Periplasmic binding protein structure and function. Refined X-ray structures of the leucine/isoleucine/valine-binding protein and its complex with leucine.
Periplasmic binding protein structure and function. Refined X-ray structures of the leucine/isoleucine/valine-binding protein and its complex with leucine.
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DOI：
10.1016/0022-2836(89)90531-7
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发表时间：
1989-03
影响因子：
5.6
通讯作者：
J. Sack;M. Saper;F. Quiocho
中科院分区：
文献类型：
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作者：
J. Sack;M. Saper;F. Quiocho
Leu/Ile/ val结合蛋白（Mr= 36,700）是大肠杆菌中支链脂肪族氨基酸高亲和主动转运系统的重要组成部分，其天然无配体形式的三维结构已被确定，并进一步细化到在2.4 Å分辨率下的晶体学r因子为0.17。整个结构由344个残基的完整序列中的2710个非氢原子和121个有序的水分子组成。改进模型中的键长和角距与理想值的均方根偏差分别为0.05 Å和0.10 Å。蛋白质的整体形状是一个长形椭球体，尺寸为35 Å × 40 Å × 70 Å。该蛋白由两个不同的球状结构域组成，由三个短肽段连接，尽管在序列上广泛分开，但在三级结构中是近端的，并形成两个结构域之间深间隙的基础。虽然每个结构域都是由位于氨基(N)和羧基(C)末端的多肽片段构建而成，但这两个结构域都表现出非常相似的超二级结构，包括一个由7条链组成的中央β-片，两侧各有2或3个螺旋。这两个域彼此相距很远，留下了大约18个空位Å。裂隙的深度约为15 Å，基底约为14 Å × 16 Å。在含有l-亮氨酸的溶液中，以2.8 Å的分辨率（r因子= 0.15）对天然Leu/Ile/ val结合蛋白晶体的结构进行独立提纯，我们已经能够定位和表征Leu/Ile/ val结合蛋白底物结合位点的初始主要部分。亮氨酸底物的结合不会改变天然晶体结构，亮氨酸位于n结构域壁上的缝隙中，该缝隙位于结构域间的间隙中。亮氨酸主要通过其α-铵基和α-羧酸基与主链肽单元和羟基侧链基之间的氢键来固定；没有盐的联系。亮氨酸两性离子上的电荷由氢键偶极子稳定。亮氨酸底物的侧链位于一个由非极性残基排列的凹陷中，包括Leu77，它通过与亮氨酸底物的侧链叠加而赋予该位点特异性。到目前为止在我们高分辨率晶体研究的几个结合蛋白(包括那些特异性forl-arabinose、d-galactose和硫酸),我们发现相关的三种结构形式灵活的领域:一种unliganded形式与敞开substrate-binding网站两个域之间的间隙,一个“开放”的结构与衬底绑定到一个域,和“封闭”形式与衬底之间的间隙,完全裹入域。结合蛋白的各种形式的存在和其他共同的结构和配体结合特征，讨论了结合蛋白在主动运输中的功能。
The three-dimensional structure of the native unliganded form of the Leu/Ile/Val-binding protein (Mr= 36,700), an essential component of the high-affinity active transport system for the branched aliphatic amino acids inEscherichia coli, has been determined and further refined to a crystallographicR-factor of 0·17 at 2·4 Å resolution. The entire structure consists of 2710 non-hydrogen atoms from the complete sequence of 344 residues and 121 ordered water molecules. Bond lengths and angle distances in the refined model have root-mean-square deviations from ideal values of 0·05 Å and 0·10 Å, respectively. The overall shape of the protein is a prolate ellipsoid with dimensions of 35 Å × 40 Å × 70 Å. The protein consists of two distinct globular domains linked by three short peptide segments which, though widely separated in the sequence, are proximal in the tertiary structure and form the base of the deep cleft between the two domains. Although each domain is built from polypeptide segments located in both the amino (N) and the carboxy (C) terminal halves, both domains exhibit very similar supersecondary structures, consisting of a central β-sheet of seven strands flanked on either side by two or three helices. The two domains are far apart from each other, leaving the cleft wide open by about 18 Å. The cleft has a depth of about 15 Å and a base of about 14 Å × 16 Å.Refining independently the structure of native Leu/Ile/Val-binding protein crystals soaked in a solution containingl-leucine at 2.8 Å resolution (R-factor = 0.15), we have been able to locate and characterize an initial, major portion of the substrate-binding site of the Leu/Ile/Val-binding protein. The binding of thel-leucine substrate does not alter the native crystal structure, and thel-leucine is lodged in a crevice on the wall of the N-domain, which is in the inter-domain cleft. Thel-leucine is held in place primarily by hydrogen-bonding of its α-ammonium and α-carboxylate groups with main-chain peptide units and hydroxyl side-chain groups; there are no salt-linkages. The charges on the leucine zwitterion are stabilized by hydrogen-bond dipoles. The side-chain of thel-leucine substrate lies in a depression lined with non-polar residues, including Leu77, which confers specificity to the site by stacking with the side-chain of the leucine substrate.So far in our high-resolution crystallographic studies of several binding proteins (including those with specificities forl-arabinose,d-galactose, and sulfate), we have observed three structural forms that are related by flexible domains: an unliganded form with a wide open substrate-binding site cleft between the two domains, an “open” structure with the substrate bound to one domain, and a “closed” form with the substrate bound in the cleft and completely entrapped between the domains. The function of binding proteins in active transport is discussed in light of the existence of the various forms and the other common structural and ligand-binding features of these proteins.