Exploring the Role of Integral Membrane Proteins in ATP-Binding Cassette Transporters: Analysis of a Collection of MalG Insertion Mutants

The maltose transport complex of Escherichia coli is a well-studied example of an ATP-binding cassette transporter. The complex, containing one copy each of the integral membrane proteins MalG and MalF and two copies of the peripheral cytoplasmic membrane protein MalK, interacts with the periplasmic maltose-binding protein to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK(2) assembly interactions and in subsequent transport interactions, we isolated and characterized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regions of MalG permitted either assembly or both assembly and transport interactions to occur. In particular, we isolated two insertions mapping to extracytoplasmic (periplasmic) regions of MalG which preserved both assembly and transport abilities, suggesting that these are permissive sites in the protein. Another periplasmic insertion seems to affect only transport-specific interactions between MalG and maltose-binding protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble into the MalFGK(2) complex. These mutants contain insertions in only two different hydrophilic regions of MalG, consistent with the notion that a restricted number of domains in this protein are critical complex assembly determinants. These MalG mutants will allow us to further explore the intermolecular interactions of this model transporter.

大肠杆菌的麦芽糖转运复合物是ATP结合盒转运蛋白（ATP-binding cassette transporter）的经典研究范例。该复合物由整合膜蛋白MalG、MalF各1拷贝，以及外周胞质膜蛋白MalK的2拷贝组装而成，可与周质麦芽糖结合蛋白相互作用，实现将麦芽糖和麦芽糊精高效跨细菌细胞质膜转运。为探究MalG在MalFGK₂组装相互作用及后续转运相互作用中的功能，我们分离并鉴定了18种不同的MalG突变体，每一种均在蛋白中带有31个残基的插入序列。其中8个插入位点位于MalG的不同亲水区域，对应的突变体可实现复合物组装，或同时具备组装与转运功能。尤为关键的是，我们获得了2个插入位点位于MalG胞外侧（周质侧）区域的突变体，二者同时保留了组装与转运能力，表明这些位点属于该蛋白的允许插入位点。另有1个周质侧插入突变体似乎仅影响MalG与麦芽糖结合蛋白之间的转运特异性相互作用，这定义了一类新型MalG突变体。最后，另有4种MalG突变体蛋白虽可稳定表达，但无法组装进入MalFGK₂复合物；这些突变体的插入位点仅分布于MalG的两个不同亲水区域，这与“该蛋白中仅有有限数量的结构域是复合物组装的关键决定因素”的观点相符。上述MalG突变体将助力我们进一步解析这一模式转运蛋白的分子间相互作用。