Krylov D M, Hurley J B
Department of Biochemistry and Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA.
J Biol Chem. 2001 Aug 17;276(33):30648-54. doi: 10.1074/jbc.M104121200. Epub 2001 May 31.
A key challenge in studying protein/protein interactions is to accurately identify contact surfaces, i.e. regions of two proteins that are in direct physical contact. Aside from x-ray crystallography and NMR spectroscopy few methods are available that address this problem. Although x-ray crystallography often provides detailed information about contact surfaces, it is limited to situations when a co-crystal of proteins is available. NMR circumvents this requirement but is limited to small protein complexes. Other methods, for instance protection from proteolysis, are less direct and therefore less informative. Here we describe a new method that identifies candidate contact surfaces in protein complexes. The complexes are first stabilized by cross-linking. They are then digested with a protease, and the cross-linked fragments are analyzed by mass spectrometry. We applied this method, referred to as COSUMAS (contact surfaces by mass spectrometry), to two proteins, retinal guanylyl cyclase 1 (RetGC1) and guanylyl cyclase-activating protein-1 (GCAP-1), that regulate cGMP synthesis in photoreceptors. Two regions in GCAP-1 and three in RetGC1 were identified as possible contact sites. The two regions of RetGC1 that are in the vicinities of Cys(741) and Cys(780) map to a kinase homology domain in RetGC1. Their identities as contact sites were independently evaluated by peptide inhibition analysis. Peptides with sequences from these regions block GCAP-1-mediated regulation of guanylyl cyclase at both high and low Ca2+ concentrations. The two regions of GCAP-1 cross-linked to these peptides were in the vicinities of Cys(17) and Cys(105) of GCAP-1. Peptides with sequences derived from these regions inhibit guanylyl cyclase activity directly. These results support a model in which GCAP-1 binds constitutively to RetGC1 and regulates cyclase activity by structural changes caused by the binding or dissociation of Ca2+.
研究蛋白质/蛋白质相互作用的一个关键挑战是准确识别接触表面，即两种蛋白质直接发生物理接触的区域。除了X射线晶体学和核磁共振光谱法外，几乎没有其他方法可用于解决这个问题。尽管X射线晶体学通常能提供有关接触表面的详细信息，但它仅限于有蛋白质共晶体的情况。核磁共振光谱法规避了这一要求，但仅限于小型蛋白质复合物。其他方法，例如蛋白酶保护法，不够直接，因此提供的信息较少。在此，我们描述了一种在蛋白质复合物中识别候选接触表面的新方法。首先通过交联使复合物稳定。然后用蛋白酶进行消化，并用质谱法分析交联片段。我们将这种称为COSUMAS（通过质谱法确定接触表面）的方法应用于两种调节光感受器中cGMP合成的蛋白质，即视网膜鸟苷酸环化酶1（RetGC1）和鸟苷酸环化酶激活蛋白-1（GCAP-1）。在GCAP-1中确定了两个区域，在RetGC1中确定了三个区域为可能的接触位点。RetGC1中靠近半胱氨酸（741）和半胱氨酸（780）的两个区域映射到RetGC1中的一个激酶同源结构域。通过肽抑制分析独立评估了它们作为接触位点的身份。来自这些区域的序列的肽在高钙和低钙浓度下均能阻断GCAP-1介导的鸟苷酸环化酶调节。与这些肽交联的GCAP-1的两个区域位于GCAP-1的半胱氨酸（17）和半胱氨酸（105）附近。来自这些区域的序列的肽直接抑制鸟苷酸环化酶活性。这些结果支持了一种模型，即GCAP-1与RetGC1组成性结合，并通过钙的结合或解离引起的结构变化来调节环化酶活性。
