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Cited by 11Complement system in diseasesVector-borne infectious diseasesBacterial Infections and Vaccines

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Outer surface lipoproteins from the Lyme disease spirochete exploit the molecular switch mechanism of the complement protease C1s

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Garrigues, Ryan J., Thomas, Sheila, Leong, John M., Garcia, Brandon L.·2022·Journal of Biological Chemistry
DOI 10.1016/j.jbc.2022.102557PMC PMC9637899

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Abstract

Proteolytic cascades comprise several important physiological systems, including a primary arm of innate immunity called the complement cascade. To safeguard against complement-mediated attack, the etiologic agent of Lyme disease, Borreliella burgdorferi, produces numerous outer surface-localized lipoproteins that contribute to successful complement evasion. Recently, we discovered a pair of B. burgdorferi surface lipoproteins of the OspEF-related protein family-termed ElpB and ElpQ-that inhibit antibody-mediated complement activation. In this study, we investigate the molecular mechanism of ElpB and ElpQ complement inhibition using an array of biochemical and biophysical approaches. In vitro assays of complement activation show that an independently folded homologous C-terminal domain of each Elp protein maintains full complement inhibitory activity and selectively inhibits the classical pathway. Using binding assays and complement component C1s enzyme assays, we show that binding of Elp proteins to activated C1s blocks complement component C4 cleavage by competing with C1s-C4 binding without occluding the active site. C1s-mediated C4 cleavage is dependent on activation-induced binding sites, termed exosites. To test whether these exosites are involved in Elp-C1s binding, we performed site-directed mutagenesis, which showed that ElpB and ElpQ binding require C1s residues in the anion-binding exosite located on the serine protease domain of C1s. Based on these results, we propose a model whereby ElpB and ElpQ exploit activation-induced conformational changes that are normally important for C1s-mediated C4 cleavage. Our study expands the known complement evasion mechanisms of microbial pathogens and reveals a novel molecular mechanism for selective C1s inhibition by Lyme disease spirochetes.

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Figure 1 Complement inhibition assays using truncations of ElpQ and ElpB.A and B, classical pathway ELISA inhibition assays were performed using C4b deposition as a marker for complement activation. A concentration series of each ElpQ and ElpB truncation protein were evaluated in triplicate and shown with SD. IC50 values are presented in Table 1. A qualitative schematic of ElpQ and ElpB truncation inhibitory activities is shown on the bottom panels: wildtype-like inhibition (green), non–wildtype-like inhibition (yellow), and no detectable inhibition (red). C, sequence alignment of ElpQ181–343 and ElpB182–378 with the N-terminal and C-terminal residue numbers indicated above (ElpQ) and below (ElpB). Alignment was performed with EMBOSS Needle and rendering performed with ESPript 3.0 (65, 66). D and E, 2 μM concentration of (D) ElpQ or (E) ElpB proteins in classical pathway (CP), lectin pathway (LP), and alternative pathway (AP) ELISA complement activation assays. Deposition products C4b (CP–LP) and C3b (AP) are presented as a function of no inhibitor controls with error bars denoting SD.
Figure 1 Complement inhibition assays using truncations of ElpQ and ElpB.A and B, classical pathway ELISA inhibition assays were performed using C4b deposition as a marker for complement activation. A concentration series of each ElpQ and ElpB truncation protein were evaluated in triplicate and shown with SD. IC50 values are presented in Table 1. A qualitative schematic of ElpQ and ElpB truncation inhibitory activities is shown on the bottom panels: wildtype-like inhibition (green), non–wildtype-like inhibition (yellow), and no detectable inhibition (red). C, sequence alignment of ElpQ181–343 and ElpB182–378 with the N-terminal and C-terminal residue numbers indicated above (ElpQ) and below (ElpB). Alignment was performed with EMBOSS Needle and rendering performed with ESPript 3.0 (65, 66). D and E, 2 μM concentration of (D) ElpQ or (E) ElpB proteins in classical pathway (CP), lectin pathway (LP), and alternative pathway (AP) ELISA complement activation assays. Deposition products C4b (CP–LP) and C3b (AP) are presented as a function of no inhibitor controls with error bars denoting SD.CC BY · PubMed Central
Figure 2 SPR binding assays using activated full-length C1s.A and B, ElpQ181–343 and ElpB182–378 were immobilized on an SPR sensor chip by amine coupling chemistry. Multicycle SPR experiments were performed using a twofold dilution series (200–0.4 nM) of purified full-length activated C1s. Reference-subtracted sensorgrams are shown as black lines. A representative injection series is shown. Each experiment was performed in triplicate. Sensorgrams were fit to steady-state (KD, ss, blue circles) and kinetic models (KD, kin, red traces) of binding using Biacore T200 Evaluation software, and calculated KD values are shown in Table 2. SPR, surface plasmon resonance.
Figure 2 SPR binding assays using activated full-length C1s.A and B, ElpQ181–343 and ElpB182–378 were immobilized on an SPR sensor chip by amine coupling chemistry. Multicycle SPR experiments were performed using a twofold dilution series (200–0.4 nM) of purified full-length activated C1s. Reference-subtracted sensorgrams are shown as black lines. A representative injection series is shown. Each experiment was performed in triplicate. Sensorgrams were fit to steady-state (KD, ss, blue circles) and kinetic models (KD, kin, red traces) of binding using Biacore T200 Evaluation software, and calculated KD values are shown in Table 2. SPR, surface plasmon resonance.CC BY · PubMed Central
Figure 3 ElpQ and ElpB bind to the C-terminal domains of activated C1s.A, an AlphaFold (42) model of full-length human C1s lacking the N-terminal signal sequence is shown (UniProt ID: P09871). The domain architecture of C1s and the C4-binding site on C1s are highlighted. B, ElpQ19–343 was immobilized on an SPR sensor chip. Selected C1s truncation proteins containing CUB2, CCP1, CCP2, and SP domains were injected over the ElpQ biosensor at 500 nM. Representative curves from a single injection are shown from an experiment performed in triplicate. C, Alpha (amplified luminescence proximity homogenous assay) competition experiment schematic. Donor beads conjugated with glutathione bound to 50 nM GST-ElpQ were added to anti-c-Myc antibody–conjugated acceptor beads bound to 500 nM activated c-Myc-C1s–CCP1–CCP2–SPS632A to produce an Alpha signal (Fig. S4). D, untagged ElpQ181–343 or ElpB182–378 was titrated (2200–1 nM) and incubated for 1 h at room temperature in the dark. Alpha signal was obtained with excitation at 680 nm, and emission was at 615 nm. Alpha signals were normalized to wells containing no competitor (100%) and no C1s (0%). Curves were fit using nonlinear regression, and IC50 values and 95% confidence intervals are reported in Table 3. Experiments were performed in triplicate, and error bars are reported with SD. CCP, complement control protein; CUB, C1r/C1s, Uegf, Bmp1; GST, glutathione-S-transferase; SP, serine protease; SPR, surface plasmon resonance.
Figure 3 ElpQ and ElpB bind to the C-terminal domains of activated C1s.A, an AlphaFold (42) model of full-length human C1s lacking the N-terminal signal sequence is shown (UniProt ID: P09871). The domain architecture of C1s and the C4-binding site on C1s are highlighted. B, ElpQ19–343 was immobilized on an SPR sensor chip. Selected C1s truncation proteins containing CUB2, CCP1, CCP2, and SP domains were injected over the ElpQ biosensor at 500 nM. Representative curves from a single injection are shown from an experiment performed in triplicate. C, Alpha (amplified luminescence proximity homogenous assay) competition experiment schematic. Donor beads conjugated with glutathione bound to 50 nM GST-ElpQ were added to anti-c-Myc antibody–conjugated acceptor beads bound to 500 nM activated c-Myc-C1s–CCP1–CCP2–SPS632A to produce an Alpha signal (Fig. S4). D, untagged ElpQ181–343 or ElpB182–378 was titrated (2200–1 nM) and incubated for 1 h at room temperature in the dark. Alpha signal was obtained with excitation at 680 nm, and emission was at 615 nm. Alpha signals were normalized to wells containing no competitor (100%) and no C1s (0%). Curves were fit using nonlinear regression, and IC50 values and 95% confidence intervals are reported in Table 3. Experiments were performed in triplicate, and error bars are reported with SD. CCP, complement control protein; CUB, C1r/C1s, Uegf, Bmp1; GST, glutathione-S-transferase; SP, serine protease; SPR, surface plasmon resonance.CC BY · PubMed Central
Figure 4 C1s–CCP1–CCP–-SP enzyme inhibition assays. Enzymatic cleavage of C4 by C1s at 37 °C for 1 h results in four chains: C4α (∼95 kDa), C4α’ (∼85 kDa), C4β (∼75 kDa), and C4γ (∼35 kDa) that can be resolved by SDS-PAGE analysis. A, M: marker (kilodalton). Lanes 1 to 2: SDS-PAGE gel of C4 cleavage in the presence (“+”) or the absence (“−”) of 6.25 nM C1s–CCP1–CCP2–SP and 620 nM C4. Lanes 3 to 10: C4 cleavage profile in the presence of 6.25 nM C1s, 620 nM C4, and a twofold dilution series (from 25 to 0.20 μM) of ElpQ181–343. B, the fraction of C4α′ relative to input C4β in the same lane and normalized to C1s + C4 positive control (lane 2) and negative control C4 (lane 1) was determined by densitometry analysis. A representative gel of three independent experiments is shown with error calculated by SD. C, enzymatic cleavage by C1s–CCP1–CCP2–SP of the small peptide substrate Z-L-Lys-sBzl was assayed with 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) in the presence of 25 μM ElpQ181–343 or Futhan (a small-molecule C1s inhibitor). Absorbance was read at 412 nm, and signals were normalized to negative control no-enzyme wells. Data were collected in triplicate with SD error bars shown. CCP, complement control protein; SP, serine protease.
Figure 4 C1s–CCP1–CCP–-SP enzyme inhibition assays. Enzymatic cleavage of C4 by C1s at 37 °C for 1 h results in four chains: C4α (∼95 kDa), C4α’ (∼85 kDa), C4β (∼75 kDa), and C4γ (∼35 kDa) that can be resolved by SDS-PAGE analysis. A, M: marker (kilodalton). Lanes 1 to 2: SDS-PAGE gel of C4 cleavage in the presence (“+”) or the absence (“−”) of 6.25 nM C1s–CCP1–CCP2–SP and 620 nM C4. Lanes 3 to 10: C4 cleavage profile in the presence of 6.25 nM C1s, 620 nM C4, and a twofold dilution series (from 25 to 0.20 μM) of ElpQ181–343. B, the fraction of C4α′ relative to input C4β in the same lane and normalized to C1s + C4 positive control (lane 2) and negative control C4 (lane 1) was determined by densitometry analysis. A representative gel of three independent experiments is shown with error calculated by SD. C, enzymatic cleavage by C1s–CCP1–CCP2–SP of the small peptide substrate Z-L-Lys-sBzl was assayed with 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) in the presence of 25 μM ElpQ181–343 or Futhan (a small-molecule C1s inhibitor). Absorbance was read at 412 nm, and signals were normalized to negative control no-enzyme wells. Data were collected in triplicate with SD error bars shown. CCP, complement control protein; SP, serine protease.CC BY · PubMed Central
Figure 5 C4 competes with ElpQ binding of C1s–CCP1–CCP2–SPS632A. The Alpha competition experiment described for Figure 3C was used to assess C4 competition. Purified C4 (1000–0.5 nM) or an untagged ElpQ19–343 control (6100–3 nM) was titrated and incubated for 1 h at room temperature in the dark. Alpha signal was obtained with excitation at 680 nm, and emission was at 615 nm. Alpha signals were normalized to wells containing no competitor (100%) and no C1s (0%). Curves were fit using nonlinear regression. Experiments were performed in triplicate, and error was reported using SD. CCP, complement control protein; SP, serine protease.
Figure 5 C4 competes with ElpQ binding of C1s–CCP1–CCP2–SPS632A. The Alpha competition experiment described for Figure 3C was used to assess C4 competition. Purified C4 (1000–0.5 nM) or an untagged ElpQ19–343 control (6100–3 nM) was titrated and incubated for 1 h at room temperature in the dark. Alpha signal was obtained with excitation at 680 nm, and emission was at 615 nm. Alpha signals were normalized to wells containing no competitor (100%) and no C1s (0%). Curves were fit using nonlinear regression. Experiments were performed in triplicate, and error was reported using SD. CCP, complement control protein; SP, serine protease.CC BY · PubMed Central
Figure 6 C1s–CCP1–CCP2–SP exosite mutagenesis SPR-binding studies.A, ribbon diagram of crystal structures of CCP1–CCP2–SP in a proenzyme (gray, Protein Data Bank [PDB] ID: 4J1Y) and activated form (blue, PDB ID: 5UBM). The catalytic triad is marked in yellow on each structure. Site-directed alanine mutagenesis was performed on residues in each of the C4 exosites: the anion-binding exosite (ABE, red loop) consisting of K575–R576–R581–K583 (magenta sticks), the region between CCP1 and CCP2 termed the CCP exosite (CCPE) consisting of E326–D358 (green sticks), or simultaneous mutation of both (CCPE/ABE). Surface representations of each site, denoted proexosites for C1s proenzyme models, are shown from another vantage with the mutated residues highlighted. B and C, ElpQ181–343 and ElpB182–378 were immobilized on an SPR sensor chip, and activated C1s–CCP1–CCP2–SP constructs were injected as a twofold dilution curve (200–0.8 nM). Symbols represent steady-state values at each concentration. D, steady-state fits are shown of the raw sensorgrams presented in B and C with colored circles corresponding to the steady-state RU. Representative curves are shown, and each injection series was performed in triplicate. Fits (solid lines) were performed using Biacore T200 Evaluation software, and KD values are presented in Table 2. ABE and CCPE/ABE mutants could not be fit and are shown as dashed lines. CCP, complement control protein; CCPE, CCP exosite; SP, serine protease; SPR, surface plasmon resonance.
Figure 6 C1s–CCP1–CCP2–SP exosite mutagenesis SPR-binding studies.A, ribbon diagram of crystal structures of CCP1–CCP2–SP in a proenzyme (gray, Protein Data Bank [PDB] ID: 4J1Y) and activated form (blue, PDB ID: 5UBM). The catalytic triad is marked in yellow on each structure. Site-directed alanine mutagenesis was performed on residues in each of the C4 exosites: the anion-binding exosite (ABE, red loop) consisting of K575–R576–R581–K583 (magenta sticks), the region between CCP1 and CCP2 termed the CCP exosite (CCPE) consisting of E326–D358 (green sticks), or simultaneous mutation of both (CCPE/ABE). Surface representations of each site, denoted proexosites for C1s proenzyme models, are shown from another vantage with the mutated residues highlighted. B and C, ElpQ181–343 and ElpB182–378 were immobilized on an SPR sensor chip, and activated C1s–CCP1–CCP2–SP constructs were injected as a twofold dilution curve (200–0.8 nM). Symbols represent steady-state values at each concentration. D, steady-state fits are shown of the raw sensorgrams presented in B and C with colored circles corresponding to the steady-state RU. Representative curves are shown, and each injection series was performed in triplicate. Fits (solid lines) were performed using Biacore T200 Evaluation software, and KD values are presented in Table 2. ABE and CCPE/ABE mutants could not be fit and are shown as dashed lines. CCP, complement control protein; CCPE, CCP exosite; SP, serine protease; SPR, surface plasmon resonance.CC BY · PubMed Central

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Authors
Garrigues, R. J., Thomas, S., Leong, J. M., & Garcia, B. L.
Affiliations
  • East Carolina University (US)
  • Tufts University (US)
Corresponding author
Garcia, Brandon L.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2022-11
Volume
298
Issue
11
Pages
102557
ISSN
0021-9258 (ISSN-L)
DOI
10.1016/j.jbc.2022.102557
PMC
PMC9637899
OpenAlex
W4298009040
Cited by
11
References
1
Citations by year
2026: 1 · 2025: 5 · 2024: 4 · 2023: 1
FWCI
0.95
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • The Scholarship East Carolina University's Institutional Repository (East Carolina University) (submittedVersion)
Funders
  • U.S. Public Health Service (R01-AI146930)
  • National Institutes of Health
  • National Institute of Allergy and Infectious Diseases (R01-AI121401)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Complement system in diseases
  • Life Sciences › Immunology and Microbiology › Parasitology › Vector-borne infectious diseases
  • Life Sciences › Immunology and Microbiology › Microbiology › Bacterial Infections and Vaccines
Keywords
Borrelia burgdorferi, Lyme disease, complement evasion, complement C1, surface lipoproteins, exosite, anion-binding exosite, serine protease, classical complement pathway, antibody-mediated complement activation, complement inhibition
MeSH terms
Complement System Proteins, Complement C1s, Complement C4, Humans, Lipoproteins, Lyme Disease, Endopeptidases, Borrelia burgdorferi, Serine Proteases

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

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How to cite

Cite CoChat for the checks on this page, and the paper for its own findings.

This page
CoChat. (2026). Citation check: Outer surface lipoproteins from the Lyme disease spirochete exploit the molecular switch mechanism of the complement protease C1s. https://app.cochat.ai/papers/10.1016/j.jbc.2022.102557
The paper
Garrigues, R. J., Thomas, S., Leong, J. M., & Garcia, B. L. (2022). Outer surface lipoproteins from the Lyme disease spirochete exploit the molecular switch mechanism of the complement protease C1s. Journal of Biological Chemistry. https://doi.org/10.1016/j.jbc.2022.102557