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首页> 外文期刊>Biochemistry >Structural and Functional Consequences of Binding Site Mutations in Transferrin: Crystal Structures of the Asp63Glu and Arg124Ala Mutants of the N-Lobe of Human Transferrin
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Structural and Functional Consequences of Binding Site Mutations in Transferrin: Crystal Structures of the Asp63Glu and Arg124Ala Mutants of the N-Lobe of Human Transferrin

机译：运铁蛋白中结合位点突变的结构和功能后果：人类运铁蛋白的N细胞的Asp63Glu和Arg124Ala突变体的晶体结构。

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摘要

Human transferrin is a serum protein whose function is to bind Fe~(3+) with very high affinity and transport it to cells, for delivery by receptor-mediated endocytosis. Structurally, the transferrin molecule is folded into two globular lobes, representing its N-terminal and C-terminal halves, with each lobe possessing a high-affinity iron binding site, in a cleft between two domains. Central to function is a highly conserved set of iron ligands, including an aspartate residue (Asp63 in the N-lobe) that also hydrogen bonds between the two domains and an arginine residue (Arg124 in the N-lobe) that binds an iron-bound carbonate ion. To further probe the roles of these residues, we have determined the crystal structures of the D63E and R124A mutants of the N-terminal half-molecule of human transferrin. The structure of the D63E mutant, determined at 1.9 A resolution (R=0.245, R_(free)=0.261), showed that the carboxyl group still binds to iron despite the large size of the Glu side chain, with some slight rearrangement of the first turn of alpha-helix residues 63-72, to which it is attached. The structure of the R124A mutant, determined at 2.4 A resolution (R=0.219, R_(free)=0.288), shows that the loss of the arginine side chain results in a 0.3 A displacement of the carbonate ion, and an accompanying movement of the iron atom. In both mutants, the iron coordination is changed slightly, the principal change being in each case a lengthening of the Fe-N(His249) bond. Both mutants also release iron more readily than the wild type, kinetically and in terms of acid lability of iron binding. We attribute this to more facile protonation of the synergistically bound carbonate ion, in the case of R124A, and to strain resulting from the accommodation of the larger Glu side chain, in the case of D63E. In both cases, the weakened Fe-N(His) bond may also contribute, consistent with protonation of the His ligand being an early intermediate step in iron release, following the protonation of the carbonate ion.
机译：人转铁蛋白是一种血清蛋白，其功能是以非常高的亲和力结合Fe〜（3+）并将其转运至细胞，以通过受体介导的内吞作用进行传递。在结构上，运铁蛋白分子折叠成两个球状小叶，分别代表其N端和C端两半，每个叶在两个结构域之间的缝隙中具有高亲和力的铁结合位点。功能的中心是一组高度保守的铁配体，包括天冬氨酸残基（N瓣中的Asp63）和两个域之间的氢键，以及精氨酸残基（N瓣中的Arg124），其结合铁结合碳酸根离子。为了进一步探究这些残基的作用，我们确定了人类运铁蛋白N端半分子的D63E和R124A突变体的晶体结构。 D1.9E突变体的结构在1.9 A的分辨率下确定（R = 0.245，R_（free）= 0.261），表明尽管Glu侧链的尺寸很大，羧基仍与铁结合，但Glu侧链有些许重排与其相连的第一轮α-螺旋残基63-72。 R2.4A突变体的结构在2.4 A分辨率下确定（R = 0.219，R_（free）= 0.288），表明精氨酸侧链的丢失导致碳酸根离子位移0.3 A，并伴随着运动铁原子。在这两个突变体中，铁的配位都略有变化，主要变化是每种情况下Fe-N（His249）键的延长。两种突变体在动力学上以及在铁结合的酸不稳定性方面也比野生型更容易释放铁。我们将其归因于在R124A情况下协同结合的碳酸根离子更容易质子化，在D63E情况下归因于更大的Glu侧链的容纳所引起的应变。在这两种情况下，削弱的Fe-N（His）键也可能起作用，这与His配体的质子化是碳酸根离子质子化之后铁释放的早期中间步骤相一致。

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