A comprehensive description is presented of the effects on two-spin coherences (i.e., superpositions of zero-and double-quantum coherences) of cross-correlation between the fluctuations of two different relaxation mechanisms in nuclear magnetic resonance (NMR). Dipole-dipole (DD) interactions between four nuclei and chemical shift anisotropy (CSA) of two of these nuclei are considered. Two complementary experiments have been designed for 15 N, 13 C-labeled proteins to quantify the effects of cross-correlation between the 13 C R -1 H R and 15 N-1 H N dipolar interactions on two-spin coherences involving 13 C R of the ith residue with the 15 N of the (i+1)th amino acid. Two other experiments allow one to quantify the effect of cross-correlation between the 13 C′ (carbonyl) CSA and the 13 C R -1 H R dipolar coupling on the relaxation of two-spin coherences involving the 13 C′ and 13 C R nuclei on the same residue of the protein. These experiments have been used to extract relevant cross-correlation rates in 15 N, 13 C-labeled human ubiquitin. These rates show a high degree of correlation with the backbone Ψ angles in proteins.

The mitogen activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and non-canonical motifs found in a variety of upstream kinases, regulating phosphatases and downstream targets. Interactions involving two of these sites, the D-recruitment site (DRS) and the F-recruitment site (FRS), have been shown to play a key role in signal transduction by ERK/MAP kinases. The dynamic nature of these recruitment events makes NMR uniquely suited to provide significant insight into these interactions. While NMR studies of kinases in general have been greatly hindered by their large size and complex dynamic behavior leading to the sub-optimal performance of standard methodologies, we have overcome these difficulties for inactive full-length ERK2 and obtained an acceptable level of backbone resonance assignments. This allowed a detailed investigation of the structural perturbations that accompany interactions involving both canonical and non-canonical recruitment events. No crystallographic information exists for the latter. We found that the chemical shift perturbations in inactive ERK2, indicative of structural changes in the presence of canonical and non-canonical motifs, are not restricted to the recruitment sites, but also involve the linker that connects the N- and C-lobes and, in most cases, a gatekeeper residue that is thought to exert allosteric control over catalytic activity. We also found that, even though the canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions, the non-canonical interactions primarily involve the latter. These results demonstrate the feasibility of solution NMR techniques for a comprehensive analysis of docking interactions in a full-length ERK/MAP kinase.

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