Solution NMR Insights into Docking Interactions Involving Inactive ERK2

Abstract

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.

The PNT domain from Drosophila pointed‐P2 contains a dynamic N‐terminal helix preceded by a disordered phosphoacceptor sequence

“…As shown in Figure 3(A), the heteronuclear 15 N-NOE values of residues in helix H0 progressively decrease toward its N-terminus. A modelfree analysis of the 15 N T 1 , T 2 , and NOE values of PntP2 142-252 shows a similar trend with lower S 2 order parameters for amides near the start of this helix, as well as at the C-terminus of helix H5 (Supporting Information Fig.…”

“…1(B)]. With the exception of Glu142, Val143, Phe166 and the N-terminal Gly-Ser-His-Met tag, almost complete 1 H N , 15 N, 13 C a , 13 C b , and 13 C 0 resonance assignments were obtained for this construct. These chemical shifts were used to identify the secondary structural elements of PntP2 142-252 with the MICS (Motif Identification by Chemical Shift) algorithm.…”

“…The dynamic properties of PntP2 142-252 were examined using 15 N relaxation and amide HX measurements. As shown in Figure 3(A), the heteronuclear 15 N-NOE values of residues in helix H0 progressively decrease toward its N-terminus.…”

“…These data are indicative of enhanced backbone mobility on a nsec-psec timescale relative to the well-ordered SAM core. However, it is difficult to estimate the nature of the conformations sampled by these fast motions from 15 N relaxation measurements alone. 30 More importantly, rapid amide HX was detected for residues throughout helices H0/H1 [ Fig.…”

“…10,11 The docking interfaces on the PNT domain and the kinase have been mapped coarsely through mutagenesis, NMR spectroscopic, chemical footprinting, and competition studies. 10,[12][13][14][15] To both accommodate the proposed docking mechanism and position the phosphoacceptors in the catalytic site of the kinase, a significant conformational change in the ETS1 PNT domain, such as the unfolding of the appended Nterminal helix H0 (residues Lys42-Thr52), appears to be required. 16 Indeed, NMR relaxation and amide hydrogen exchange (HX) measurement have shown that this helix is only marginally stable and structurally flexible.…”