Heinemann B, Andersen K V, Nielsen P R, Bech L M, Poulsen F M
Carlsberg Laboratorium, Kemisk Afdeling, Copenhagen, Denmark.
Protein Sci. 1996 Jan;5(1):13-23. doi: 10.1002/pro.5560050103.
Because of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three-dimensional structure in solution of bLTP as determined by NMR spectroscopy. The 1H NMR spectrum of the 91-residue protein was assigned for more than 97% of the protein 1H atoms, and the structure was calculated on the basis of 813 distance restraints from 1H-1H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 phi-angles, 61 chi 1 angles, and 2 chi 2 angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well-defined alpha-helices A-D (A, Cys 3-Gly 19; B, Gly 25-Ala 38; C, Arg 44-Gly 57; D, Leu 63-Cys 73), separated by three short loops that are less well defined and concluded by a well defined C-terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 A +/- 0.04 A for backbone atoms and 0.93 A +/- 0.06 A for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur J Biochem 226:413-422; Shin DH et al, 1995, Structure 3:189-199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.
由于脂质在水中的溶解度较低，因此脂质转移的细胞间和细胞内途径是必需的，例如用于膜的形成。体内脂质从其生物合成部位（内质网和叶绿体）运输到其作用部位的机制尚不清楚。已经分离出几种能够在体外介导放射性标记的磷脂从脂质体供体膜转移到线粒体和叶绿体受体膜的小植物蛋白，并且在这里研究了一种具有这种能力的蛋白，即从大麦种子中分离出的非特异性脂质转移蛋白（nsLTP）（bLTP）。脂质转移蛋白的结构和蛋白质-脂质相互作用对于理解其功能至关重要，在这里我们展示了通过核磁共振光谱法测定的bLTP在溶液中的三维结构。91个残基的蛋白质的1H NMR谱图中超过97%的蛋白质1H原子已被归属，并且基于来自1H-1H核Overhauser效应的813个距离约束、四个二硫键约束、66个φ角、61个χ1角和2个χ2角的二面角约束以及31组氢键约束计算了结构。bLTP的溶液结构由四个定义明确的α螺旋A-D组成（A，Cys 3-Gly 19；B，Gly 25-Ala 38；C，Arg 44-Gly 57；D，Leu 63-Cys 73），由三个定义不太明确的短环隔开，并以一个定义明确的无明显规则二级结构的C末端肽段结束。对于用于表示bLTP溶液结构的17个结构，主链原子相对于平均结构的均方根偏差为0.63 ű0.04 Å，所有重原子的均方根偏差为0.93 ű0.06 Å。二级结构元件及其在序列中的位置类似于先前已确定结构的另外两种植物小麦和玉米的nsLTP（Gincel E等人，1995，欧洲生物化学杂志226：413-422；Shin DH等人，1995，结构3：189-199）。在bLTP中，与构成棕榈酸结合位点的玉米nsLTP中类似的残基形成了一个类似的疏水腔和一个潜在的酰基结合位点。分析bLTP的溶液结构以及与配体结合的bLTP可能会提供有关配体结合后蛋白质构象变化的信息，并随后提供有关配体摄取和释放模式的信息。在这项工作中，我们希望为进一步确定与合适配体棕榈酰辅酶A结合的bLTP的溶液结构奠定基础，并随后概述配体结合模式。
