Won Hyung-Sik, Low Lieh Yoon, Guzman Roberto De, Martinez-Yamout Maria, Jakob Ursula, Dyson H Jane
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
J Mol Biol. 2004 Aug 20;341(4):893-9. doi: 10.1016/j.jmb.2004.06.046.
The Escherichia coli chaperone Hsp33 contains a C-terminal zinc-binding domain that modulates activity by a so-called "redox switch". The oxidized form in the absence of zinc is active, while the reduced form in the presence of zinc is inactive. X-ray crystal structures of Hsp33 invariably omit details of the C-terminal domain, which is truncated in protein constructs that are capable of forming crystals. We report the solution structure of a recombinant 61-residue protein containing the zinc-binding domain (residues 227-287) of Hsp33, in the presence of stoichiometric amounts of Zn2+. The zinc-bound protein is well folded, and forms a novel structure unlike other published zinc-binding domains. The structure consists of two helices at right-angles to each other, a two-stranded B-hairpin and a third helix at the C terminus. The zinc site comprises the side-chains of the conserved cysteine residues 232, 234, 262 and 265, and connects a short sequence before the first helix with the tight turn in the middle of the B-hairpin. The structure of the C-terminal zinc-binding domain suggests a mechanism for the operation of the redox switch: loss of the bound zinc ion disrupts the folded structure, allowing the ligand cysteine residues to be oxidized, probably to disulfide bonds. The observation that the C-terminal domain is poorly structured in the active oxidized form suggests that the loss of zinc and unfolding of the domain precedes the oxidation of the thiolate groups of the cysteine residues, since the formation of disulfides between distant parts of the domain sequence would presumably promote the formation of stable three-dimensional structure in the oxidized form.Hsp33 provides an example of a redox signaling system that utilizes protein folding and unfolding together with chemical modification for transduction of external stimuli, in this case oxidative stress, to activate the machinery of the cell that is designed to deal with that stress.
大肠杆菌伴侣蛋白Hsp33含有一个C端锌结合结构域，该结构域通过所谓的“氧化还原开关”调节活性。在没有锌的情况下，氧化形式具有活性，而在有锌的情况下，还原形式无活性。Hsp33的X射线晶体结构总是省略C端结构域的细节，该结构域在能够形成晶体的蛋白质构建体中被截断。我们报道了一种重组61个残基的蛋白质的溶液结构，该蛋白质含有Hsp33的锌结合结构域（残基227 - 287），存在化学计量的Zn2 +。锌结合蛋白折叠良好，形成一种与其他已发表的锌结合结构域不同的新结构。该结构由彼此成直角的两个螺旋、一个双链β - 发夹和C端的第三个螺旋组成。锌位点由保守的半胱氨酸残基232、234、262和265的侧链组成，并将第一个螺旋之前的短序列与β - 发夹中间的紧密转角相连。C端锌结合结构域的结构提示了氧化还原开关的作用机制：结合的锌离子的丢失会破坏折叠结构，使配体半胱氨酸残基被氧化，可能形成二硫键。C端结构域在活性氧化形式中结构不佳的观察结果表明，锌的丢失和结构域的去折叠先于半胱氨酸残基硫醇盐基团的氧化，因为结构域序列远处部分之间二硫键的形成可能会促进氧化形式中稳定三维结构的形成。Hsp33提供了一个氧化还原信号系统的例子，该系统利用蛋白质的折叠和去折叠以及化学修饰来转导外部刺激，在这种情况下是氧化应激，以激活细胞中旨在应对该应激的机制。
