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 (13)C/(15)N-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 (13)C 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 (13)C(1)H3-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 (13)C(1)H3-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.

Figure 1

Hierarchical strategy for structure-based assignment of the ILV methyl resonances in ERK2. (a) Identification of methyl residue type (I, L, or V) utilizes both the ¹³C chemical shifts and through-bond correlation experiments. (b) The predicted and observed Ile–Ile NOE clusters are analyzed first using a hierarchy of largest to smallest clusters. If there is only a single observed and predicted Ile cluster of a particular size, then this cluster can be uniquely mapped onto the X-ray structure. If a cluster cannot be uniquely mapped using only Ile–Ile NOEs then additional Ile–Val and Ile–Leu NOEs are progressively analyzed to try to uniquely map each cluster onto the structure. (c) Once specific clusters have been mapped onto the X-ray structure, the patterns and sizes of the predicted and observed methyl–methyl NOEs are qualitatively analyzed to assign individual methyl resonances within a cluster to specific residues in the sequence. Examples for various criteria are given in italics.

Figure 2

Identification of methyl residue type in ERK2 using ¹³C chemical shifts and through-bond 3D HMCMCBCA spectra. (a) The pattern of ¹³C-labeling for Ile, Leu, and Val residues in the ERK2 samples used in this 3D experiment, where, as previously described, several of the side chain carbons in Ile, and backbone carbons in Leu, originate from the ¹²C-labeled glucose and not from the ¹³C-labeled precursor (see Methods). The aliphatic carbons colored blue and magenta have opposite signs for their resonances in the 3D spectrum. (b) Strip plots of the 3D HMCMCBCA spectrum for 0P-ERK2 showing distinct chemical shifts for Leu and Val aliphatic carbons. In the ¹³C aliphatic dimension, Ile residues have one peak, whereas two peaks of opposite sign (colored blue and magenta) are observed for Leu (C^γ and C^β) and Val (C^β and C^α). The brackets denote the typical range of chemical shift (average ± 3σ, 99.7%) for a specific aliphatic carbon in ILV using data from the BMRB. The identity of Leu and Val was primarily determined by the distinct chemical shifts of the Val C^α and the Leu C^β resonances (see text).

Figure 3

Mapping of Ile-methyl-NOE clusters onto the X-ray structure of ERK2. (a) Strip plots of the 3D (¹³C, ¹³C, ¹H) HMQC-NOESY-HMQC spectra for 0P-ERK2 illustrating the Ile-methyl-NOE clusters in the protein. Diagonal peaks are marked with squares, the horizontal solid lines indicate NOE cross peaks to other methyls, and dashed lines indicate where a cross peak is not observed. Asterisks mark peaks that arise from bleed-through in the third dimension from a different methyl resonance, and double asterisks indicate noise peaks or artifacts. The numbers at the bottom of each strip plot are the ¹³C chemical shift for this methyl plane. (b) Geometry models illustrating the various Ile-methyl-NOE clusters in ERK2. The numbers at the vertex represent the methyl for that strip plot in (a) and a line connecting two vertices indicates an observed NOE between these two methyls with single and double headed arrows indicating that one and two NOE cross peaks, respectively, were observed between the two resonances. (c) Ile-methyl clusters in the X-ray structure of ERK2 that fit the geometry models in (b). The upper panel shows the only six-residue Ile-methyl cluster (using an 8 Å cutoff), the middle panel shows the only three-residue Ile-methyl cluster, and the bottom two panels show two of the five 2-residue Ile-methyl clusters. Ile side chains are shown as red lines, their C^δ1 methyls as red spheres and the backbone is a gray ribbon.

Figure 4

Ile, Val, or Leu to Ala mutations used to confirm methyl assignments. (a) Structure of ERK2 showing the sites of Ala mutations. The backbone is represented as a gray ribbon and side chains of mutation sites are shown in red with their methyls as spheres. (b) Overlay of the Ile region of the 2D (¹³C, ¹H) HMQC spectra of mutant I124A (red) and wild-type 0P-ERK2 (black). The missing peak was unambiguously assigned to I124. (c) Overlay of the Ile region of the HMQC spectra of mutant L161A (red) and wild-type 0P-ERK2 (black). (d) The L161 region of the X-ray structure of 0P-ERK2 where the distances from the L161 methyl groups to nearby Ile methyls are shown.

Figure 5

Binding of the peptide Elk1D to inactive and active ERK2. (a) Titration of Elk1D into 0P- or 2P-ERK2 as monitored by chemical shift changes of methyl resonances for L155 and L119 in the 2D HMQC spectra. The arrows show the change in the position of the methyl resonance upon titration with Elk1D, where the free ERK2 is in black, and the saturated peak ([0P-ERK2]:[Elk1D] = 1:7, [2P-ERK2]:[Elk1D] = 1:10) is in red. The CSP_C,H values for these methyl resonances are similar in 0P and 2P-ERK2 (Table S2). (b) The CSP_C,H values for L155 and L119 are plotted against the concentration of Elk1D, where the solid lines are the global fits to a single-site binding curve. (c) The CSP_C,H values for 2P-ERK2 derived from the binding curves are mapped onto the X-ray structure of 0P-ERK2 in complex with a DEJL-motif peptide (colored in gold) derived from hematopoietic tyrosine phosphatase (HePTP, PDB 2GPH). The color scale for the CSP_C,H values is on the right, with blue indicating no measurable perturbation and red indicating the largest perturbation.

Figure 6

Binding of AMP-PNP to inactive and active ERK2. (a) A portion of the Leu/Val region of the 2D HMQC spectra of 0P- and 2P-ERK2 upon titration with AMP-PNP. These spectra show that one of the methyl resonances of L26 is in slow exchange on the NMR chemical shift time scale between free and bound forms for both 0P-and 2P-ERK2. The arrows point to the peak in the free form of ERK2, and the dashed lines outline the position of the free peak. (b) The CSP_C,H for methyls between the free and AMP-PNP-bound form is mapped onto the X-ray structure of 0P-ERK2 bound to ATP (PDB 4GT3). The color scale for the CSP_C,H values is on the right, with blue indicating no measurable perturbation, red indicating the largest perturbations, and black indicating that these peaks were not observed in the spectra with saturated AMP-PNP, possibly due to chemical exchange broadening in the bound state. The ligand ATP is highlighted with orange sticks. The side chains of the eight catalytic spine residues were shown in magenta sticks. Six out of the eight these residues were I/L/V, with their methyls highlighted with magenta circles.