Competitive Interactions of Ligands and Macromolecular Crowders with Maltose Binding Protein

Cellular signaling involves a cascade of recognition events occurring in a complex environment with high concentrations of proteins, polysaccharides, and other macromolecules. The influence of macromolecular crowders on protein binding affinity through hard-core repulsion is well studied, and possible contributions of protein-crowder soft attraction have been implicated recently. Here we present direct evidence for weak association of maltose binding protein (MBP) with a polysaccharide crowder Ficoll, and that this association effectively competes with the binding of the natural ligand, maltose. Titration data over wide ranges of maltose and Ficoll concentrations fit well with a three-state competitive binding model. Broadening of MBP 1H­15N TROSY spectra by the addition of Ficoll indicates weak protein-crowder association, and subsequent recovery of sharp NMR peaks upon addition of maltose indicates that the interactions of the crowder and the ligand with MBP are competitive. We hypothesize that, in the Escherichia coli periplasm, the competitive interactions of polysaccharides and maltose with MBP could allow MBP to shuttle between the peptidoglycan attached to the outer membrane and the ATP-binding cassette transporter in the inner membrane.

细胞信号转导是一系列识别事件的级联过程，发生在富含蛋白质、多糖及其他大分子的复杂微环境中。大分子拥挤剂通过硬核排斥作用影响蛋白质结合亲和力的机制已得到充分研究，而蛋白质与拥挤剂间的软吸引作用的潜在贡献近年来才受到关注。本研究提供了麦芽糖结合蛋白（maltose binding protein, MBP）与多糖类拥挤剂Ficoll之间存在弱相互作用的直接证据，并证实该相互作用可与天然配体麦芽糖的结合产生有效竞争。在麦芽糖与Ficoll的宽浓度范围内采集的滴定数据，与三态竞争性结合模型拟合度极佳。Ficoll的加入使MBP的1H-15N横向弛豫优化谱（Transverse Relaxation Optimized Spectroscopy, TROSY）峰发生宽化，提示蛋白质与拥挤剂间存在弱相互作用；而后续加入麦芽糖后，尖锐的NMR峰得以恢复，表明拥挤剂与配体对MBP的结合具有竞争性。我们提出假说：在大肠杆菌（Escherichia coli）周质空间中，多糖与麦芽糖对MBP的竞争性结合，可使MBP在附着于外膜的肽聚糖与内膜ATP结合盒转运体（ATP-binding cassette transporter）之间实现穿梭转运。