with equal affinity in the presence and in the absence of its three proline residues. The good inhibition by a non-folding variant of ribonuclease T1 that lacks Pro39 showed that this proline, at which the catalysis of folding occurs, is dispensable for substrate binding. The trigger factors cannot catalyze prolyl isomerization when proteins are partially folded already. They preferentially recognize unstructured protein chains, which bind with high affinity to a site distinct from the catalytic prolyl isomerase center in the FKBP domain.
触发因子与细菌核糖体相关联，并催化脯氨酸受限的蛋白质折叠反应。其折叠活性非常高且在进化过程中保守，如大肠杆菌和生殖支原体的同源酶所示。折叠的蛋白质底物（核糖核酸酶T1的变体）与触发因子具有高亲和力结合，而永久未折叠的蛋白质是强竞争性抑制剂。我们利用这种抑制作用来表征触发因子的底物结合位点。未折叠的α-乳白蛋白结合非常紧密，以50 nM的KI值抑制生殖支原体的触发因子。抑制性蛋白质的结合与脯氨酸残基无关，如未折叠的抑肽酶所示，它在存在和不存在其三个脯氨酸残基的情况下以相同亲和力结合触发因子。缺乏Pro39的核糖核酸酶T1非折叠变体的良好抑制作用表明，发生折叠催化的这个脯氨酸对于底物结合是可有可无的。当蛋白质已经部分折叠时，触发因子不能催化脯氨酰异构化。它们优先识别非结构化的蛋白质链，这些链以高亲和力结合到FKBP结构域中与催化脯氨酰异构酶中心不同的位点。
