Structure of a variable lymphocyte receptor-like protein from the amphioxus branchiostoma floridae

ABSTRACT Discovery of variable lymphocyte receptors (VLRs) in agnathans (jawless fish) has brought the origin of adaptive immunity system (AIS) forward to 500 million years ago accompanying

with the emergence of vertebrates. Previous findings indicated that amphioxus, a representative model organism of chordate, also possesses some homologs of the basic components of

TCR/BCR-based AIS, but it remains unknown if there exist any components of VLR-based AIS in amphioxus. Bioinformatics analyses revealed the amphioxus _Branchiostoma floridae_ encodes a group

of putative VLR-like proteins. Here we reported the 1.79 Å crystal structure of Bf66946, which forms a crescent-shaped structure of five leucine-rich repeats (LRRs). Structural comparisons

indicated that Bf66946 resembles the lamprey VLRC. Further electrostatic potential analyses showed a negatively-charged patch at the concave of LRR solenoid structure that might be

responsible for antigen recognition. Site-directed mutagenesis combined with bacterial binding assays revealed that Bf66946 binds to the surface of Gram-positive bacteria _Staphylococcus

aureus_ and _Streptococcus pneumonia_ via a couple of acidic residues at the concave. In addition, the closest homolog of Bf66946 is highly expressed in the potential immune organ gill of

_Branchiostoma belcheri_. Altogether, our findings provide the first structural evidence for the emergence of VLR-like molecules in the basal chordates. SIMILAR CONTENT BEING VIEWED BY

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CARTILAGINOUS AND BONY FISH LYMPHOCYTES Article Open access 03 September 2024 INTRODUCTION Exploration of the origin and evolution of vertebrate adaptive immune system (AIS) is helpful to

better understand the molecular mechanism of auto-immunity diseases. The B-cell and T-cell antigen receptors (BCRs and TCRs) based immunity has long been regarded as a unique character of

AIS. It presents only among jawed vertebrates which evolved in Silurian about 430 million years ago when the first jawed fish emerged1. Recent discovery of novel antigen receptors termed

variable lymphocyte receptors (VLRs) in jawless fishes including lamprey and hagfish reveals that AIS has evolved in a deeper time about 500 million years ago in Late Cambrian when the first

jawless fish appeared2. The VLRs in jawless fishes are composed of tandem arrays of leucine-rich repeats (LRRs) that might be diversified by a gene conversion mechanism involving

lineage-specific cytosine deaminases3,4. Three VLR genes have been identified in lampreys and hagfish (VLRA, VLRB and VLRC)2,3,5,6,7. VLRA is displayed on the surface of T-like lymphocytes

that develop in a thymus-like tissue at the tip of gill filaments, whereas VLRB is expressed and secreted as a multivalent protein in B-like lymphocytes that develop in hematopoietic

tissues8,9. In contrast, VLRC is expressed in a distinct lymphocyte lineage that may be equivalent to mammalian γδ T cells10,11. The discovery of VLR-based AIS has provided new insight into

the emergence of AIS, but it is still uncertain whether AIS exists in invertebrates. Genome sequencing of the amphioxus _Branchiostoma floridae_, a living representative of basal chordates

in the evolutionary proximity to the ancestor of vertebrates, provides crucial insights into the basal chordate condition12. Moreover, the amphioxus genome exhibits considerable synteny with

vertebrate genomes13,14, making amphioxus a valuable model for understanding the development and evolution of vertebrate immunity. In the past decades, a series of findings indicated that

amphioxus also possesses some homologs of the basic components of TCR/BCR-based AIS that have been previously identified only in jawed vertebrates. These components include the

lymphocyte-like cells15, a proto major histocompatibility complex region16 and homolog of recombination-activating gene 117, as well as two immunoglobulin superfamily members (V

region-containing chitin-binding proteins, V and C domain-bearing protein)18,19,20. Nevertheless, it remains unknown if any components of AIS specifically encoded by jawless vertebrates,

such as VLRs, exist in amphioxus. VLRs are featured with a LRR domain of multiple LRR modules and an invariant stalk region rich in threonine and proline residues2. Genomics studies have

revealed that the amphioxus _B. floridae_ genome contains a great number of LRR-containing gene models, of which 948 contain LRR only, a feature of VLRs21. Such a huge arsenal of LRR-only

models indicated that VLR-like proteins may evolve in amphioxus. Thus, we performed domain analysis using the Interpro website (http://www.ebi.ac.uk/interpro/) and obtained nine hypothetical

VLR-like candidates. To investigate whether these putative VLRs possess any characteristics of VLRs, two proteins Bf66946 and Bf289081 were applied to further structural and functional

studies. Finally, only the protein Bf66946 yielded a crystal structure at 1.79 Å resolution, which indeed shares an overall structure quite similar to VLRs. Like other VLRs, Bf66946

specifically adheres to the Gram-positive bacteria _Staphylococcus aureus_ and _Streptococcus pneumonia_ using the concave surface as the antigen-binding region. Moreover, tissue-specific

expression profiling revealed that Bb24355, the closest homolog of Bf66946 in _Branchiostoma belcheri_, is highly expressed in the potential immune organ gill. These findings indicated that

VLR-like molecules and VLR-based adaptive immunity might have evolved at a very early history of the chordate evolution. RESULTS AND DISCUSSION BIOINFORMATICS ANALYSES OF PUTATIVE VLR-LIKE

PROTEINS FROM AMPHIOXUS _B. FLORIDAE_ The extracellular domain of VLRs usually comprise of several modules: an N-terminal LRR-capping module (LRRNT), the first LRR (LRR1), up to nine

24-residue variable LRRs (LRRVs), a variable end LRR (LRRVe), a truncated LRR designated the connecting peptide (CP) and a C-terminal LRR-capping module (LRRCT)2. Searching against the

database of Interpro website (http://www.ebi.ac.uk/interpro/), we obtained 207 unique outputs containing LRRNT module (IPR000372) and 237 unique ones containing LRRCT module (IPR000483) in

amphioxus _B. floridae_. Among these outputs, 101 unique proteins contain both LRRNT and LRRCT. As VLRs are LRR-containing proteins without any other domain21, we subsequently performed

domain analysis and then obtained 40 proteins for further analyzed. As the LRRV modules conservatively possess a consensus sequence of XLXXLXXLXLXXNXLXXLPXXXFX (where X stands for any amino

acid)22, nine hypothetical proteins were finally selected as VLR-like candidates. Finally, the coding sequences of the two shortest proteins, Bf66946 (GenBank accession No. EEN54567.1) and

Bf289081 (GenBank accession No. EEN46450.1), were synthesized by Sangon Biotech (Shanghai) for further investigation. OVERALL STRUCTURE OF BF66946 We applied both the recombinant Bf66946 and

Bf289081 to crystallization trials; however, only the crystal structure of Bf66946 was successfully determined at a resolution of 1.79 Å in the space group P212121 (Table 1). Each

asymmetric unit contains two molecules with a total buried interface area of 567 Å2, which is not sufficient to maintain a stable dimer. In fact, gel-filtration chromatography also indicated

that Bf66946 exists as a monomer in solution. The overall structure of Bf66946 (residues Cys21–Asp202) adopts a crescent-shaped solenoid conformation, rich of conserved leucine and

isoleucine residues involved in formation of the hydrophobic core, which is typical for the LRR family23,24. The structure contains a 36-residue LRRNT, an 18-residue LRR1, two 24-residue

LRRVs (LRRV1 and LRRVe), a 20-residue CP and a 51-residue LRRCT (Fig. 1a). The LRRNT and LRRCT cap the ends of the solenoid to protect the hydrophobic core. The inner concave surface of

Bf66946 is formed by a β-sheet made up of six β-strands (β1, β2, β3, β5, β7 and β8), with β1 anti-parallel to the remaining β-strands that are arranged in a parallel fashion (Fig. 1b). The

outer convex surface is composed of more diverse secondary structure elements, including several loops of different length, one η-helix, two α-helices and three 310-helices (Fig. 1b).

Notably, the 20-residue CP in Bf66946 folds into an α-helix that shares no identity with the 13-residue CP of previously identified VLRs. BF66946 RESEMBLES VLRC RATHER THAN VLRA OR VLRB To

explore the molecular function of Bf66946, we compared this structure against structures deposited in the Protein Data Bank using the Dali server (http://www.ebi.ac.uk/dali/fssp/) and

obtained three closest homologs (PDB code 3TWI, 3G3A, 2R9U), all of which are VLRs from the lamprey _Petromyzon marinus_. Structural comparisons showed that the Cα atoms from LRRNT to LRRCT

of Bf66946 could be well superimposed onto these three VLRs (Supplementary Fig. 1a). The hydrophobic core of Bf66946 is flanked by the cysteine-rich N- and C-terminal modules called LRRNTs

and LRRCTs, respectively (Fig. 1), resembling the previously identified VLRs2,5,6,25. Moreover, the N- and C-terminal caps of Bf66946 also have four strictly conserved disulfide bonds

presumably important for the structural integrity (Supplementary Fig. 1b): two within LRRNT (Cys21-Cys27, Cys25-Cys39) and the other two within LRRCT (Cys146-Cys171, Cys148-Cys192). These

disulfide pairs are structurally equivalent to those previously reported VLRs26. In sum, the similar overall structure and key features strongly suggested that Bf66946 should represent the

first characterized VLR-like protein in amphioxus. In consequence, the structure of Bf66946 was superimposed onto three types of VLR, termed VLRA (PDB ID: 3M19, RMSD = 1.87 Å for 138 Cα

atoms)27, VLRB (PDB ID: 3TWI, RMSD = 1.81 Å for 156 Cα atoms)28 and VLRC (PDB ID: 3WO9, RMSD = 1.88 Å for 139 Cα atoms)29, respectively. The LRRCT region in lamprey VLRA and VLRB molecules

contains a stretch of amino acids that displays marked variations in length, amino acid composition and secondary structure (Fig. 2a,b). This stretch of residues, known as a hyper-variable

insertion, can form a loop structure or protrusion important for antigen recognition in both lamprey VLRA and VLRB molecules27,28,30,31. However, a corresponding insertion stretch of Bf66946

exists at LRRNT, but not LRRCT. This insertion of residues 29–36 covers a part of two strands β1–2 and the connecting loop (Fig. 2d). In fact, the N-terminal cap of lamprey VLRC also has a

long loop protruding towards the concave surface (Fig. 2c); and moreover, the stretch of residues constituting LRRNT protrusion is highly conserved in length and amino acid composition in

all known lamprey VLRC sequences29,32. Structure-based sequence alignment showed the LRRNT modules of VLRA and VLRB are too short to form protrusions (Fig. 2d). Previous studies also

revealed all VLRA molecules and most VLRB molecules lack the potential to form a corresponding protrusion at LRRNT region29,32. Altogether, lamprey VLRA and VLRB possess an insertion in

LRRCT, whereas Bf66946 and lamprey VLRC possess a protruding loop in LRRNT, suggesting that Bf66946 is more likely a putative VLRC rather than either VLRA or VLRB. Notably, among the nine

VLR-like candidates that we initially selected, three proteins (Bf66946, Bf289081 and Bf212402) may contain an LRRNT insertion, whereas none of them has an LRRCT insertion (Supplementary

Fig. 2). However, the numbers of LRR repeats in these candidates vary a lot, from 2 repeats in Bf66946 to 9 repeats in Bf69561 and Bf104848. BF66946 BINDS TO GRAM-POSITIVE BACTERIA VIA A

NEGATIVELY-CHARGED PATCH AT THE CONCAVE SURFACE OF SOLENOID STRUCTURE Jawless vertebrate AIS employs VLRs as antigen receptors to recognize invaded pathogens, such as viruses and bacteria.

As Bf66946 is similar to VLRs, it should recognize some ligands displayed on the surface of pathogens. Therefore, we detected the bacterial binding activity of Bf66946 towards some bacteria,

using bovine serum albumin (BSA) as the negative control. The results showed that Bf66946 specifically adheres to Gram-positive bacteria _S. aureus_ and _S. pneumoniae_, but not

Gram-negative bacteria _Escherichia coli_ and _Salmonella enterica_ (Fig. 3a,b and Supplementary Fig. 3a,b). To identify the precise ligand-binding site of Bf66946, we compared the binding

capacity of Bf66946 with the mutants. The concave surface of the LRR solenoid structure has been proved to be the antigen-binding surface of VLRs22,27,28,29,30,31, indicating that the

concave surface of Bf66946 is most likely the major ligand binding region. Indeed, the electrostatic potentials analysis displays a negatively-charged patch within the concave surface of the

LRR solenoid structure (Fig. 3c), implying it might recognize a positively-charged ligand. A couple of mutants related to the negatively-charged region within the concave surface were

designed, such as Bf66946-Y32A/E34A (located at the LRRNT insertion), Bf66946-D105K/D107K (located at the β-strand of LRRVe). These mutants were cloned, overexpressed and purified by the

same way as the wild-type Bf66946 and circular dichroism spectra confirmed these mutatants are well folded as the wild-type protein (Supplementary Fig. 4). Subsequent bacterial binding

assays revealed that Bf66946-D105K/D107K completely lost the binding capacity towards _S. aureus_ and _S. pneumonia_ (Fig. 3a,b), whereas the double mutation Y32A/E34A did not alter the

binding capacity. Thus, we identified that the residues Asp105 and Asp107 at the concave are key to recognition of Gram-positive bacteria (Fig. 3c). These results suggested that Bf66946 is

indeed VLR-like protein using the concave surface of the LRR solenoid structure to recognize antigens. As we know, the Gram-positive bacteria differ from the Gram-negative bacteria with the

peptidoglycan exposed. In fact, after treatment with 50 mM EDTA for 10 min, the Gram-negative bacteria _E. coli_ and _S. enterica_ also possess the binding affinity towards Bf66946

(Supplementary Fig. 3a-d). Moreover, _in vitro_ binding assays also indicated Bf66946 specifically binds to the peptidoglycan purified from both Gram-positive and negative bacteria; however,

the Gram-negative bacterial peptidoglycan has a much lower binding affinity (Fig. 3d,e and Supplementary Fig. 3e,f). THE CLOSEST HOMOLOG OF BF66946 IS SPECIFICALLY EXPRESSED IN THE

POTENTIAL IMMUNE ORGAN GILL OF _B. BELCHERI_ Due to the difficulty of raising the amphioxus _B. floridae_ in the laboratory, we alternatively detected the expression profile of the homolog

of Bf66946 in _B. belcheri_. Searching against the database of Transcriptome from v4.0 Online (data unpublished), Bbelcheri_v4_101980.1|cds_24355 (Bb24355) was identified as the closest

sequence relative of Bf66946. Sequence comparisons showed that Bf66946 is of 86% sequence identity to that of Bb24355 (Supplementary Fig. 5). The method of 2−ΔΔCt for histogram chart was

used to analyse the gene expression levels of Bb24355 in various tissues (notochord, gill, intestine, hepatic cecum, nerve and muscle), respectively. Each tissue has a 2−ΔΔCt value to

represent the gene expression level of Bb24355. The expression level of Bb24355 in gill was the highest and significantly higher than that in other tissues (Fig. 4). Moreover, a cluster of

cells with morphological similarity to vertebrate lymphocytes has been identified in amphioxus gill15, suggesting the gill is a potential immune organ. In summary, we identified Bf66946 from

_B. floridae_ as an analog of variable lymphocyte receptor VLRC using bioinformatics analysis and structural comparison combined with _in vitro_ bacterial binding assays. This is the first

characterized VLR-like protein in amphioxus, suggesting the emergence of VLR-based AIS in basal chordates. However, further investigations are needed to define the bona fide substrate of

Bf66946 and other basic components of VLR-based AIS. METHODS PROTEIN EXPRESSION, PURIFICATION AND CRYSTALLIZATION The coding sequence of Bf66946 was synthetized by Sangon Biotech (Shanghai).

Recombinant Bf66946 containing residues 21 to 202 was cloned into the NdeI/XhoI site of pET29b-derived expression vector and expressed as inclusion bodies in _E. coli_ strain BL21 (DE3)

(Novagen, Madison) using 2 × YT (yeast extract and tryptone) culture medium with 30 μg/ml kanamycin. Bacteria were grown at 37 °C to an absorbance of 0.8 at 600 nm and then induced with 0.2 

mM isopropyl-β-D-1-thiogalactopyranoside for an additional 4 hr. The bacteria were harvested by centrifugation and resuspended in buffer A (50 mM Tris-HCl pH 8.0, 0.1 M NaCl and 2 mM EDTA).

After sonication for 20 min, inclusion bodies were collected and washed with buffer B (50 mM Tris-HCl pH 8.0, 0.1 M NaCl, 2 mM EDTA and 0.5% (v/v) Triton X-100), then solubilized in

denaturing buffer (50 mM Tris-HCl pH 8.0, 10 mM DTT, 5 mM EDTA, 8 M urea). For _in vitro_ refolding, denaturing buffer containing proteins were diluted into refolding buffer (0.8 M arginine,

50 mM Tris-HCl pH 8.0, 2 mM EDTA, 3.7 mM cystamine, 6.6 mM cysteamine) to a final concentration of 10 mg/l and stirred for 72 hr at 4 °C. Afterwards, the refolding mixture was concentrated,

dialyzed against 50 mM MES pH 6.0 at 4 °C overnight to remove arginine. After centrifugation at 12,000 × g for 30 min, the supernatant containing the soluble target protein was dialyzed

against 20 mM Tris-HCl pH 8.0 and applied to a QHP column (GE Healthcare, UK) followed by gel filtration with a HiLoad 26/60 Superdex 75 prep-grade column (GE Healthcare, UK) for further

purification. Fractions containing the target protein were pooled and concentrated to 10 mg/ml for crystallization. The concentration of protein was measured using absorbance spectroscopy at

280 nm (OD-1000 Spectrophotometer, One Drop). The purity of protein was assessed by electrophoresis and the protein samples were stored at −80 °C. Site-directed mutagenesis was performed

using the PCR-based site-directed mutagenesis with the plasmid encoding the wild-type Bf66946 as the template. The mutants were expressed, purified and stored in the same manners as the

wild-type Bf66946. Selenomethionine (SeMet)-labeled Bf66946 (Se-Bf66946) was expressed in _E. coli_ strain B834 (DE3) (Novagen). Transformed cells were inoculated into LB medium and

incubated at 37 °C. The cells were harvested when the A600 nm reached 0.2 and were then washed twice with M9 medium. The cells were then cultured in SeMet medium (M9 medium with 25 μg/ml

L-SeMet and 50 μg/ml other essential amino acids) containing 30 μg/ml kanamycin to the absorbance of 0.8 at 600 nm. The remaining steps of protein expression, purification and storage of

Se-Bf66946 were the same as those for native Bf66946. CRYSTALLIZATION, DATA COLLECTION AND PROCESSING Se-Bf66946 was concentrated to 10 mg/ml by ultrafiltration (Millipore Amicon) for

crystallization. Screening for the crystallization conditions of Se-Bf66946 was performed using screening kits of Crystal Screen I and II, Index, Grid screens and SaltRx (Hampton Research)

with the hanging drop vapor-diffusion method in 96-well plates at 16 °C. Crystals of Se-Bf66946 were grown with the initial condition of mixing 1 μl protein sample with an equal volume of

reservoir solution (15% (w/v) polyethylene glycol 8000, 0.1 M Bis-Tris pH 6.2) against 0.5 ml reservoir solution. Typically, crystals appeared in 1–2 days and reached the maximum size in one

week. The crystals were transferred to cryoprotectant (reservoir solution supplemented with 25% glycerol) and flash-cooled with liquid nitrogen. SeMet-derivative data sets were collected

from single crystal at 100 K in a liquid-nitrogen-gas stream using an ADSC Q315r CCD detector (Area Detector Systems Corporation) on beamline BL17U at Shanghai Synchrotron Radiation Facility

(SSRF). The data were collected at a radiation wavelength of 0.97915 Å. All diffraction data were indexed, integrated and scaled with the program HKL200033. STRUCTURE DETERMINATION AND

REFINEMENT The crystal structure of Bf66946 was determined by the single-wavelength anomalous dispersion (SAD) phasing method using data from a single SeMet-substituted protein crystal to a

maximum resolution of 1.79 Å. The AutoSol program from PHENIX34 was used to locate the selenium atoms and to calculate the initial phases. Automatic model building was carried out using

AutoBuild in PHENIX34. Refinement was carried out using the maximum likelihood method implemented in REFMAC535 as part of CCP4i36 program suite and the model was rebuilt interactively using

the program COOT37 until the free R-factor converged. The final model was evaluated with MOLPROBITY38 and PROCHECK39. Crystallographic parameters were listed in Table 1. Structural

superpositions were performed by the program Superpose Molecules as implemented in CCP4i program suite. _IN VITRO_ BACTERIAL BINDING ASSAYS Labeling of protein was performed with fluorescein

isothiocyanate (FITC, Sigma-Aldrich) according to a conventional protocol. Briefly, the protein sample at a concentration of 5 mg/ml was prepared in 0.1 M sodium carbonate buffer, pH 9.0.

Then 50 μl of FITC solution (1 mg/ml FITC in anhydrous dimethyl sulfoxide solution) was added into each 1 ml of protein solution, very slowly in 5 μl aliquots while gently and continuously

stirring the protein solution. The reaction was incubated in the dark for 8 hr at 4 °C. After that, a final concentration of 50 mM NH4Cl was added and incubated for 2 hr at 4 °C to terminate

the reaction. The desalting column (GE Healthcare) was used to separate the unbound FITC from the conjugate with phosphate buffered saline (PBS) solution (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 

mM NaCl, 2.7 mM KCl. pH 7.4) and fractions containing the conjugate were pooled and stored in PBS in a lightproof container at 4 °C. The ratio of fluorescein to protein of the product was

estimated by measuring the absorbance at 495 nm and 280 nm. _E. coli_ BL21 (DE3) strain (Novagen), _S. enterica_, _S. aureus_ (capsular type S strain Reynolds) and _S. pneumonia_ Strains

(rough, type 6; LytA knockout) were used in this assays. _E. coli_, _S. enterica_ and _S. aureus_ were grown at 37 °C using 2 × YT culture medium, whereas _S. pneumonia_ was cultured in 2 × 

THY (3% Todd-Hewitt Broth, 0.2% Yeast Extract) under aerobic conditions (37 °C, 5% CO2). When the absorbance at 600 nm of bacteria reached about 0.6, bacterial strains were harvested by

centrifugation at 1,500 × g for 10 min. After washed three times, the bacterial pellets were resuspended in PBS and adjusted to an optical density (OD) of 0.6 (1 cm light path) at 600 nm. In

each reaction, the pellets of 400 μl bacteria were resuspended with the FITC-labeled protein and incubated in the dark for 1 hr at 25 °C with shaking gently. Afterwards, the bacterial

pellets were washed in PBS for five times to remove the unbound protein. The bacterial samples were observed and photographed with con-focal laser scanning microscope (Zeiss LSM 710).

PEPTIDOGLYCAN PURIFICATION Peptidoglycan from Gram-positive/negative bacteria was purified as described previously40 with modifications. Briefly, Cells of a 2-L culture were harvested at an

OD620 of 0.5 by centrifugation for 30 min at 4 °C and 7,500 × g. The cell pellet was resuspended in 40 ml of ice-cold 50 mM Tris–HCl (pH 7.0). The cell suspension was introduced dropwise

into a flask with 150 ml of boiling 5% sodium dodecyl sulfate (SDS) solution. The solution was boiled for another 30 min. The cell suspension was pelleted by ultracentrifugation at 25 °C for

30 min at 130,000 × g. The pellet was washed twice with 30 ml of 1 M NaCl and repeatedly with deionized water (dH2O) until it was free of SDS, confirmed by a previously published assay. The

pellet was resuspended in 20 ml of 100 mM Tris–HCl (pH 7.5) containing 20 mM MgSO4. DNase A and RNase I were added to final concentrations of 10 and 50 μg/ml, respectively and the sample

was stirred for 2 hr at 37 °C. CaCl2 (10 mM) and trypsin (100 μg/ml) were added and the sample was stirred for 18 hr at 37 °C. Then, the sample was incubated for 15 min at 80 °C to

inactivate the enzymes. The cell wall was recovered by centrifugation for 45 min at 130,000 × g at 25 °C, resuspended with 20 ml of 8 M LiCl and incubated for 15 min at 37 °C. After another

centrifugation (see above), the pellet was resuspended in 20 mM EDTA (pH 7.0) and incubated at 37 °C for 1 hr. The cell wall was washed with dH2O before finally being resuspended in 2 to 4 

ml of dH2O prior to lyophilization. Cell wall samples were stored at −20 °C. Next, for Gram-positive bacteria only, 5 mg of cell wall from was stirred with 48% hydrofluoric acid (HF) for 48 

hr at 16 °C in an ultracentrifuge tube made of polyallomer tightly closed by Parafilm. The volatile, aggressive and toxic HF was handled with great caution to avoid any contact with the skin

and spillage. The peptidoglycan was recovered by centrifugation (45 min, 130,000 × g, 4 °C) and washed with ice-cold dH2O, 100 mM Tris–HCl (pH 7.0) and then twice with dH2O. The pellet of

peptidoglycan was resuspended in 1 ml dH2O and stored at 4 °C. PEPTIDOGLYCAN BINDING ASSAYS Peptidoglycan binding of the recombinant proteins was assessed using an assay described

previously41 with modifications. In short, the peptidoglycan was mixed at 10 mg/ml concentration with the tested proteins (Bf66946-WT and Bf66946-D105K/D107K) at 1 mg/ml concentration in PBS

and incubated for 30 min at 25 °C to allow binding. BSA were used as negative controls. After incubation, the mixtures were centrifuged at 12,000 × g for 10 min to sediment the

peptidoglycan and the supernatants were collected. The peptidoglycan pellet fraction was washed with PBS for three times and dissolved in SDS loading buffer. Samples from the supernatant

(unbound protein) and the resuspended pellet (bound protein) both were analyzed in SDS-PAGE and stained with Coomassie Brilliant Blue. CIRCULAR DICHROISM (CD) SPECTROSCOPY CD spectra (250 to

190 nm) of protein (4 μM) in 0.1 M sodium phosphate buffer (pH 8.0) were recorded at 25 °C on a Jasco J810 Spectrophotometer using a quartz cuvette with a 0.1 cm path length and instrument

setting of 1 nm band-width, 0.5 nm data-interval and 30 second averaging time. Spectra were recorded in triplicate, averaged and background subtracted42. ANIMAL CULTIVATION The adults of

amphioxus _B. belcheri_ were obtained from Zhanjiang, Guangdong Province, China, cultured at 24–25 °C with air-pumped circulating seawater and fed with green alga _Chlorella_. QUANTITATIVE

REAL-TIME PCR Total RNA samples were extracted from distinct tissues (every kind of tissues was collected from five amphioxus) of _B. belcheri_ using Trizol Reagent (Invitrogen, Carlsbad,

CA, USA) according to the manufacturer’s protocols. The total RNA samples were then treated with RNase-free DNase (TaKaRa, Dalian, China) using a standardized protocol. Then 1 μg of total

RNA were reverse transcribed to cDNA with oligodT (TaKaRa, Dalian, China). _B. belcheri_ VLR-like homolog (Bb24355) was identified by using _B. floridae_ Bf66946 as the BLAST query against

the database of Transcriptome from v4.0 Online (data unpublished). For the analysis of Bb24355, quantitative real-time PCR was performed on an Applied Biosystems 7300 Sequence Detection

System (Applied Biosystems, Foster City, CA, USA) using a SYBR Green RT-PCR kit (DRR420A, TaKaRa, Dalian, China). Amplification reactions were incubated in a 96-well plate with a thermal

cycling profile of beginning at 95 °C for 30 sec, followed by 40 cycles of 95 °C for 5 sec and 60 °C for 31 sec43. All of the reactions were performed in triplicate. The sequences of the

sense and antisense primers used for the amplification of Bb24355 gene were as follows: 5′- TGTCGCATGGCCTGGTTGG-3′ (sense), 5′- CGGAGCACACGAAGGAAGCC-3′ (antisense). The 18S RNA levels were

used as internal control. Data were quantified using the 2−ΔΔCt method based on Ct Values44. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Cao, D.-D. _et al._ Structure of a variable

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protein domain structures. Cell Res 19, 271–3 (2009). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Prof. Baolin Sun for providing the _S. enterica_

strain. This work was supported by the Ministry of Science and Technology of China (Grants No. 2013CB835300 and 2014CB910100), the Scientific Research Grants of Hefei Science Center of CAS

(2015SRG-HSC045 and 2015SRG-HSC046) and the Program for Changjiang Scholars and Innovative Research Team in University. We thank the assistance of the staff at the Shanghai Synchrotron

Radiation Facility (SSRF) and the Core Facility Center for Life Sciences in University of Science and Technology of China. We are grateful to all the developers of CCP4 Suit, ESPript,

MolProbity and PyMOL. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and

Technology of China, Hefei, Anhui, 230027, China Dong-Dong Cao, Wang Cheng, Yong-Liang Jiang, Wen-Jie Wang, Qiong Li, Yuxing Chen & Cong-Zhao Zhou * Beihai Marine Station, Evo-devo

Institute, School of Life Sciences, Nanjing University, Hankou Road 22#, Nanjing, 210093, Jiangsu, China Xin Liao & Jun-Yuan Chen Authors * Dong-Dong Cao View author publications You can

also search for this author inPubMed Google Scholar * Xin Liao View author publications You can also search for this author inPubMed Google Scholar * Wang Cheng View author publications You

can also search for this author inPubMed Google Scholar * Yong-Liang Jiang View author publications You can also search for this author inPubMed Google Scholar * Wen-Jie Wang View author

publications You can also search for this author inPubMed Google Scholar * Qiong Li View author publications You can also search for this author inPubMed Google Scholar * Jun-Yuan Chen View

author publications You can also search for this author inPubMed Google Scholar * Yuxing Chen View author publications You can also search for this author inPubMed Google Scholar * Cong-Zhao

Zhou View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS D.D.C., J.Y.C., Y.C. and C.Z.Z. designed the study; D.D.C. and X.L. performed

experiments; D.D.C., X.L., W.C., Y.L.J., J.Y.C., Y.C. and C.Z.Z. analyzed data; W.J.W. and Q.L. provided reagents; D.D.C., W.C., Y.C. and C.Z.Z. wrote the manuscript; J.Y.C. revised the

manuscript. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY INFORMATION RIGHTS AND PERMISSIONS

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Cheng, W. _et al._ Structure of a variable lymphocyte receptor-like protein from the amphioxus _Branchiostoma floridae_. _Sci Rep_ 6, 19951 (2016). https://doi.org/10.1038/srep19951 Download

citation * Received: 04 November 2015 * Accepted: 21 December 2015 * Published: 29 January 2016 * DOI: https://doi.org/10.1038/srep19951 SHARE THIS ARTICLE Anyone you share the following

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