Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2

Abstract

Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly 13C/15N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap. The challenges involved in studies of larger proteins led to efficient methods for 13C labeling of side chain methyl groups, which have favorable relaxation properties and high signal-to-noise. These methyls are often still assigned by linking them to the previously assigned backbone, thus limiting the applications for larger proteins. Here, a structure-based procedure is described for assignment of 13C1H3-labeled methyls by comparing distance information obtained from three-dimensional methyl–methyl nuclear Overhauser effect (NOE) spectroscopy with the X-ray structure. The Ile, Leu, or Val (ILV) methyl type is determined by through-bond experiments, and the methyl–methyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed “NOE-methyl cluster” onto the structure. The combination of identification of ILV methyl type with mapping of the NOE-methyl clusters greatly simplifies the assignment process. This method was applied to the inactive and active forms of the 42-kDa ILV 13C1H3-methyl labeled extracellular signal-regulated kinase 2 (ERK2), leading to assignment of 60% of the methyls, including 90% of Ile residues. A series of ILV to Ala mutants were analyzed, which helped confirm the assignments. These assignments were used to probe the local and long-range effects of ligand binding to inactive and active ERK2.

Sparse multidimensional iterative lineshape-enhanced (SMILE) reconstruction of both non-uniformly sampled and conventional NMR data

“…Of the many 4D pulse schemes in use, 4D NOESY measurements are arguably the most critical to structural studies, as they can dramatically reduce the spectral crowding of 2D and 3D NOESY spectra (Kay et al 1990; Clore et al 1991; Zuiderweg et al 1991). 4D NOESY of methyl–methyl interactions is particularly useful for deriving distance restraints in larger proteins, which often require perdeuteration with ILV-protonation of methyl groups (Tugarinov et al 2005; Hiller et al 2009; Sheppard et al 2009; Coggins et al 2012; Linser et al 2014; Xiao et al 2015). Here, we therefore include an example of the use of SMILE for processing of such a spectrum.…”

NMR assignments of sparsely labeled proteins using a genetic algorithm

“…Metabolic labeling in methyl groups of isoleucine, leucine and valine, combined with perdeuteration, has become a popular approach to NMR-based structural work on large proteins (Tugarinov et al 2006). Some alternate assignment strategies have also been developed for these systems (Chao et al 2014; John et al 2007; Mishra and Frueh 2015; Xiao et al 2015), but we believe the approach described here could offer some advantages. Labeling with 13 C-methyl alanine provides the same excellent sensitivity and resolution, but it also provides RDCs that are backbone centered, much like 15 N- 1 H amide RDCs.…”

“…However, these approaches are labor intensive. 13 C-methy labeling of large proteins can also be seen as a type of sparse labeling, and this has fostered the development of assignment strategies that depend on data that can be collected via these sites, primarily methyl-methyl NOEs (Xiao et al 2015) and paramagnetic effects (John et al 2007). It has also stimulated exploration of probabilistic approaches to assignment using these and other types of data (Chao et al 2014; Mishra and Frueh 2015).…”

Backbone resonance assignments of the catalytic and regulatory domains of Ca2+/calmodulin-dependent protein kinase 1D

“…However, protein kinases are challenging to study by NMR, often yielding poor spectra due to their intrinsic dynamics and large molecular weights. Thus few protein kinases have been assigned (Langer et al 2004 ; Vogtherr et al 2005 , 2006 ; Gelev et al 2006 ; Vajpai et al 2008 ; Masterson et al 2009 ; Xiao et al 2015 ; Serimbetov et al 2017 ; Sanfelice et al 2018 ). Following optimization of solution conditions, the autoinhibited human CaMK1D was found to be amenable to NMR analysis, yielding resolved spectra under physiological buffer conditions.…”