Sequence dependence of BNIP3 transmembrane domain dimerization implicates side-chain hydrogen bonding and a tandem GxxxG motif in specific helix-helix interactions.
Sequence dependence of BNIP3 transmembrane domain dimerization implicates side-chain hydrogen bonding and a tandem GxxxG motif in specific helix-helix interactions.
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DOI：
10.1016/j.jmb.2006.09.065
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发表时间：
2006-12
影响因子：
5.6
通讯作者：
E. Sulistijo;K. MacKenzie
中科院分区：
文献类型：
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作者：
E. Sulistijo;K. MacKenzie
促凋亡蛋白 BNIP3 的跨膜结构域在膜和去污剂中具有很强的自关联性。我们使用定点诱变来分析 BNIP3 跨膜结构域二聚化的序列依赖性，从中我们推断出该系统中强且特异的螺旋-螺旋相互作用的物理基础。疏水取代确定了六个对二聚化至关重要的残基，敏感残基的模式表明 BNIP3 螺旋以右手交叉角相互作用。基于单点突变体的二聚化倾向，我们提出：极性残基His173和Ser172通过其侧链彼此形成单体间氢键； Ala176、Gly180 和 Gly184 形成串联 GxxxG 基序，允许螺旋靠近； Ile183 产生单体间范德华接触。由于串联 GxxxG 基序和氢键对都不足以驱动二聚化，因此我们的结果证明了序列背景对于氢键或 GxxxG 基序参与 BNIP3 跨膜螺旋-螺旋相互作用的重要性。在这项研究中，远离六个界面位置的疏水取代对二聚化几乎没有影响，证实了疏水取代仅在干扰界面残基的堆积或氢键时才会影响螺旋-螺旋相互作用的预期。然而，即使远离界面，轻微极性残基的变化也会产生一定程度的破坏，并且破坏程度与疏水性的降低相关。改变 BNIP3 跨膜结构域的疏水性会改变其螺旋性和主链酰胺的保护。我们认为极性取代通过稳定跨膜跨度的未折叠单体状态来减少二聚体的分数，而不是通过影响螺旋-螺旋相互作用。这一结果对于解释去污剂中膜蛋白稳定性的序列依赖性具有广泛的意义。
The transmembrane domain of the pro-apoptotic protein BNIP3 self-associates strongly in membranes and in detergents. We have used site-directed mutagenesis to analyze the sequence dependence of BNIP3 transmembrane domain dimerization, from which we infer the physical basis for strong and specific helix–helix interactions in this system. Hydrophobic substitutions identify six residues as critical to dimerization, and the pattern of sensitive residues suggests that the BNIP3 helices interact at a right-handed crossing angle. Based on the dimerization propensities of single point mutants, we propose that: polar residues His173 and Ser172 make inter-monomer hydrogen bonds to one another through their side-chains; Ala176, Gly180, and Gly184 form a tandem GxxxG motif that allows close approach of the helices; and Ile183 makes inter-monomer van der Waals contacts. Since neither the tandem GxxxG motif nor the hydrogen bonding pair is sufficient to drive dimerization, our results demonstrate the importance of sequence context for either hydrogen bonding or GxxxG motif involvement in BNIP3 transmembrane helix–helix interactions. In this study, hydrophobic substitutions away from the six interfacial positions have almost no effect on dimerization, confirming the expectation that hydrophobic replacements affect helix–helix interactions only if they interfere with packing or hydrogen bonding by interfacial residues. However, changes to slightly polar residues are somewhat disruptive even when located away from the interface, and the degree of disruption correlates with the decrease in hydrophobicity. Changing the hydrophobicity of the BNIP3 transmembrane domain alters its helicity and protection of its backbone amides. We suggest that polar substitutions decrease the fraction of dimer by stabilizing an unfolded monomeric state of the transmembrane span, rather than by affecting helix–helix interactions. This result has broad implications for interpreting the sequence dependence of membrane protein stability in detergents.