National Center for Structural Biology and Bioimaging (CENABIO)/National Center for Nuclear Magnetic Resonance (CNRMN), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Institute of Medical Biochemistry - IBqM, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Methods Mol Biol. 2023;2705:135-151. doi: 10.1007/978-1-0716-3393-9_8.
Protein interactions are at the essence of life. Proteins evolved not to have stable structures, but rather to be specialized in participating in a network of interactions. Every interaction involving proteins comprises the formation of an encounter complex, which may have two outcomes: (i) the dissociation or (ii) the formation of the final specific complex. Here, we present a methodology to characterize the encounter complex of the Grb2-SH2 domain with a phosphopeptide. This method can be generalized to other protein partners. It consists of the measurement of N CPMG relaxation dispersion (RD) profiles of the protein in the free state, which describes the residues that are in conformational exchange. We then acquire the dispersion profiles of the protein at a semisaturated concentration of the ligand. At this condition, the chemical exchange between the free and bound state leads to the observation of dispersion profiles in residues that are not in conformational exchange in the free state. This is due to fuzzy interactions that are typical of the encounter complexes. The transient "touching" of the ligand in the protein partner generates these new relaxation dispersion profiles. For the Grb2-SH2 domain, we observed a wider surface at SH2 for the encounter complex than the phosphopeptide (pY) binding site, which might explain the molecular recognition of remote phosphotyrosine. The Grb2-SH2-pY encounter complex is dominated by electrostatic interactions, which contribute to the fuzziness of the complex, but also have contribution of hydrophobic interactions.
蛋白质相互作用是生命的本质。蛋白质的进化不是为了形成稳定的结构，而是为了专门参与相互作用网络。每一个涉及蛋白质的相互作用都包括形成一个遭遇复合物，它可能有两种结果：（i）解离或（ii）最终特定复合物的形成。在这里，我们提出了一种方法来描述 Grb2-SH2 结构域与磷酸肽的遭遇复合物。这种方法可以推广到其他蛋白质伴侣。它包括测量蛋白质在自由状态下的 N CPMG 弛豫分散（RD）谱，该谱描述了处于构象交换中的残基。然后，我们在配体的半饱和浓度下获取蛋白质的分散谱。在这种条件下，自由态和结合态之间的化学交换导致在自由态中不处于构象交换的残基中观察到分散谱。这是由于遭遇复合物中典型的模糊相互作用。配体在蛋白质伴侣中的短暂“接触”产生了这些新的弛豫分散谱。对于 Grb2-SH2 结构域，我们观察到在 SH2 上的遭遇复合物比磷酸肽（pY）结合位点具有更广泛的表面，这可能解释了远程磷酸酪氨酸的分子识别。Grb2-SH2-pY 遭遇复合物主要由静电相互作用主导，这些相互作用有助于复合物的模糊性，但也有疏水相互作用的贡献。
