Tyrosine Phosphorylation within the Intrinsically Disordered Cytosolic Domains of the B-Cell Receptor: An NMR-Based Structural Analysis

<div><p>Intrinsically disordered proteins are found extensively in cell signaling pathways where they often are targets of posttranslational modifications e.g. phosphorylation. Such modifications can sometimes induce or disrupt secondary structure elements present in the modified protein. CD79a and CD79b are membrane-spanning, signal-transducing components of the B-cell receptor. The cytosolic domains of these proteins are intrinsically disordered and each has an immunoreceptor tyrosine-based activation motif (ITAM). When an antigen binds to the receptor, conserved tyrosines located in the ITAMs are phosphorylated which initiate further downstream signaling. Here we use NMR spectroscopy to examine the secondary structure propensity of the cytosolic domains of CD79a and CD79b <i>in vitro</i> before and after phosphorylation. The phosphorylation patterns are identified through analysis of changes of backbone chemical shifts found for the affected tyrosines and neighboring residues. The number of the phosphorylated sites is confirmed by mass spectrometry. The secondary structure propensities are calculated using the method of intrinsic referencing, where the reference random coil chemical shifts are measured for the same protein under denaturing conditions. Our analysis revealed that CD79a and CD79b both have an overall propensity for α-helical structure that is greatest in the C-terminal region of the ITAM. Phosphorylation of CD79a caused a decrease in helical propensity in the C-terminal ITAM region. For CD79b, the opposite was observed and phosphorylation resulted in an increase of helical propensity in the C-terminal part.</p></div

Secondary structure propensity of CD79b and the effects of phosphorylation.

<p>The ITAM region in the sequence is underlined and all shifts are plotted against their corresponding residue number (<b>A</b>) positive values of secondary chemical shifts (Δδ^Cα–Δδ^Cβ) indicate an overall tendency for α-helical structure with an increased propensity in the region Thr206 to Gly216. (<b>B</b>) change of secondary chemical shifts upon phosphorylation (Δδ^Cα–Δδ^Cβ)_P–(Δδ^Cα–Δδ^Cβ). Positive values for residues Tyr207 to Gly216 indicates an increased helical content in this region following phosphorylation. (<b>C</b>) Δδ^CO secondary chemical shifts. The Δδ^CO shift pattern agrees well with the pattern of (Δδ^Cα–Δδ^Cβ) indicating an overall tendency for α-helical structure (<b>D</b>) change of secondary chemical shifts (Δδ_P^CO–Δδ^CO). Phosphorylation increases the tendency for α-helical structure in the C-terminal part of the ITAM region.</p

Secondary structure propensity of CD79a and the effects of phosphorylation.

<p>The ITAM region in the sequence is underlined and all shifts are plotted against their corresponding residue number (<b>A</b>) (Δδ^Cα–Δδ^Cβ) secondary chemical shifts. CD79a has an overall tendency for α-helical structure with an increased propensity in the regions Arg166 to Gly175 and Asp194 to Gly205. (<b>B</b>) (Δδ^Cα–Δδ^Cβ)_P–(Δδ^Cα–Δδ^Cβ) secondary chemical shifts. Negative values in the C-terminal part of the ITAM indicate decreased helicity in this region following phosphorylation. (<b>C</b>) Δδ^CO secondary chemical shifts. The Δδ^CO shift pattern agrees well with the pattern of (Δδ^Cα–Δδ^Cβ) indicating an overall tendency for α-helical structure (<b>D</b>) (Δδ_P^CO–Δδ^CO) secondary chemical shifts. Phosphorylation decreases the tendency for α-helical structure in the C-terminal part of the ITAM region.</p

Chemical shift changes induced by tyrosine phosphorylation.

<p>(<b>A</b>) δ−δ_P (black bars) and (δ−δ_P)_UREA (gray bars) of CD79a and CD79b calculated from Cα chemical shifts. For CD79a, significant δ−δ_P values can be observed surrounding Tyr188, Tyr199 and Tyr210 indicating phosphorylation of these sites. For CD79b, such values are observed surrounding Tyr196 and Tyr207. A comparison between δ−δ_P and (δ−δ_P)_UREA reveals that a dominating part of the chemical shift changes induced by phosphorylation is still present in 6 M urea. (<b>B</b>) δ−δ_P (black bars) and (δ−δ_P)_UREA (gray bars) of CD79a and CD79b calculated from CO chemical shifts. Analysis using CO chemical shifts results in similar patterns as Cα. (<b>C</b>) Overlays of ¹H-¹⁵N-HSQC spectra of CD79a_P and CD79b_P (red) and the corresponding spectra of CD79a and CD79b (black). Phosphorylation induces changes in the amide chemical shifts of targeted tyrosines as well as surrounding residues. Phosphorylated tyrosines show a ¹H downfield shift while the direction of the ¹⁵N shifts varies. Upon phosphorylation, the amide peaks of the tyrosines tend to move into already crowded areas of the spectra.</p

Estimate of the secondary structure in MALT1_Casp-Ig3(338–719).

<p>Secondary chemical shifts (Δδ) were calculated by subtracting random coil chemical shifts corrected for nearest-neighbour effects from ¹³C’, ¹³Cα and ¹³Cβ chemical shifts corrected for deuterium isotope shifts. Consecutive values above 0.7 indicates alpha helix, while consecutive values below -0.7 indicates beta strand for Δδ¹³C’ and Δδ¹³Cα. The opposite is true for Δδ¹³Cβ. The CSI for the three nuclei were averaged and reported as a “consensus” CSI. β3, β3A and β3B are denoted β3 AB in the Fig. The star (*) indicates that the secondary structure is part of the Ig3 domain.</p

Estimated secondary structure from NMR experiments (in black) compared to secondary structure from the in-house X-ray structure (in dark grey) and from the published X-ray structure of <i>apo</i> MALT1_Casp-Ig3, PDB ID: 3V55 (in light grey).

<p>Alpha helices are indicated with a greater symbol size than the beta-sheets. β3, β3A and β3B are denoted β3 AB in the Fig. The star (*) indicates that the secondary structure is part of the Ig3 domain.</p

¹H-¹⁵N TROSY spectrum of MALT1_Casp-Ig3(338–719) with the assigned amino acid residue number annotated.

<p>¹H-¹⁵N TROSY spectrum of MALT1_Casp-Ig3(338–719) with the assigned amino acid residue number annotated.</p

Peak appearance progress during the course of the TA procedure for the MALT1 sample.

<p>The horizontal axis shows the total measurement time excluding the HNCO experiment, which was recorded prior to the TA. The spectral processing and analysis was done automatically during the course of the data acquisition.</p

Zoomed region of the ¹⁵N-HSQC of CD79a in different conditions.

<p>(<b>A</b>) NaPi buffer, (<b>B</b>) 6 M urea, (<b>C</b>) 20% TFE, (<b>D</b>) reduced spin label (MTSL) attached to CD79a, (<b>E</b>) K4C/C35S form, (<b>F</b>) Y25E/Y36E form. Selected peaks are annotated to show rearrangements of the signal position for the different conditions (19Y, 35M) or position of the specific mutations (4K, 33C, 25Y, 36Y). Peaks outside the zoomed region are shown as arrows pointing towards the correct position.</p

Cell-free expressed cytosolic constructs of the T cell- and B cell receptor.

<p>(<b>A</b>) Cartoon of the receptors with indicated immunoreceptor tyrosine-based activation motifs (ITAMs), (<b>B</b>) SDS-PAGE gel of <i>in vitro</i> expressed disordered constructs in levels suitable for NMR experiments.</p