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Production of Chemokine/Chemokine Receptor Complexes for Structural Biophysical Studies - PubMed

Production of Chemokine/Chemokine Receptor Complexes for Structural Biophysical Studies

Martin Gustavsson et al. Methods Enzymol. 2016.

Abstract

The development of methods for expression and purification of seven-transmembrane receptors has led to an increase in structural and biophysical data and greatly improved the understanding of receptor structure and function. For chemokine receptors, this has been highlighted by the determination of crystal structures of CXCR4 and CCR5 in complex with small-molecule antagonists, followed recently by two receptor/chemokine complexes; CXCR4 in complex with vMIP-II and US28 in complex with the CX3CL1. However, these studies cover only a few of the many chemokines and chemokine receptors and production of stable receptor/chemokine complexes remains a challenging task. Here, we present a method for producing purified complexes between chemokine receptors and chemokines by coexpression in Sf9 cells. Using the complex between atypical chemokine receptor 3 and its native chemokine CXCL12 as an example, we describe the virus production, protein expression, and purification process as well as reconstitution into different membrane mimics. This method provides an efficient way of producing pure receptor/chemokine complexes and has been used to successfully produce receptor/chemokine complexes for CXC as well as CC receptors.

Keywords: ACKR3; CXCL12; Chemokine; Chemokine receptor; Coexpression; Membrane protein; Protein complex; Sf9.

© 2016 Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1

Flow chart for production of complexes.

Figure 2
Figure 2

Construct design of receptor and chemokine samples. A.) Schematic overview of chemokine and receptor constructs. B.) SDS-PAGE of purified ACKR3 showing the effect of promoter and tag placement on the final yield of ACKR3 protein. The doubling and fuzziness of the ACKR3 band is due to glycosylation.

Figure 3
Figure 3

Baculovirus production detected by flow cytometry. A-B.) P0 and P1 virus expression detected by PE conjugated anti-GP64 antibody. C.) Interpretation of FITC assay for expression of receptor and chemokine. D.) FITC assay results for receptor and chemokine samples. Control experiments (columns 1 and 2) were acquired with an anti-FLAG FITC conjugated antibody, identical results were obtained with anti-HA FITC antibody (data not shown). Note that due to their larger size, cells expressing untagged chemokine have higher non-specific antibody binding than untransfected cells. Receptor and chemokine experiments (columns 3-5) were acquired with FITC conjugated FLAG or HA antibodies as indicated in the figure. Samples in the bottom row contained 0.0075% triton X-100 to permeabilize the cells. E.) Titer of P1 virus using the GP64 assay and serial virus dilutions as indicated above each plot. The % infected cells of each dilution is shown in red above the infected population in each plot.

Figure 4
Figure 4

Expression of ACKR3/CXCL12 complex. A.) Detection of total receptor expression in co-expressed samples using anti-FLAG FITC antibody in the presence of triton X-100 to a final concentration of 0.0075% (v/v). B.) Detection of chemokine binding to receptor at the cell surface using anti-HA FITC antibody. Co-expression with the receptor gives a large signal increase for CXCL12-HA, indicating specific binding of the chemokine to the receptor.

Figure 5
Figure 5

Purification of ACKR3/CXCL12 complex. A.) 10% SDS-PAGE of an bril-ACKR3 fusion protein extracted with the small molecule CCX777 or with a mutant of CXCL12 where KPV at the N-terminus was replaced with LRHQ (LRHQ-CXCL12) (Hanes, et al., 2015). B.) Different stages of purification characterized by SDS-PAGE (10%). C.) 18% SDS-PAGE of a co-expressed and co-purified ACKR3/CXCL12 sample (left lane). ACKR3 expressed alone and purified in complex with a small molecule compound is shown in the right lane. D.) Western blot detecting FLAG-tagged ACKR3 and HA-tagged CXCL12 in samples from C.

Figure 6
Figure 6

Characterization of receptor/chemokine complexes. A.) Analytical SEC traces of varying quality. The top trace has a sharp, symmetric peak while the peak in the bottom trace has a shoulder (indicated by arrow), which is a sign that the sample is partially aggregated. B.) Analytical SEC trace of the ACKR3/CXCL12 complex. C.) CPM measurements are used to determine the midpoint of thermal unfolding (Tm) of the receptor. Data can be plotted either as CPM fluorescence as a function of temperature (top) or as the derivative of the fluorescence (bottom) D.) CPM experiments with apo ACKR3 and the ACKR3/CXCL12 complex.

Figure 7
Figure 7

The ACKR3/CXCL12 complex reconstituted into different membrane mimics. A.) The ACKR3/CXCL12 complex is stable in all three membrane mimics as indicated by the sharp peak (derivative of the CPM unfolding curves, see Fig 6C) and the Tm above 60°C. B.) SDS-PAGE of ACKR3/CXCL12 sample in nanodiscs and DDM/CHS micelles.

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References

    1. Alexandrov AI, Mileni M, Chien EY, Hanson MA, Stevens RC. Microscale fluorescent thermal stability assay for membrane proteins. Structure. 2008;16:351–9. - PubMed
    1. Allen SJ, Crown SE, Handel TM. Chemokine: receptor structure, interactions, and antagonism. Annu Rev Immunol. 2007;25:787–820. - PubMed
    1. Allen SJ, Hamel DJ, Handel TM. A rapid and efficient way to obtain modified chemokines for functional and biophysical studies. Cytokine. 2011;55:168–73. - PMC - PubMed
    1. Allen SJ, Ribeiro S, Horuk R, Handel TM. Expression, purification and in vitro functional reconstitution of the chemokine receptor CCR1. Protein Expr Purif. 2009;66:73–81. - PMC - PubMed
    1. Bannert N, Craig S, Farzan M, Sogah D, Santo NV, Choe H, et al. Sialylated O-glycans and sulfated tyrosines in the NH2-terminal domain of CC chemokine receptor 5 contribute to high affinity binding of chemokines. J Exp Med. 2001;194:1661–73. - PMC - PubMed

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