Person M D, Brown K C, Mahrus S, Craik C S, Burlingame A L
Department of Pharmaceutical Chemistry, University of California at San Francisco, 94143-0446, USA.
Protein Sci. 2001 Aug;10(8):1549-62. doi: 10.1110/ps.ps.46601.
In the presence of a suitable oxidizing agent, the Ni(II) complex of glycyl-glycyl-histidine (GGH) mediates efficient and specific oxidative protein cross-linking. The fusion of GGH to the N terminus of a protein allows for the cross-linking reagent to be delivered in a site-specific fashion, making this system extremely useful for analyzing protein-protein contacts in complicated mixtures of biomolecules. Tyrosine residues have been postulated to be the primary amino acid target of this reaction, and using the dimeric serine protease inhibitor ecotin, we previously demonstrated that engineering a tyrosine at the protein interface of a dimer dramatically increased cross-linking efficiency. Cross-linking increased four-fold for GGH-ecotin D137Y in comparison to wild-type GGH-ecotin, presumably through bityrosine formation at the dimer interface. Here we report the first complete structural analysis of the cross-linked GGH-ecotin D137Y dimer. Using a combination of mass spectrometric and chemical derivatization methods, a sole novel cross-link between the N-terminal glycine residues and the engineered tyrosine at position 137 has been characterized. The dimer cross-link is localized to a single site without other protein modifications, but different reaction pathways produce structurally related products. We propose a mechanism that involves covalent bond formation between the protein backbone and a dopaquinone moiety derived from a specific tyrosine residue. This finding establishes that it is not necessary to have two tyrosine residues within close proximity in the protein interface to obtain high protein cross-linking yields, and suggests that the cross-linking reagent may be of more general utility than previously thought.
在合适的氧化剂存在下，甘氨酰 - 甘氨酰 - 组氨酸（GGH）的镍（II）配合物介导高效且特异性的氧化蛋白质交联。将GGH融合到蛋白质的N末端可使交联试剂以位点特异性方式递送，这使得该系统对于分析生物分子复杂混合物中的蛋白质 - 蛋白质相互作用极为有用。酪氨酸残基被认为是该反应的主要氨基酸靶点，我们先前使用二聚体丝氨酸蛋白酶抑制剂依考汀证明，在二聚体的蛋白质界面处工程化一个酪氨酸可显著提高交联效率。与野生型GGH - 依考汀相比，GGH - 依考汀D137Y的交联增加了四倍，推测是通过在二聚体界面形成双酪氨酸实现的。在此，我们报告了交联的GGH - 依考汀D137Y二聚体的首次完整结构分析。通过结合质谱和化学衍生化方法，已鉴定出N末端甘氨酸残基与137位工程化酪氨酸之间唯一的新型交联。二聚体交联定位于单个位点，无其他蛋白质修饰，但不同的反应途径产生结构相关的产物。我们提出了一种机制，该机制涉及蛋白质主链与源自特定酪氨酸残基的多巴醌部分之间形成共价键。这一发现表明，在蛋白质界面中无需两个酪氨酸残基紧密相邻即可获得高蛋白交联产率，并表明交联试剂的通用性可能比先前认为的更高。
