Probing Transient Release of Membrane-Sequestered Tyrosine

Aug 1, 2017 - To solve the severe resonance overlap problem in the 2D spectra of mIgG-tail, a non-uniformly sampled pseudo-4D hydrogen exchange NMR ex...
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Probing Transient Release of Membrane-Sequestered TyrosineBased Signaling Motif by Solution NMR Spectroscopy Hui Wang,† Shengli Wang,† Changting Li,‡ Hua Li,‡ Yunyun Mao,† Wanli Liu,§ Chenqi Xu,*,‡ and Dong Long*,† †

Hefei National Laboratory for Physical Sciences at the Microscale & School of Life Sciences, University of Science and Technology of China, 443 Huangshan Street, Hefei 230027, China ‡ State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China § School of Life Sciences, Tsinghua University, Beijing 100084, China S Supporting Information *

ABSTRACT: Membrane sequestration of tyrosine-based signaling motifs of antigen receptors effectively restricts the signaling activities in resting lymphocytes. However, low level of basal signaling in resting cells is required for lymphocyte survival and antigen responsiveness, of which the molecular mechanism remains obscure. Here we probe the transient release of the cytoplasmic domain of the membrane-bound IgG heavy chain (mIgG-tail) by hydrogen exchange NMR spectroscopy, illustrating a dynamic molecular basis for its basal signaling activity. To solve the severe resonance overlap problem in the 2D spectra of mIgG-tail, a non-uniformly sampled pseudo-4D hydrogen exchange NMR experiment has been exploited to quantitatively measure site-specific hydrogen exchange rates. Our solution NMR study reveals transient solvent exposure of the ITT signaling motif that can be further enhanced by calcium ion, and provides insight into the mechanism of lymphocyte basal signaling.

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levels has shown that membrane sequestration of the ITT motif effectively restricts the access of the key tyrosine, while the induced dissociation by antigen engagement or calcium mobilization leads to its phosphorylation and eventual activation of cells.7 Interestingly, the activity of the ITT motif is not fully blocked in resting B cells and a low level of basal ITT phosphorylation can be faithfully observed.7,13−15 Structurally, mIgG-tail falls into the category of intrinsically disordered regions (IDRs)16,17 in which each residue samples very diverse conformations in solution.7 The binding kinetics between the unstructured ensemble of mIgG-tail and the membrane is therefore inherently complex and may not be depicted as a global two-state model. The details of interplay between mIgG-tail and the plasma membrane on a per-residue basis, as well as shift of populations in the presence of Ca2+, remain obscure. A site-specific examination of the transient solvent-exposed state of membrane-sequestered mIgG-tail, as well as the enhanced dissociation from the membrane by calcium ions, will extend our understanding of the mechanism of receptor basal signaling and activation. In this work, we probe the transient release of membranesequestered mIgG-tail using hydrogen exchange rates that are

n essential step of adaptive immune response is the recognition of antigen molecules by antigen receptors, i.e., B cell receptor (BCR) and T cell receptor (TCR), and the subsequent phosphorylation of tyrosine-based signaling motifs in the cytoplasmic domains of antigen receptors that activates sophisticated downstream signaling pathways.1 Increasing evidence suggests that acidic phospholipid molecules play highly active roles in regulating the membrane protein functions.2−6 Deep insertion of tyrosine-based signaling motifs into the membrane bilayer sequesters key tyrosine residues from cytosolic solution, and represents a safety control mechanism to limit BCR/TCR signaling in resting lymphocytes. Antigen stimulation releases these sequestered signaling motifs from the membrane, which are then phosphorylated by tyrosine kinases to trigger downstream signaling cascades.6,7 It has been long recognized that a low level of BCR/TCR basal signaling is required for lymphocyte survival and antigen responsiveness.8−10 However, the molecular mechanism of BCR/TCR basal signaling remains elusive. Memory B cells express isotype-switched IgG-B cell receptors (IgG-BCRs).7 The membrane-bound IgG contains a cytoplasmic domain (mIgG-tail) of 28 amino acid residues with a key immunoglobulin tail tyrosine (ITT) motif that is indispensable for the enhanced signaling of IgG-BCRs.11,12 Moreover, recent studies also demonstrated that the ITT improves the survival of IgG-BCR expressing memory B cells.13 Experimental evidence at both molecular and cell © XXXX American Chemical Society

Received: June 28, 2017 Accepted: July 27, 2017

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experiment often limits its practical application, particularly to biological samples with low stability. Non-uniform sampling (NUS) technique27−33 provides a tool to significantly accelerate NMR data collection, and allows the measurement of high dimensionality spectra in comparable times with lower dimensionality spectra. Despite the advantage offered by NUS, it has not found wide application in quantitative NMR analyses, partly owing to the concerns of potential distortion of peak intensities in NUS spectral reconstruction. We have recently demonstrated a general NUS processing method for quantitative analysis of relaxation-based NMR experiments,34,35 in which accurate signal intensities can be extracted through modeling of the non-uniformly sampled time-domain data without reconstruction of frequency dimensions, exploiting the fact that the frequencies and lineshapes of resonances remain invariant with respect to relaxation delays or radio frequency fields. In the present study, this approach is further extended to the pseudo-4D CLEANEX-PM experimental scheme (Figure S1). In order to obtain the intrinsic hydrogen exchange rates (krc, eq 1) of mIgG-tail as well as to validate the NUS pseudo-4D CLEANEX-PM experiment, we measured the kH‑EX of lipidfree mIgG-tail, which assumes a “random coil” conformation in solution, using both the “standard” uniformly sampled HSQC-based experiment and non-uniformly sampled HNCObased experiment. Figure 2A compares the relative signal intensities measured in the pseudo-3D and pseudo-4D experiments. Due to the small difference in the fractions of water (f w) preserved at the beginning of the mixing period, which were measured to be 0.7 (pseudo-4D) and 0.64 (pseudo-3D) respectively, the observed relative intensities in the pseudo-4D experiment are slightly (∼9%) higher than those in the pseudo-3D experiment. Fitting of the experimental data to the theoretical model (eq S2) extracted the respective hydrogen exchange rates from the two experiments, which show a high level of consistency (Figure 2C). It is noteworthy that the kH‑EX rates of the overlapped residues as shown in Figure 1 are also available in the pseudo4D approach (Figure 2D). The bicelle constituted by 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) and dihexanoyl-phosphatidylcholine (DHPC) at a molar ratio of 0.8:1 was previously shown to be a physiologically relevant membrane analogue that binds mIgG-tail in its lipid bilayer.7 In order to measure the residual hydrogen exchange rates in bicelle-bound mIgG-tail contributed by the transient solvent-exposed state as well as to improve spectral quality, the pseudo-4D CLEANEX-PM experiment was carried out at pH 7.34 and 40 °C. Under this condition, distinct signals of the C-terminal region (residues 21−28) including the critical ITT motif, originated from the water magnetization, could be detected (Figure 3A,B), attesting to the transient release of the C-terminal region. Based on the kH‑EX values of lipid-free and -bound mIgG-tail (see the Supporting Information (SI) for details), the population of the transiently solvent-exposed state (pSE) of the ITT motif is estimated to be ∼10% (Figure 3C). Variation of pSE among residues in the C-terminal region is observed with higher pSE for residues closer to the C-terminal end, which is in accord with the intrinsically disordered nature of mIgG-tail and highlights its residue-specific interactions with the lipid bilayer. The signals of the N-terminal region, in contrast, were hardly built up during the mixing period, with the extracted kH‑EX values being few s−1 and the errors on the

sensitive probes reporting on the solvent accessibility of individual residues,

where krc is the intrinsic “random coil” hydrogen exchange rate. Equation 1 is a special representation of the general Linderstrøm−Lang Model for amide hydrogen exchange,18 where proteins are pictured to undergo a closed-to-open reaction with hydrogen exchange occurring in the open state. NMR spectroscopy has emerged as a powerful tool for measuring hydrogen exchange rates (kH‑EX) of individual amide sites in proteins.19−25 In the phase-modulated CLEAN chemical exchange (CLEANEX-PM) experiment,19 the buildup of transferred magnetization is monitored as a function of the mixing time during which a spin-lock sequence is applied to suppress cross-relaxation and TOCSY effects, thereby allowing accurate extraction of kH‑EX. The signals of the hydrogen exchange NMR experiments are typically detected in the form of 2D 1H−15N correlation maps for application to biomolecules. Nevertheless, the narrow chemical shift dispersions (less than 0.8 ppm along the proton dimension) of mIgG-tail result in severe overlapping of 24% and 48% of the backbone resonances in the 2D spectra of lipid-free and -bound mIgG-tail (Figure 1). To facilitate site-

Figure 1. 1H−15N HSQC spectra of lipid-free (A) and POPG/ DHPC bicelle-bound (B) mIgG-tail recorded at 25 °C, 16.4 T (A) and 40 °C, 16.4 T (B), respectively. Assignments of severely overlapped resonances are labeled in green.

specific measurement of hydrogen exchange rates in mIgGtail, we describe herein a non-uniformly sampled pseudo-4D CLEANEX-PM experiment (Figure S1) with superior spectral resolution. This sparsely sampled pseudo-4D approach is first applied to the lipid-free mIgG-tail as a proof-of-principle study, and the subsequent application to POPG bicelle-bound mIgG-tail reveals transient solvent exposure of the C-terminal region that includes the ITT motif critical for signaling, which can be augmented by addition of Ca2+. Although measurement of kH‑EX using an HNCO-based detection scheme is technically straightforward,26 the substantially increased experimental time in a pseudo-4D 3766

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Figure 3. Hydrogen exchange rates of POPG/DHPC bicelle-bound mIgG-tail in the absence (blue) and presence (red) of CaCl2 ([Ca2+]/[POPG] = 0.05) measured with the non-uniformly sampled pseudo-4D CLEANEX-PM at 40 °C, pH 7.34. (A) Relative intensities of representative residues at the C-terminal region as a function of τm in the absence (blue) and presence (red) of Ca2+. (B) Comparison of extracted hydrogen exchange rates of bicelle-bound mIgG-tail with (red) and without (blue) Ca2+. (C) Populations of the solvent-exposed conformation (pSE) for individual residues estimated based on the measured solvent exchange rates (see the SI for details). The orange-shaded areas in panels B and C highlight the ITT motif (residues 21−24).

Figure 2. Measurement of the hydrogen exchange rates of the lipidfree mIgG-tail using the pseudo-3D HSQC-based versus nonuniformly sampled pseudo-4D HNCO-based CLEANEX-PM experiments, at 25 °C, pH 6.68. (A) Gradual buildup of the signals as a function of the mixing time (τm) in the pseudo-3D (filled circles; left panel) and pseudo-4D (open circles; right panel) experiments. I and I0 in the left (right) panel are signal intensities obtained from the pseudo-3D (pseudo-4D) CLEANEX-PM experiment and a reference HSQC (HNCO) spectrum, respectively (see the SI for details). The relative intensities of representative residues (S8, T16, V18, K22, and A28) are shown in black, blue, green, magenta, and red, respectively. The data of V18 and K22 are not available in the HSQC-based approach due to resonance overlap (Figure 1A). (B) A strip taken from the HNCO spectrum (15N = 122.7 ppm) showing separation of the resonances from V18 and K22 along the 13C dimension. The carbonyl shifts correspond to the 13CO nuclei of the preceding residues. (C) Correlation plot of the kH‑EX values obtained from the pseudo-4D experiment versus those from the pseudo-3D experiment, where R is the Pearson correlation coefficient. (D) Hydrogen exchange rates of lipid-free mIgG-tail plotted as a function of the residue number.

ionic interaction between mIgG-tail and the acidic lipid molecules.7 It is noted, however, that titration of Ca2+ to lipidbound mIgG-tail causes significant broadening and eventual disappearance of most resonances, indicating additional intermediate exchange processes that occur inside of the membrane, since binding/unbinding of mIgG-tail is fast on the chemical shift time scale (Figure S2) and the fast tumbling of the dissociated peptide in solution is expected to sharpen instead of broaden the spectral lines (Figure 1A). In this context, hydrogen exchange rates, which specifically respond to the binding/unbinding process, represent a desirable probe to quantitatively examine the effect of Ca2+ on the release of the ITT motif, avoiding complications from additional in-membrane conformational changes. To further demonstrate the utility of hydrogen exchange NMR spectroscopy, we measured kH‑EX of bicelle-bound mIgG-tail in the presence of CaCl2 at a molar ratio ([Ca2+]/[POPG]) of 0.05, which causes minimal line broadening. At this much lower concentration of Ca2+, very subtle chemical shift perturbations could be observed (Figure S3). Yet, the small increase in kH‑EX of the C-terminal residues was well captured (Figure 3A,B), showing the sensitivity of this experiment to change in pSE. The hydrogen exchange rates of residues 21−28 on average

same scale, making accurate determination of pSE difficult. Nevertheless, qualitatively different hydrogen exchange rates of the N- and C-terminal regions are clear and point out distinctly weakened affinity of the C-terminal region. The intrinsic dissociation propensity of the ITT motif provides a mechanistic explanation of the basal level signaling of mIgGtail observed in the quiescent B cells in the absence of antigen stimulation.13,14 Influx of Ca2+ from the calcium release-activated channel (CRAC) is a key event in the B cell signaling. As a second messenger, Ca2+ transmits signals involved in a variety of intracellular processes, such as kinase signaling and trafficking of transcription factors.36 It was previously shown that titration of Ca2+ to the POPG bicelle-bound mIgG-tail up to a molar ratio ([Ca2+]/[POPG]) of 0.6 could disrupt the 3767

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The Journal of Physical Chemistry Letters increased by ∼20%, while those in the N-terminal part remained very small, of which we could not tell any changes within the limit of experimental accuracy. It is noteworthy that kH‑EX as a probe of transient solvent exposed states shows comparable sensitivity to, and higher specificity than, that of chemical shift perturbations (see the SI for details). In summary, we have explored the residue-specific interaction between mIgG-tail and membrane, which is important to the regulation of BCR activity. Hydrogen exchange rates have been demonstrated as a quantitative tool for probing the transient solvent-exposed, signalingcompetent state of mIgG-tail, and a non-uniformly sampled pseudo-4D experiment is described to facilitate site-specific measurement with superior spectral resolution. The transient release of the ITT signaling motif in the membranesequestered mIgG-tail, which can be further enhanced by Ca2+, has been unambiguously detected, illustrating a biophysical basis for basal level signaling in resting B cells. Ionic protein−lipid interaction is known as a critical factor for controlling functions and activities of many signaling proteins in addition to lymphocyte antigen receptors,2 and is likely a general regulatory mechanism cells have adopted during evolution. The hydrogen exchange NMR spectroscopy could serve as a broadly applicable approach for characterizing protein−lipid interaction in quantitative and site-specific details.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.7b01662. Details of sample preparation, NMR experiments, data processing, and supporting figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D.L.). *E-mail: [email protected] (C.X.). ORCID

Dong Long: 0000-0002-0705-5499 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (2016YFA0501202), the National Natural Science Foundation of China (21673217), the Fundamental Research Funds for the Central Universities (WK2070000069), and the National “1000 Talents Program” for Young Scholars to D.L.

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