Department of Physics, University of Illinois, Urbana, Illinois 61801, USA.
Proteins. 2010 Apr;78(5):1153-62. doi: 10.1002/prot.22635.
BtuB is a beta-barrel membrane protein that facilitates transport of cobalamin (vitamin B12) from the extracellular medium across the outer membrane of Escherichia coli. It is thought that binding of B12 to BtuB alters the conformation of its periplasm-exposed N-terminal residues (the TonB box), which enables subsequent binding of a TonB protein and leads to eventual uptake of B12 into the cytoplasm. Structural studies determined the location of the B12 binding site at the top of the BtuB's beta-barrel, surrounded by extracellular loops. However, the structure of the loops was found to depend on the method used to obtain the protein crystals, which-among other factors-differed in calcium concentration. Experimentally, calcium concentration was found to modulate the binding of the B12 substrate to BtuB. In this study, we investigate the effect of calcium ions on the conformation of the extracellular loops of BtuB and their possible role in B12 binding. Using all-atom molecular dynamics, we simulate conformational fluctuations of several X-ray structures of BtuB in the presence and absence of calcium ions. These simulations demonstrate that calcium ions can stabilize the conformation of loops 3-4, 5-6, and 15-16, and thereby prevent occlusion of the binding site. Furthermore, binding of calcium ions to extracellular loops of BtuB was found to enhance correlated motions in the BtuB structure, which is expected to promote signal transduction. Finally, we characterize conformation dynamics of the TonB box in different X-ray structures and find an interesting correlation between the stability of the TonB box structure and calcium binding.
BtuB 是一种β桶膜蛋白，可促进钴胺素（维生素 B12）从细胞外介质穿过大肠杆菌外膜的运输。人们认为，B12 与 BtuB 的结合改变了其周质暴露的 N 端残基（TonB 盒）的构象，从而使随后与 TonB 蛋白结合，并最终将 B12 摄取到细胞质中。结构研究确定了 B12 结合位点位于 BtuB 的β桶顶部，周围是细胞外环。然而，发现环的结构取决于获得蛋白质晶体的方法，而这些方法除其他因素外，钙离子浓度也不同。实验发现，钙离子浓度可调节 B12 底物与 BtuB 的结合。在这项研究中，我们研究了钙离子对 BtuB 细胞外环构象的影响及其在 B12 结合中的可能作用。我们使用全原子分子动力学模拟了存在和不存在钙离子的几种 BtuB X 射线结构的构象波动。这些模拟表明，钙离子可以稳定环 3-4、5-6 和 15-16 的构象，从而防止结合位点被封闭。此外，发现钙离子与 BtuB 细胞外环的结合增强了 BtuB 结构中的相关运动，这有望促进信号转导。最后，我们对不同 X 射线结构中的 TonB 盒构象动力学进行了表征，并发现 TonB 盒结构的稳定性与钙离子结合之间存在有趣的相关性。
