Byrne Lee J, Sidhu Ateesh, Wallis A Katrine, Ruddock Lloyd W, Freedman Robert B, Howard Mark J, Williamson Richard A
Department of Biosciences, University of Kent, Canterbury, UK.
Biochem J. 2009 Sep 25;423(2):209-17. doi: 10.1042/BJ20090565.
PDI (protein disulfide-isomerase) catalyses the formation of native disulfide bonds of secretory proteins in the endoplasmic reticulum. PDI consists of four thioredoxin-like domains, of which two contain redox-active catalytic sites (a and a'), and two do not (b and b'). The b' domain is primarily responsible for substrate binding, although the nature and specificity of the substrate-binding site is still poorly understood. In the present study, we show that the b' domain of human PDI is in conformational exchange, but that its structure is stabilized by the addition of peptide ligands or by binding the x-linker region. The location of the ligand-binding site in b' was mapped by NMR chemical shift perturbation and found to consist primarily of residues from the core beta-sheet and alpha-helices 1 and 3. This site is where the x-linker region binds in the X-ray structure of b'x and we show that peptide ligands can compete with x binding at this site. The finding that x binds in the principal ligand-binding site of b' further supports the hypothesis that x functions to gate access to this site and so modulates PDI activity.
蛋白质二硫键异构酶（PDI）催化内质网中分泌蛋白天然二硫键的形成。PDI由四个硫氧还蛋白样结构域组成，其中两个含有氧化还原活性催化位点（a和a'），另外两个则没有（b和b'）。b'结构域主要负责底物结合，尽管对底物结合位点的性质和特异性仍知之甚少。在本研究中，我们发现人PDI的b'结构域处于构象交换状态，但其结构可通过添加肽配体或结合交联区而得以稳定。通过核磁共振化学位移扰动对b'中配体结合位点的位置进行了定位，发现其主要由核心β折叠以及α螺旋1和3的残基组成。该位点正是交联区在b'x的X射线结构中结合的位置，并且我们表明肽配体可在此位点与交联区的结合形成竞争。交联区在b'的主要配体结合位点结合这一发现进一步支持了如下假说：交联区起到控制进入该位点的作用，从而调节PDI的活性。
