Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.
Department of Biochemistry, University of Bayreuth, Germany.
FEBS J. 2023 Sep;290(18):4496-4512. doi: 10.1111/febs.16819. Epub 2023 May 29.
Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.
基质结合蛋白（SBPs）被来自生命三个域的生物体用于运输和信号传递。SBPs 由两个共同捕获高亲和力和选择性配体的结构域组成。为了探索结构域的作用以及它们之间铰链区的完整性在 SBPs 功能和构象中的作用，我们在这里描述了来自沙门氏菌的赖氨酸精氨酸鸟氨酸（LAO）结合蛋白及其两个独立结构域的配体结合、构象稳定性和折叠动力学。LAO 是由连续和不连续结构域组成的 II 类 SBP。与基于它们的连接性预期的行为相反，不连续结构域显示出稳定的类似天然的结构，可适度结合 l-精氨酸，而连续结构域几乎不稳定，且未检测到配体结合。关于折叠动力学，对整个蛋白质的研究揭示了至少存在两种中间体。虽然连续结构域的展开和重折叠只显示出一个中间体，且动力学比 LAO 更简单和更快，但不连续结构域的折叠机制复杂且涉及多个中间体。这些发现表明，在完整的蛋白质中，连续结构域可引发折叠，并且其存在可引导不连续结构域的折叠，避免非生产性相互作用。功能、稳定性和折叠途径的结构域强烈依赖于它们的共价缔合，这很可能是两个结构域作为单个单元共同进化的结果。
