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Assignment of backbone resonances in a eukaryotic protein kinase - ERK2 as a representative example.

ABSTRACT: A first step toward the analysis of the structure, dynamics, and interactions of proteins by NMR is obtaining an acceptable level of resonance assignments. This process is nontrivial in most eukaryotic kinases given their size and suboptimal behavior in solution. Using inactive ERK2 as a representative example, we describe the procedures we utilized to achieve a significant degree of completeness of backbone resonance assignment.

SUBMITTER: Piserchio A

PROVIDER: S-EPMC3305997 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Assignment of backbone resonances in a eukaryotic protein kinase - ERK2 as a representative example.

Piserchio Andrea A Dalby Kevin N KN Ghose Ranajeet R

Methods in molecular biology (Clifton, N.J.) 20120101

A first step toward the analysis of the structure, dynamics, and interactions of proteins by NMR is obtaining an acceptable level of resonance assignments. This process is nontrivial in most eukaryotic kinases given their size and suboptimal behavior in solution. Using inactive ERK2 as a representative example, we describe the procedures we utilized to achieve a significant degree of completeness of backbone resonance assignment. ...[more]

PMID: 22167683

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Project description:Experimental residual dipolar couplings (RDCs) in combination with structural models have the potential for accelerating the protein backbone resonance assignment process because RDCs can be measured accurately and interpreted quantitatively. However, this application has been limited due to the need for very high-resolution structural templates. Here, we introduce a new approach to resonance assignment based on optimal agreement between the experimental and calculated RDCs from a structural template that contains all assignable residues. To overcome the inherent computational complexity of such a global search, we have adopted an efficient two-stage search algorithm and included connectivity data from conventional assignment experiments. In the first stage, a list of strings of resonances (CA-links) is generated via exhaustive searches for short segments of sequentially connected residues in a protein (local templates), and then ranked by the agreement of the experimental (13)C(α) chemical shifts and (15)N-(1)H RDCs to the predicted values for each local template. In the second stage, the top CA-links for different local templates in stage I are combinatorially connected to produce CA-links for all assignable residues. The resulting CA-links are ranked for resonance assignment according to their measured RDCs and predicted values from a tertiary structure. Since the final RDC ranking of CA-links includes all assignable residues and the assignment is derived from a "global minimum", our approach is far less reliant on the quality of experimental data and structural templates. The present approach is validated with the assignments of several proteins, including a 42 kDa maltose binding protein (MBP) using RDCs and structural templates of varying quality. Since backbone resonance assignment is an essential first step for most of biomolecular NMR applications and is often a bottleneck for large systems, we expect that this new approach will improve the efficiency of the assignment process for small and medium size proteins and will extend the size limits assignable by current methods for proteins with structural models.

Project description:Five hundred protein kinases phosphorylate 10 000 proteins in human cells. Frequently, more than one site in a protein is phosphorylated, and often by more than one protein kinase. The mechanistic basis underlying the overlapping specificity of the phospho-proteome is not well understood. We are interested in understanding why ERK2, a proline-directed protein kinase, phosphorylates only a fraction of the (S/T-P) sites found in the surface loops of proteins, at an appreciable rate. To address this fundamental question, we utilized a well-established protein substrate EtsDelta138, which comprises a globular ERK2-recognition domain (pnt domain) and an unstructured peptide-like N-terminal tail. This tail contains T38, the sole ERK2 phosphorylation site. We mutated the TP motif, which is recognized by the active site and found that mutagenesis of the T-38/P-39 motif to TD, TR, TA, TG, and TV has no effect on the stability of the ternary complex but does decrease kcat. We also investigated the effect of perturbing the binding between ERK2 and the pnt domain, which occurs outside the active site, to find that mutation of the pnt domain (F120A) leads to a 10-fold decrease in binding but the kcat remains the same. The data support a mechanism of proximity-mediated catalysis, where the docking of the pnt domain, outside the active site, increases the effective concentration of the TP motif near the active site. The data are consistent with the notion that the interaction between ERK2 and the pnt domain provides uniform binding energy and stabilizes each enzyme intermediate and transition state to an equal extent. While other steps on the reaction pathway contribute towards the specificity of the ERK2 reaction, a docking interaction provides the initial basis for substrate recognition. Those residues within the docked complex, which have the ability to access the active site with an appropriate geometry, can be phosphorylated at an efficient rate if followed by a proline or small hydrophobic amino acid.

Dataset Information

Assignment of backbone resonances in a eukaryotic protein kinase - ERK2 as a representative example.

Publications

Assignment of backbone resonances in a eukaryotic protein kinase - ERK2 as a representative example.

Similar Datasets

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