Distinct activation mechanisms of CXCR4 and ACKR3 revealed by single-molecule analysis of their conformational landscapes
- Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmacology, University of California San Diego, United States
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, United States
Figures
Figure 1 with 2 supplements
Experimental design of the smFRET system.
(A) A single receptor molecule (blue) was labeled with donor (D) and acceptor (A) fluorophores, inserted into a phospholipid (yellow) nanodisc (green), and immobilized on a quartz slide via biotin …
Figure 1—figure supplement 1
Cysteine mutations on TM4 and TM6 of CXCR4 and ACKR3 do not impair CXCL12 mediated β-arrestin2 recruitment observed by BRET.
(A, B) Locations of the cysteines (magenta spheres) on CXCR4 (A) and ACKR3 (B). (C, D) Dose-response BRET-based arrestin recruitment to WT receptors and Cys-containing (C) CXCR4 and (D) ACKR3. Data …
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Figure 1—figure supplement 1—source data 1
Arrestin recruitment to WT and Cys-engineered CXCR4 and ACKR3 across CXCL12 concentrations.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Example single-molecule traces of apo-ACKR3 and apo-CXCR4.
Donor intensity, acceptor intensity and apparent FRET efficiency time traces are colored green, magenta, and black, respectively.
Figure 2 with 5 supplements
ACKR3 exhibits greater conformational flexibility compared to CXCR4.
(A) Apparent FRET efficiency histogram of apo-CXCR4 (left, black trace) resolved into three distinct conformational states: a high-FRET state corresponding to the inactive receptor conformation (R, …
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Figure 2—source data 1
Histograms for Apo CXCR4 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-data1-v1.csv
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Figure 2—source data 2
Contour map for Apo CXCR4 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-data2-v1.csv
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Figure 2—source data 3
Histograms for CXCL12 CXCR4 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-data3-v1.csv
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Figure 2—source data 4
Contour map for CXCL12 CXCR4 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-data4-v1.csv
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Figure 2—source data 5
Histograms for Apo ACKR3 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-data5-v1.csv
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Figure 2—source data 6
Contour map for Apo ACKR3 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-data6-v1.csv
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Figure 2—source data 7
Histograms for CXCL12 ACKR3 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-data7-v1.csv
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Figure 2—source data 8
Contour map for CXCL12 ACKR3 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-data8-v1.csv
Figure 2—figure supplement 1
Quantitative evaluation of the appropriate number of FRET states required to model the CXCR4 smFRET distributions.
SmFRET data for CXCR4 recorded in the apo-state (first row), and in the presence of agonist CXCL12WT (second row) and small-molecule ligand IT1t (third row) were globally analyzed by Hidden Markov …
Figure 2—figure supplement 2
Comparison of the CXCR4 intermediate states for the conditions detailed in Figure 2—figure supplement 1 using three-state and four-state models revealed over fitting artifacts using four states.
(Top) With the three-state model, the R’ states for apo-CXCR4 and for CXCL12- and IT1t-bound receptor overlapped well with similar apparent FRET values across all of the tested conditions. In the …
Figure 2—figure supplement 3
Evaluation of the number of discrete FRET states present in ACKR3 under various conditions.
SmFRET data for ACKR3 recorded in the apo-state (first row), and in the presence of agonist CXCL12WT (second row) and inverse agonist VUF16840 (third row) were globally analyzed by Hidden Markov …
Figure 2—figure supplement 4
Comparison of intermediate state FRET histograms of ACKR3 from the three- and four-state models shown in Figure 2—figure supplement 3.
(Top) The R’ state histogram from the three-state model for apo-ACKR3, and CXCL12WT- and VUF16840-treated ACKR3 showed a spread across intermediate FRET values. The R’ distribution of the …
Figure 2—figure supplement 5
The natural and engineered agonists CXCL11 and VUF15485, respectively, both promoted low-FRET, active-like ACKR3 conformations.
(A) Repeated results of apo-ACKR3 from Figure 2B for comparison of the apparent FRET envelope and modeled states and the TDP. (B) Treatment with CXCL11 leads to a predicted shift in population and …
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Figure 2—figure supplement 5—source data 1
Histograms for CXCL11 ACKR3 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-figsupp5-data1-v1.csv
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Figure 2—figure supplement 5—source data 2
Contour map for CXCL11 ACKR3 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-figsupp5-data2-v1.csv
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Figure 2—figure supplement 5—source data 3
Histograms for VUF15485 ACKR3 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-figsupp5-data3-v1.csv
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Figure 2—figure supplement 5—source data 4
Contour map for VUF15485 ACKR3 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig2-figsupp5-data4-v1.csv
Figure 3 with 1 supplement
A small molecule inhibitor shifts the ACKR3 conformational population to the inactive FRET state, while CXCR4 is largely unaffected.
(A) FRET distributions and TDP of apo-CXCR4 repeated from Figure 2A for comparison. (B) Treatment of CXCR4 with the inhibitor IT1t had little impact on the FRET distribution, but increased …
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Figure 3—source data 1
Histograms for IT1t CXCR4 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig3-data1-v1.csv
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Figure 3—source data 2
Contour map for IT1t CXCR4 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig3-data2-v1.csv
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Figure 3—source data 3
Histograms for VUF16840 ACKR3 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig3-data3-v1.csv
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Figure 3—source data 4
Contour map for VUF16840 ACKR3 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig3-data4-v1.csv
Figure 3—figure supplement 1
Change in the population percentages of individual FRET states due to ligand treatment of CXCR4 and ACKR3.
Data used for this analysis are presented as full apparent FRET distributions in Figures 2—4, Figure 2—figure supplement 5.
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Figure 3—figure supplement 1—source data 1
Change in proportion of CXCR4 and ACKR3 FRET populations with ligand treatments.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig3-figsupp1-data1-v1.csv
Figure 4
CXCL12 variants containing mutations to the N-terminus promoted active receptor conformations in both CXCR4 and ACKR3.
(A) FRET distributions and TDP for apo-CXCR4 repeated from Figure 2A for reference. (B) Addition of CXCL12P2G to CXCR4 promoted a shift to the low-FRET active (R*) conformation and an increase in …
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Figure 4—source data 1
Histograms for P2G CXCL12 CXCR4 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig4-data1-v1.csv
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Figure 4—source data 2
Contour map for P2G CXCL12 CXCR4 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig4-data2-v1.csv
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Figure 4—source data 3
Histograms for LRHQ CXCL12 CXCR4 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig4-data3-v1.csv
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Figure 4—source data 4
Contour map for LRHQ CXCL12 CXCR4 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig4-data4-v1.csv
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Figure 4—source data 5
Histograms for P2G CXCL12 ACKR3 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig4-data5-v1.csv
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Figure 4—source data 6
Contour map for P2G CXCL12 ACKR3 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig4-data6-v1.csv
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Figure 4—source data 7
Histograms for LRHQ CXCL12 ACKR3 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig4-data7-v1.csv
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Figure 4—source data 8
Contour map for LRHQ CXCL12 ACKR3 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig4-data8-v1.csv
Figure 5 with 1 supplement
Replacement of Y2576.40 with the corresponding residue in CXCR4 (leucine) reduces conformational heterogeneity of ACKR3.
(A) Structure of ACKR3 bound with CXCL12WT (PDBID: 7SK3) highlighting the location of Y2576.40 (purple; Yen et al., 2022). (B) Apparent FRET efficiency distributions and TDP of WT ACKR3 in the …
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Figure 5—source data 1
Histograms for Apo Y257L ACKR3 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig5-data1-v1.csv
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Figure 5—source data 2
Contour map for Apo Y257L ACKR3 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig5-data2-v1.csv
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Figure 5—source data 3
Histograms for CXCL12 Y257L ACKR3 FRET states.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig5-data3-v1.csv
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Figure 5—source data 4
Contour map for CXCL12 Y257L ACKR3 TDP.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig5-data4-v1.csv
Figure 5—figure supplement 1
The mutation Y2576.40L reduces the constitutive activity of ACKR3.
β-arrestin2 recruitment to ACKR3 was detected by BRET between the rluc-tagged receptor and GFP10-tagged arrestin. The data represent the mean of three independent experiments measured in triplicate …
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Figure 5—figure supplement 1—source data 1
Arrestin recruitment measurements to WT and Y257L ACKR3 across CXCL12 concentrations.
- https://cdn.elifesciences.org/articles/100098/elife-100098-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 1 supplement
Schematic illustration of the conformational energy landscapes for CXCR4 and ACKR3, highlighting the differences in the responsiveness of the two receptors to ligands.
CXCR4 populates three distinct conformations, shown here as wells on the energy landscape. Apo-CXCR4 is predominantly in the inactive R state. The receptor is converted incompletely to R* with …
Figure 6—figure supplement 1
Agonist-receptor interaction networks in ACKR3 and CXCR4.
(A) Structures of ACKR3 (blue) bound to CXCL12 (gold, PDB ID 7SK3) or the small molecule agonist CCX662 (green, PDB ID 7SK8) reveal multiple modes of receptor activation (Yen et al., 2022). (B) When …
