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Solution NMR insights into docking interactions involving inactive ERK2.

ABSTRACT: The mitogen-activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and noncanonical 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 suboptimal 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 noncanonical 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 noncanonical 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 noncanonical 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.

SUBMITTER: Piserchio A

PROVIDER: S-EPMC3103835 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Solution NMR insights into docking interactions involving inactive ERK2.

Piserchio Andrea A Warthaka Mangalika M Devkota Ashwini K AK Kaoud Tamer S TS Lee Sunbae S Abramczyk Olga O Ren Pengyu P Dalby Kevin N KN Ghose Ranajeet R

Biochemistry 20110419 18

The mitogen-activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and noncanonical 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 int ...[more]

PMID: 21449613

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Project description:ERK2 primarily recognizes substrates through two recruitment sites, which lie outside the active site cleft of the kinase. These recruitment sites bind modular-docking sequences called docking sites and are potentially attractive sites for the development of non-ATP competitive inhibitors. The D-recruitment site (DRS) and the F-recruitment site (FRS) bind D-sites and F-sites, respectively. For example, peptides that target the FRS have been proposed to inhibit all ERK2 activity (Galanis, A., Yang, S. H., and Sharrocks, A. D. (2001) J. Biol. Chem. 276, 965-973); however, it has not been established whether this inhibition is steric or allosteric in origin. To facilitate inhibitor design and to examine potential coupling of recruitment sites to other ligand recognition sites within ERK2, energetic coupling within ERK2 was investigated using two new modular peptide substrates for ERK2. Modeling shows that one peptide (Sub-D) recognizes the DRS, while the other peptide (Sub-F) binds the FRS. A steady-state kinetic analysis reveals little evidence of thermodynamic linkage between the peptide substrate and ATP. Both peptides are phosphorylated through a random-order sequential mechanism with a k(cat)/K(m) comparable to Ets-1, a bona fide ERK2 substrate. Occupancy of the FRS with a peptide containing a modular docking sequence has no effect on the intrinsic ability of ERK2 to phosphorylate Sub-D. Occupancy of the DRS with a peptide containing a modular docking sequence has a slight effect (1.3 ± 0.1-fold increase in k(cat)) on the intrinsic ability of ERK2 to phosphorylate Sub-F. These data suggest that while docking interactions at the DRS and the FRS are energetically uncoupled, the DRS can exhibit weak communication to the active site. In addition, they suggest that peptides bound to the FRS inhibit the phosphorylation of protein substrates through a steric mechanism. The modeling and kinetic data suggest that the recruitment of ERK2 to cellular locations via its DRS may facilitate the formation of F-site selective ERK2 signaling complexes, while recruitment via the FRS will likely inhibit ERK2 through a steric mechanism of inhibition. Such recruitment may serve as an additional level of ERK2 regulation.

Project description:Many multidomain proteins and ribonucleic acids consist of domains that autonomously fold and that are linked together by flexible junctions. This architectural design allows domains to sample a wide range of positions with respect to one another, yet do so in a way that retains structural specificity, since the number of sampled conformations remains extremely small compared to the total conformations that would be sampled if the domains were connected by an infinitely long linker. This "tuned" flexibility in interdomain conformation is in turn used in many biochemical processes. There is great interest in characterizing the dynamic properties of multidomain systems, and moving beyond conventional descriptions in terms of static structures, toward the characterization of population-weighted ensembles describing a distribution of many conformations sampled in solution. There is also great interest in understanding the design principles and underlying physical and chemical interactions that specify the nature of interdomain flexibility. NMR spectroscopy is one of the most powerful techniques for characterizing motions in complex biomolecules and has contributed greatly toward our basic understanding of dynamics in proteins and nucleic acids and its role in folding, recognition, and signaling. Here, we review methods that have been developed in our laboratories to address these challenges. Our approaches are based on the ability of one domain of the molecule to self-align in a magnetic field, or to dominate the overall orientation of the molecule, so that the conformational freedom of other domains can be assessed by their degree of alignment induced by the aligned part. In turn, this self-alignment ability can be intrinsic or can be caused by tagging appropriate constructs to the molecule of interest. In general, self-alignment is due to magnetic susceptibility anisotropy. Nucleic acids with elongated helices have this feature, as well as several paramagnetic metal centers that can be found in, or attached to, a protein domain.

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Solution NMR insights into docking interactions involving inactive ERK2.

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Solution NMR insights into docking interactions involving inactive ERK2.

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