BioCeV - Institute of Microbiology, The Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague, Czech Republic.
BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France; IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
Biochim Biophys Acta Gen Subj. 2017 Feb;1861(2):157-167. doi: 10.1016/j.bbagen.2016.11.016. Epub 2016 Nov 13.
Cellobiose dehydrogenase (CDH) is a fungal extracellular oxidoreductase which fuels lytic polysaccharide monooxygenase with electrons during cellulose degradation. Interdomain electron transfer between the flavin and cytochrome domain in CDH, preceding the electron flow to lytic polysaccharide monooxygenase, is known to be pH dependent, but the exact mechanism of this regulation has not been experimentally proven so far.
To investigate the structural aspects underlying the domain interaction in CDH, hydrogen/deuterium exchange (HDX-MS) with improved proteolytic setup (combination of nepenthesin-1 with rhizopuspepsin), native mass spectrometry with ion mobility and electrostatics calculations were used.
HDX-MS revealed pH-dependent changes in solvent accessibility and hydrogen bonding at the interdomain interface. Electrostatics calculations identified these differences to result from charge neutralization by protonation and together with ion mobility pointed at higher electrostatic repulsion between CDH domains at neutral pH. In addition, we uncovered extensive O-glycosylation in the linker region and identified the long-unknown exact cleavage point in papain-mediated domain separation.
Transition of CDH between its inactive (open) and interdomain electron transfer-capable (closed) state is shown to be governed by changes in the protein surface electrostatics at the domain interface. Our study confirms that the interdomain electrostatic repulsion is the key factor modulating the functioning of CDH.
The results presented in this paper provide experimental evidence for the role of charge repulsion in the interdomain electron transfer in cellobiose dehydrogenases, which is relevant for exploiting their biotechnological potential in biosensors and biofuel cells.
纤维二糖脱氢酶（CDH）是一种真菌细胞外氧化还原酶，在纤维素降解过程中为溶细胞多糖单加氧酶提供电子。已知 CDH 中黄素和细胞色素结构域之间的域间电子转移，在电子流向溶细胞多糖单加氧酶之前，依赖于 pH，但迄今为止，这种调节的确切机制尚未通过实验证明。
为了研究 CDH 中结构域相互作用的结构基础，使用了改进的蛋白水解设置（组合使用 Nepenthesin-1 和 Rhizopuspepsin）的氢/氘交换（HDX-MS）、与离子淌度和静电计算相结合的天然质谱。
HDX-MS 揭示了在域间界面处溶剂可及性和氢键的 pH 依赖性变化。静电计算表明，这些差异是由质子化引起的电荷中和引起的，与离子淌度一起，表明在中性 pH 下 CDH 结构域之间存在更高的静电排斥。此外，我们在连接区发现了广泛的 O-糖基化，并确定了在木瓜蛋白酶介导的结构域分离中一直未知的确切切割点。
CDH 从其非活性（打开）状态到具有域间电子转移能力的（关闭）状态的转变被证明受结构域界面处蛋白质表面静电的变化控制。我们的研究证实，结构域间静电排斥是调节 CDH 功能的关键因素。
本文介绍的结果为电荷排斥在纤维二糖脱氢酶的域间电子转移中的作用提供了实验证据，这对于利用其在生物传感器和生物燃料电池中的生物技术潜力具有重要意义。
