Hopfner K P, Kopetzki E, Kresse G B, Bode W, Huber R, Engh R A
Abteilung Strukturforschung, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.
Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9813-8. doi: 10.1073/pnas.95.17.9813.
Protein functions have evolved in part via domain recombination events. Such events, for example, recombine structurally independent functional domains and shuffle targeting, regulatory, and/or catalytic functions. Domain recombination, however, can generate new functions, as implied by the observation of catalytic sites at interfaces of distinct folding domains. If useful to an evolving organism, such initially rudimentary functions would likely acquire greater efficiency and diversity, whereas the initially distinct folding domains would likely develop into single functional domains. This represents the probable evolution of the S1 serine protease family, whose two homologous beta-barrel subdomains assemble to form the binding sites and the catalytic machinery. Among S1 family members, the contact interface and catalytic residues are highly conserved whereas surrounding surfaces are highly variable. This observation suggests a new strategy to engineer viable proteins with novel properties, by swapping folding subdomains chosen from among protein family members. Such hybrid proteins would retain properties conserved throughout the family, including folding stability as single domain proteins, while providing new surfaces amenable to directed evolution or engineering of specific new properties. We show here that recombining the N-terminal subdomain from coagulation factor X with the C-terminal subdomain from trypsin creates a potent enzyme (fXYa) with novel properties, in particular a broad substrate specificity. As shown by the 2.15-A crystal structure, plasticity at the hydrophobic subdomain interface maintains activity, while surface loops are displaced compared with the parent subdomains. fXYa thus represents a new serine proteinase lineage with hybrid fX, trypsin, and novel properties.
蛋白质功能部分是通过结构域重组事件进化而来的。例如，此类事件会重组结构上独立的功能结构域，并改变靶向、调节和/或催化功能。然而，结构域重组能够产生新功能，这一点已从不同折叠结构域界面处存在催化位点的观察结果中得到暗示。对于不断进化的生物体而言，如果这些最初的基本功能有用，它们可能会获得更高的效率和多样性，而最初不同的折叠结构域可能会发展成单一功能结构域。这代表了S1丝氨酸蛋白酶家族可能的进化过程，该家族的两个同源β-桶状亚结构域组装形成结合位点和催化机制。在S1家族成员中，接触界面和催化残基高度保守，而周围表面则高度可变。这一观察结果提示了一种设计具有新特性的可行蛋白质的新策略，即通过交换从蛋白质家族成员中选择的折叠亚结构域来实现。此类杂合蛋白将保留整个家族保守的特性，包括作为单结构域蛋白的折叠稳定性，同时提供适合定向进化或特定新特性工程改造的新表面。我们在此表明，将凝血因子X的N端亚结构域与胰蛋白酶的C端亚结构域重组，可产生一种具有新特性的强效酶（fXYa），特别是具有广泛的底物特异性。如2.15埃的晶体结构所示，疏水亚结构域界面处的可塑性维持了活性，而与亲本亚结构域相比，表面环发生了位移。因此，fXYa代表了一个具有fX、胰蛋白酶杂合特性和新特性的新丝氨酸蛋白酶谱系。
