Mandacaru Samuel C, do Vale Luis H F, Vahidi Siavash, Xiao Yiming, Skinner Owen S, Ricart Carlos A O, Kelleher Neil L, de Sousa Marcelo Valle, Konermann Lars
Department of Chemistry, Western University , London, Ontario, Canada N6A 5B7.
Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia , Brasilia, Brazil.
Biochemistry. 2017 Mar 21;56(11):1645-1655. doi: 10.1021/acs.biochem.7b00020. Epub 2017 Mar 7.
Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue α-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP1 apisimin) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20-265. Marginally stable secondary structure (likely antiparallel β-sheet) exists around residues 266-432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a "dimer of dimers" quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment VLFFGLV. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP1 apisimin) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.
蜂王浆（RJ）促使雌性蜜蜂幼虫发育成为蜂后。这种作用归因于蜂王浆中存在的主要蜂王浆蛋白1（MRJP1）。从蜂王浆中分离出的MRJP1与蜂毒明肽紧密结合，蜂毒明肽是一种由54个残基组成的α-螺旋肽，可促进MRJP1非共价组装成多聚体。目前尚无这些复合物的高分辨率结构数据，其结合化学计量比仍不确定。我们使用一系列生物物理技术研究了MRJP1/蜂毒明肽。我们还研究了去糖基化样品以及蜂毒明肽含量降低的样品的行为。我们的质谱（MS）数据表明，天然复合物主要以（MRJP1蜂毒明肽）化学计量比存在。氢/氘交换质谱显示，这些复合物中的MRJP1在20-265位残基范围内广泛无序。在266-432位残基周围存在略微稳定的二级结构（可能是反平行β-折叠）。这些弱结构区域与广泛展开的构象异构体相互交换，产生双峰（EX1）同位素分布。我们提出，天然复合物具有“二聚体的二聚体”四级结构，其中MRJP1链由蜂毒明肽桥接。具体而言，我们的数据表明，蜂毒明肽作为连接体，形成涉及MRJP1片段VLFFGLV的疏水接触。去糖基化产生大量可溶性聚集体，突出了聚糖作为聚集抑制剂的作用。蜂毒明肽含量降低的样品形成化学计量比为（MRJP1蜂毒明肽）的二聚体复合物。这项工作中揭示的信息将有助于为更好地理解MRJP1在蜂后分化过程中所起的独特生理作用铺平道路。
