1. Biochemistry and Chemical Biology
Download icon

De novo macrocyclic peptides dissect energy coupling of a heterodimeric ABC transporter by multimode allosteric inhibition

  1. Erich Stefan
  2. Richard Obexer
  3. Susanne Hofmann
  4. Khanh Vu Huu
  5. Yichao Huang
  6. Nina Morgner
  7. Hiroaki Suga  Is a corresponding author
  8. Robert Tampé  Is a corresponding author
  1. Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Germany
  2. Department of Chemistry, Graduate School of Science, The University of Tokyo, Japan
  3. Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Germany
Research Article
Cite this article as: eLife 2021;10:e67732 doi: 10.7554/eLife.67732
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Selection of macrocyclic peptides (CPs) by random nonstandard peptide integrated discovery (RaPID).

(A) RaPID selection of CPs. Starting from a DNA library, macrocyclic peptides were generated through transcription and ribosomal translation using the Flexible In-vitro Translation (FIT) system. Cognate mRNA was covalently attached to the nascent peptide through incorporation of mRNA-linked puromycin. ABC transporter TmrAEQB reconstituted in lipid nanodiscs (Nds) was immobilized on streptavidin matrices and used as bait during affinity selections. After several iterative rounds of selection, high-affinity binders were isolated and identified by deep sequencing. (B) CPs targeting TmrAB. Enriched CPs were produced by solid-phase synthesis and conjugated with a short linear extension (colored in blue). Head-to-side chain thioether cyclization was mediated between the N-terminal N-chloroacetyl-D-tyrosine and a cysteine residue. The C-terminal lysine was used for site-directed labeling of fluorescein (F) or biotin (B). (C) Enrichment of CPs targeting TmrAB. The 5000 most abundant macrocyclic peptides per selection round based on deep sequencing were utilized to generate sequence similarity networks (Gerlt et al., 2015). Nodes represent unique peptide sequences, node sizes depict peptide frequency, and node colors exhibit peptide length. Sequence alignments of peptide clusters were generated using WebLogo (Crooks et al., 2004).

Figure 1—figure supplement 1
Selection and synthesis of CPs.

(A) TmrAEQB was reconstituted in lipid nanodiscs composed of biotinylated (B) MSP. Reconstituted nanodiscs were isolated by size-exclusion chromatography (SEC). Empty nanodiscs without TmrAEQB were isolated by SEC and served as bait for negative selections. (B) Purification of macrocyclic peptides (CPs). Selected CPs were synthesized on solid support followed by on-resin labeling and cyclization. Macrocycles were purified by C18 reversed-phase HPLC. (C) Identity of CPs. After synthesis, cyclization, and purification, molecular masses of macrocycles were confirmed by MALDI-TOF mass spectrometry. Derived masses, CP6F, calculated: 2708.18 Da, observed: 2707.85 Da; CP12F, calculated: 2788.24 Da, observed: 2788.19 Da; CP13F, calculated: 2574.19 Da, observed: 2574.61 Da; CP14F, calculated: 2579.14 Da, observed: 2579.41 Da.

Figure 2 with 2 supplements
CPFs specifically interact with TmrAB displaying nanomolar binding affinities.

(A, B) Equilibrium binding analysis. Fluorescence anisotropy of CPFs (50 nM) was determined at λex/em = 485/520 nm with increasing concentrations of TmrAB which was reconstituted in liposomes. TmrAB concentrations were calculated based on the random (50/50%) orientation in proteoliposomes (Stefan et al., 2020). As a control, equal amounts of empty liposomes were added to CPFs (dashed lines, open symbols). The difference in fluorescence polarization was normalized to free (0%) and 100% bound CPF. Mean values ± SD (n = 3) are shown, and data were analyzed by a one-site binding model. (C) TmrAB binds specifically to CP14B-loaded matrices. Streptavidin beads loaded with CP14B (1 µM) were mixed with purified TmrAB (60 nM) for 1 hr at 4°C. Beads were washed and bound TmrAB was eluted by adding SDS loading buffer for 10 min at 95°C. Amounts of TmrA in input (I), flow-through (FT), wash fraction (W), and eluate (E) were analyzed by SDS-PAGE and immunoblotting (α-His). Beads without CPB served as negative control (right panel). (D) One-step purification of TmrAB via immobilized CP14B matrices. Streptavidin-agarose beads were loaded with CP14B (1 µM) and mixed with DDM-solubilized membranes of E. coli containing TmrAB as described in (C). Bound TmrAB was eluted in SDS loading buffer and analyzed by SDS-PAGE (silver stain). (E) Native mass spectrometry. TmrAB (4 µM) was buffer exchanged to ESI buffer and incubated with a 2-fold molar excess of CP14F for 10 min on ice. Protein complexes were investigated by ESI-TOF-mass spectrometry. Derived masses, TmrAB: 134.9 kDa, TmrAB +CP14F: 137.4 kDa, TmrAB +2 CP14F:140.0 kDa.

Figure 2—figure supplement 1
CPs specifically bind to TmrAB.

(A) CPBs-mediated isolation of solubilized TmrAB. Streptavidin matrices were loaded with CPBs (1 µM) and incubated with purified TmrAB (60 nM) for 1 hr at 4°C. Beads without CPBs served as control (see Figure 2C). Bound TmrAB complexes were eluted by the addition of SDS loading buffer for 10 min at 95°C. TmrAB in input (I), flow-through (FT), washing fraction (W), and eluate (E) was analyzed by SDS-PAGE and immunoblotting (α-His). (B) Isolation of TmrAB using CP13B. Streptavidin matrices were loaded with CP13B (1 µM) and mixed with DDM-solubilized membranes of E. coli expressing TmrAB as described in (A). Bound TmrAB complexes were eluted in SDS loading buffer and analyzed by SDS-PAGE (silver stain). (C) Formation of CPF-TmrAB complexes. DDM-solubilized TmrAB (1 µM) was incubated with CP12F, CP13F, CP14F (2 µM each), or CP6F (5 µM) for 10 min on ice. CPF-TmrAB complexes were analyzed by gel filtration. Non-specific peptide aggregation was not observed. (D) Conformational epitopes. CPFs (50 nM) were incubated with TmrAB (0.6 µM for CP6F and CP13F, 0.7 µM for CP12F, 0.5 µM for CP14F) in the presence of 1% (v/v) SDS. Fluorescence anisotropy was determined at λex/em = 485/520 nm. Mean values ± SD (n = 3) are displayed.

Figure 2—figure supplement 2
Analysis of CPF-TmrAB complexes by native mass spectrometry.

TmrAB (4 µM) was buffer exchanged to ESI buffer and incubated with a 2-fold molar excess of CPFs for 10 min on ice. Protein complexes were investigated by ESI-TOF-mass spectrometry. (A) CP6F-TmrAB complexes. Derived masses, TmrAB: 135.1 kDa, TmrAB +CP6F: 137.7 kDa. (B) CP12F-TmrAB complexes. Derived masses, TmrAB +CP12F: 137.6 kDa, TmrAB +2 CP12F: 140.5 kDa. (C) CP13F-TmrAB complexes. Derived masses, TmrAB +CP13F: 137.4 kDa, TmrAB +2 CP13F: 140.0 kDa.

CPFs are potent inhibitors of ATP turnover and substrate transport.

(A) Experimental scheme of ATP hydrolysis and peptide transport by TmrAB. (B) CPFs inhibit ATP hydrolysis. TmrAB in liposomes (100 nM) was incubated with ATP (2 mM, traced with [γ32P]-ATP), and MgCl2 (5 mM) in the presence and absence of CPFs (1 µM) for 10 min at 45°C. Autohydrolysis was determined in the absence of TmrAB, and background turnover was conducted in the presence of EDTA (10 mM). Release of [γ32P] was quantified by thin layer chromatography. (C) CPFs are not transported by TmrAB. TmrAB (0.4 µM) reconstituted in liposomes was incubated with C4F peptide (3 µM) or CPFs (1 µM) in the presence of ATP/ADP (3 mM) and MgCl2 (5 mM) for 5 min at 45°C. Since CPFs were dissolved in maximal 0.5% (v/v) of DMSO, C4F plus 0.5% DMSO served as control. Proteoliposomes were washed on filter plates, and transported peptides were quantified at λex/em = 485/520 nm. (D) Inhibition of substrate translocation. TmrAB in liposomes (0.4 µM) were incubated with C4F peptide (3 µM), ATP/ADP (3 mM), and MgCl2 (5 mM) in the presence and absence of CPFs (1 µM) for 15 min at 45°C. Transported peptides were quantified as described in (C). In (B–C), mean values ± SD (n = 3) are shown.

CPs do not affect peptide or ATP binding of TmrAB.

(A) CPFs binding TmrAB is not affected by substrate peptide binding. After the addition of TmrAB (0.6 µM for CP6F and CP13F, 0.7 µM for CP12F, 0.5 µM for CP14F), fluorescence anisotropy of CPFs (50 nM) were monitored at λex/em = 485/520 nm. For competition, C4ATTO655 peptide (2 µM) or R9LQK peptide (200 µM) were added. (B) Binding of C4F peptide is not affected by CPBs. C4F peptides (50 nM) were mixed with TmrAB (4 µM), and fluorescence anisotropy was monitored as described in (A). For competition, C4ATTO655 (10 µM) or CPBs (6 µM) were added. (C) CPBs do not largely affect ATP binding. TmrAB (0.2 µM) were immobilized on SPA beads and incubated with ATP (3 μM, traced with 3H-ATP) in the presence and absence of CPBs (1 µM each) for 30 min on ice. ATP binding was monitored by SPA. Background values were determined after releasing TmrAB complexes from the beads by adding imidazole (200 mM). The background signal in the absence of CPBs was set to 0 cpm. In the case of CP13, the value of bound ATP exceeds the control without CPs. As this is an equilibrium experiment, this can be rationalized by the fact that CP13 stabilizes the ATP-bound OF conformer.

Figure 5 with 1 supplement
CPs bind preferentially to IF- und OF conformation and stabilize nucleotide occlusion.

Conformation-specific preference of CPs. (A, B) CP6F (A) or CP13F (B, 50 nM each) were incubated with increasing concentrations of detergent-solubilized inward-facing TmrAB (in the absence of Mg-ATP) or outward-facing TmrAEQB, which was trapped with Mg-ATP (1 mM) for 5 min at 45°C as described (Hofmann et al., 2019; Stefan et al., 2020). Immediately after, the fluorescence anisotropy was assayed at λex/em = 485/520 nm. The difference in fluorescence polarization was normalized to free and fully bound CPF. Data were fitted by a one-site binding model. (C) Nucleotide occlusion promoted by CPs. TmrAB (2 µM each) were mixed with CPFs (4 µM), ATP (1 mM, traced with [α32P]-ATP), and MgCl2 (5 mM) for 5 min at 4°C or 45°C. Cold ATP (10 mM) was added, and freely exchangeable nucleotides were removed by rapid gel filtration. Occluded nucleotides were analyzed by thin layer chromatography and autoradiography. Representative radiograms for independent triplicates are shown. (D) Stably occluded nucleotides, [α32P]-ATP and [α32P]-ADP, were quantified by autoradiography. Data were normalized to the vanadate-trapped state. In (A, B, D), mean values ± SD (n = 3) are shown.

Figure 5—figure supplement 1
Conformational arrest of TmrAB by CPFs.

(A) CPFs (50 nM each) were incubated with increasing concentrations of detergent-solubilized IF TmrAB in the inward-facing (IF) conformation (absence of Mg-ATP) or TmrAEQB populated in the outward-facing (OF) conformation, which was trapped in the OF conformation by Mg-ATP (1 mM) after 5 min incubation at 45°C. The difference in fluorescence polarization was normalized to unbound (0%) and 100% bound CPF. Data were fitted by a one-site binding model. Fluorescence anisotropy was recorded at λex/em = 485/520 nm. (B) CPFs (50 nM each) were incubated with detergent-solubilized IF or OF TmrAEQB (150 nM for CP6F, 170 nM for CP12F, 50 nM for CP13F, and 120 nM for CP14F). (C) Nucleotide occlusion of the catalytically reduced TmrAB variant. TmrAEQB (2 µM) were incubated in the absence and presence of CPFs (4 µM each), ATP (1 mM, traced with [α32P]-ATP), and MgCl2 (5 mM) for 5 min at 4°C or 45°C. Cold ATP (10 mM) was added, and free nucleotides were removed by rapid gel filtration. Occluded nucleotides were analyzed by thin layer chromatography and autoradiography. Representative radiograms for independent triplicates are shown. In (A, B), mean values ± SD (n = 3) are displayed.

Figure 6 with 1 supplement
CPs block multiple-turnover transport monitored by quantitative flow cytometry.

(A) Single turnover by IF-to-OF switch monitored by single-liposome flow cytometry. TmrAB in liposomes (0.4 µM) were incubated with C4ATTO655 peptide (1 µM), ATP/ADP (3 mM), MgCl2 (5 mM), and CPFs (1 µM) for 5 min at 45°C. Transport reactions were stopped by the addition of EDTA (10 mM). 100,000 proteoliposomes were analyzed by flow cytometry monitoring fluorescein and ATTO655 intensities. (B) Mean fluorescence intensities of C4ATTO655 were converted into the number of peptides per liposome using the regression analysis described above (two-tailed T-test, ***p<0.0001). (C, D) Slowdown of multiple-turnover substrate transport. TmrAB reconstituted in liposomes (0.4 µM) was incubated with C4ATTO655 peptide (1 µM), ATP/ADP (3 mM), MgCl2 (5 mM), and CPFs (1 µM) for various periods of time at 45°C. Transported peptides per liposomes were evaluated and corrected by ADP background levels as described in (B). Transport kinetics were fitted monoexponentially. In (C), mean fluorescence intensities of transported C4ATTO655 were converted into the number of peptides per liposome as described above. (E) Two consecutive transport cycles. TmrAB reconstituted in liposomes was incubated with C4ATTO655 peptide, ATP/ADP, MgCl2, and CPFs as described in (A) for 5 min at 45°C, 60 min at 4°C, 5 min at 45°C, and 60 min at 4°C. Transported peptides per liposome were evaluated as described in (B) and corrected by background levels in the presence of ADP. In (B–E), mean values ± SD (n = 3) are displayed.

Figure 6—figure supplement 1
Linear regression of quantitative flow cytometry analysis.

(A) Gating strategy. Liposomes were selected according to side and forward scatter areas. Single liposomes were gated based on the height of forward scatter correlated to the area of forward scatter. Fluorescence intensities of 20,000 to 100,000 single liposomes were evaluated. (B) Liposomes without reconstituted TmrAB were destabilized by Triton X-100 and incubated with defined amounts of C4ATTO655 peptide (left). Detergent was removed, and liposomes were extensively washed by centrifugation. Mean fluorescence intensities of 100,000 liposomes were analyzed by flow cytometry. Equal concentrations of fluorescein served as loading control (right). (C) Linear regression. Encapsulated C4ATTO655 peptide intensities were correlated with the number of peptides per liposome. Data were analyzed by linear regression yielding y = 68.7 x±1.7 x and R2 = 0.99. Mean values ± SD (n = 3) are shown.

Substrate translocation precedes ATP hydrolysis in a heterodimeric ABC transporter.

In the resting IF state, TmrAB binds nucleotides and substrates independently, which is not affected by CPs. ATP binding induces an IF-to-OF switch, which drives unidirectional substrate translocation. CP6F and CP12F bind preferentially to the IF state and block the transition to the OF state, preventing ATP occlusion and ATP hydrolysis. CP13F and CP14F favor and stabilize a pre-hydrolysis OF state after the IF-to-OF conformation switch and peptide translocation. CPs block ATP hydrolysis at different steps of the transport cycle. In the absence of CPs, ATP hydrolysis and phosphate release initiate the OF-to-IF return restoring transporter function.

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
AntibodyMonoclonal α-His antibodySigma-AldrichSAB1305538Mouse origin. Final dilution: 1/2,000 (v/v)
Antibodyα-Mouse-HRP conjugateSigma-AldrichAP130PGoat origin. Final dilution: 1/20,000 (v/v)
Chemical compound, drugβ-n-Dodecyl β-D-maltoside (DDM)Carl RothCN26.5
Chemical compound, drugBovine brain lipid extractSigma-AldrichB1502
Chemical compound, drug[2,5’,8-3H(N)]-ATP (3H-ATP)PerkinElmerNET118900
Chemical compound, drug32P]-ATPHartmann AnalyticFP-207
Chemical compound, drugCopper-chelated PVT SPA beadsPerkinElmerRPNQ0095
Chemical compound, drugClAc-D-Tyr-CMESynthesized according to
DOI: 10.1021/cb200388k
Chemical compound, drugFmoc-protected amino acidsMerck Millipore/Watanabe Chemical Industriesvarious
Chemical compound, drugHBTUWatanabe Chemical IndustriesA00149
Chemical compound, drugHOBtWatanabe Chemical IndustriesA00014
Chemical compound, drugNovaPEG Rink Amide resinMerck Millipore855047
Chemical compound, drugN,N-DiisopropylethylamineNacalai Tesque14014–55
Chemical compound, drugN,N-DimethylformamideNacalai Tesque13016–23
Chemical compound, drug5/6-Carboxyfluorescein succinimidyl esterThermo Fisher Scientific46410
Chemical compound, drugAcetonitrileWako Chemicals015–08633
Chemical compound, drugTrifluoroacetic acidNacalai Tesque3483305
Chemical compound, drugD-BiotinNacalai Tesque04822–91
Chemical compound, drugPluronic F127Sigma-AldrichP2443
Chemical compound, drugAlbumin, Bovine, AcetylatedNacalai Tesque01278–44
Chemical compound, drugNTPsJena BioscienceNU-1010 NU-1011 NU-1012 NU-1013
Chemical compound, drugDynabeads M-280 StreptavidinThermo Fisher Scientific11206
Sequence-based reagentT7g10M.F46Eurofins Genomics K.K. (Japan)PCR primerTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATA
Sequence-based reagentNNK(n).R(3n + 45)n = 10–15Eurofins Genomics K.K. (Japan)PCR primer for DNA libraryGCTGCCGCTGCCGCTGCCGCA(MNN)nCATATGTATATCTCCTTCTTAAAG
Sequence-based reagentCGS3an13.R36Eurofins Genomics K.K. (Japan)PCR primerTTTCCGCCCCCCGTCCTAGCTGCCGCTGCCGCTGCC
Sequence-based reagentIni-3'.R20-MeGene Design Inc (Japan)PCR primer for tRNAfMetCAU assemblyTGmGTTGCGGGGGCCGGATTT (Gm = 2'-Methoxylated G)
Sequence-based reagentIni-3'.R38Eurofins Genomics K.K. (Japan)PCR primer for tRNAfMetCAU assemblyTGGTTGCGGGGGCCGGATTTGAACCGACGATCTTCGGG
Sequence-based reagentIni1-1G-5'.F49Eurofins Genomics K.K. (Japan)PCR primer for tRNAfMetCAU assemblyGTAATACGACTCACTATAGGCGGGGTGGAGCAGCCTGGTAGCTCGTCGG
Sequence-based reagentIni cat.R44Eurofins Genomics K.K. (Japan)PCR primer for tRNAfMetCAU assemblyGAACCGACGATCTTCGGGTTATGAGCCCGACGAGCTACCAGGCT
Sequence-based reagentFx5'.F36Eurofins Genomics K.K. (Japan)PCR primer for eFx assemblyGTAATACGACTCACTATAGGATCGAAAGATTTCCGC
Sequence-based reagenteFx.R45Eurofins Genomics K.K. (Japan)PCR primer for eFx assemblyACCTAACGCTAATCCCCTTTCGGGGCCGCGGAAATCTTTCGATCC
Sequence-based reagenteFx.R18Eurofins Genomics K.K. (Japan)PCR primer for eFx assemblyACCTAACGCTAATCCCCT
Sequence-based reagentT7e × 5 .F22Eurofins Genomics K.K. (Japan)PCR primer for eFx assemblyGGCGTAATACGACTCACTATAG
Sequence-based reagentDNA-PEG-puromycinGene Design Inc, Osaka, Japanlinker for mRNA displayCTCCCGCCCCCCGTCC-(PEG18)5-CC-Pu
GeneTmrAQ72J05TTC0976Species: Thermus thermophilus
GeneTmrBQ72J04TTC0977Species: Thermus thermophilus
Peptide, recombinant proteinRRY-C*-KSTELThis study (methods and material)C* denotes fluorescein-labeled Cys
Peptide, recombinant proteinMacrocyclic peptides CP6, CP12, C13 and CP14This study (methods and material)
Peptide, recombinant proteinKOD DNA PolymerasePrepared in house (methods and material)
Peptide, recombinant proteinT7 RNA polymerasePrepared in house (methods and material)
Peptide, recombinant proteinT4 RNA ligasePrepared in house (methods and material)
Peptide, recombinant proteinFIT systemPrepared in house according to DOI: 10.1038/nprot.2015.08250 mM HEPES-KOH (pH 7.6), 12 mM magnesium acetate, 100 mM potassium acetate, 2 mM spermidine, 20 mM creatine phosphate, 2 mM DTT,2 mM ATP, 2 mM GTP, 1 mM CTP,1 mM UTP, 0.5 mM 19 proteinogenic amino acids other than Met, 1.5 mg/ml E. coli total tRNA, 0.73 µM AlaRS, 0.03 µM ArgRS, 0.38 µM AsnRS, 0.13 µM AspRS,0.02 µM CysRS, 0.06 µM GlnRS, 0.23 µM GluRS, 0.09 µM GlyRS, 0.02 µM HisRS,0.4 µM IleRS, 0.04 µM LeuRS, 0.11 µM LysRS, 0.03 µM MetRS, 0.68 µM PheRS,0.16 µM ProRS, 0.04 µM SerRS, 0.09 µM ThrRS, 0.03 µM TrpRS, 0.02 µM TyrRS,0.02 µM ValRS, 0.6 µM MTF, 2.7 µM IF1,0.4 µM IF2, 1.5 µM IF3,0.26 µM EF-G, 10 µM EF-Tu,10 µM EF-Ts, 0.25 µM RF2,0.17 µM RF3, 0.5 µM RRF,0.1 µM T7 RNA polymerase, 4 µg/ml creatine kinase, 3 µg/ml myokinase,0.1 µM pyrophosphatase, 0.1 µM nucleotide-diphosphatase kinase, 1.2 µM ribosome
Recombinant DNA reagentpET-22bMerck Millipore69744Vector for protein expression in E. coli
Strain, strain background (Escherichia coli)BL21(DE3)Thermo FisherC600003Chemically competent cells
Software, AlgorithmPrism 5GraphPad
Software, algorithmCytoscapeShannon P et al. Genome Research 2003 13(11) 2498–504
Software, algorithmEFI-ESTGerlt JA et al. Biochim Biophys Acta 1854: 1019-37
Software, algorithmWebLogoCrooks GE et al. Genome 561 Res 14: 1188–90

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)