Protein and Lipid Interactions Driving Molecular Mechanisms of <i>in meso</i> Crystallization

The recent advances in the in meso crystallization technique for the structural characterization of G-protein coupled receptor (GPCR) proteins have established the usefulness of the lipidic-cubic phases (LCPs) in the field of crystallography of membrane proteins. It is surprising that despite the success of the approach, the molecular mechanisms of the in meso method are still not well understood. Therefore, the approach must rely on extensive screening for a suitable protein construct, for host and additive lipids, and for the appropriate precipitants and temperature. To shed light on the in meso crystallization mechanisms, we used extensive coarse-grained molecular dynamics simulations to study, in molecular detail, LCPs under different conditions (compositions and temperatures relevant to crystallogenesis) and their interactions with different types of GPCR constructs. The results presented show how the modulation of the lattice constant of the LCP (triggered by the addition of precipitant during the in meso assay), or of the host lipid type, can destabilize monomeric proteins in the bilayer of the LCP and thus drive their aggregation into the stacked lamellae, where the residual hydrophobic mismatch between the protein and the membrane can drive the formation of lateral contacts leading to nucleation and crystal growth. Moreover, we demonstrate how particular protein designs (such as transmembrane proteins engineered to contain large polar regions) can promote protein stacking interactions in the third, out-of-plane, dimension. The insights provided by the new aspects of the specific molecular mechanisms responsible for protein–protein interactions inside the cubic phase presented here should be helpful in guiding the rational design of future in meso trials with successful outcomes.近年来用于G蛋白偶联受体（G-protein coupled receptor, GPCR）结构表征的介相结晶（in meso crystallization）技术取得进展，确立了脂立方相（lipidic-cubic phases, LCPs）在膜蛋白晶体学研究中的应用价值。令人意外的是，尽管该方法已取得成功，介相结晶法的分子机制仍未得到充分阐明。因此，当前该方法仍需通过大规模筛选来适配合适的蛋白构建体、宿主脂质与添加剂脂质，以及适宜的沉淀剂与温度条件。为阐明介相结晶的分子机制，本研究采用大规模粗粒度分子动力学模拟，从分子细节层面详细探究了与结晶过程相关的不同组分、温度条件下的脂立方相，及其与不同类型GPCR蛋白构建体的相互作用。本研究结果揭示：在介相检测实验中通过添加沉淀剂引发的脂立方相晶格常数调控，或是宿主脂质种类的改变，均可破坏脂立方相双分子层内单体蛋白的稳定性，进而促使其聚集为堆叠的片层结构；此时蛋白与膜之间残存的疏水错配效应，可进一步驱动侧向接触的形成，最终促成成核与晶体生长。此外，本研究还证实，特定的蛋白设计（如经工程改造带有大极性区域的跨膜蛋白）可如何促进蛋白在第三维（平面外维度）上的堆叠相互作用。本研究揭示了脂立方相内蛋白-蛋白相互作用的特定分子机制的新视角，所获得的相关认知可为未来介相结晶实验的理性设计与成功实施提供指导。