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BBA - General Subjects

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Functional and structural investigations of fibronectin-binding protein Apafrom Mycobacterium tuberculosis

Chih-Jung Kuoa, Jian Gaob,c, Jian-Wen Huangb, Tzu-Ping Kod, Chao Zhaib, Lixin Mab,Weidong Liub,c, Longhai Daib, Yung-Fu Change, Ter-Hsin Chenf, Yumei Hub, Xuejing Yub,⁎,Rey-Ting Guob,c,⁎, Chun-Chi Chenb,⁎

a Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwanb State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratoryof Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, Chinac Tianjin Institute of Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308,Chinad Institute of Biological Chemistry, Academia Sinica, Taiwane Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States of AmericafGraduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung 402, Taiwan

A R T I C L E I N F O

Keywords:Crystal structureMycobacterium tuberculosisAlanine and proline-rich proteinFibronectinAdhesin

A B S T R A C T

Background: Alanine and proline-rich protein (Apa) is a secreted antigen of Mycobacterium spp. which involvesin stimulating immune responses and adhering to host cells by binding to fibronectin (Fn). Here, we report thecrystal structure of Apa from Mycobacterium tuberculosis (Mtb) and its Fn-binding characteristics.Methods: The crystal structure of Mtb Apa was determined at resolutions of 1.54 Å. The dissociation constants(KD) of Apa and individual modules of Fn were determined by surface plasmon resonance and enzyme-linkedimmunosorbent assay. Site-directed mutagenesis was performed to investigate the putative Fn-binding motif ofApa.Results: Mtb Apa folds into a large seven-stranded anti-parallel β-sheet which is flanked by three α-helices. Thebinding affinity of Mtb Apa to individual Fn modules was assessed and the results indicated that the Mtb Apabinds to FnIII-4 and FnIII-5 of Fn CBD segment. Notably, structure analysis suggested that the previously pro-posed Fn-binding motif 258RWFV261 is buried within the protein and may not be accessible to the bindingcounterpart.Conclusions: The structural and Fn-binding characteristics we reported here provide molecular insights into themultifunctional protein Mtb Apa. FnIII-4 and FnIII-5 of CBD are the only two modules contributing to Apa-Fninteraction.General significance: This is the first study to report the structure and Fn-binding characteristics of mycobacterialApa. Since Apa plays a central role in stimulating immune responses and host cells adhesion, these results are ofgreat importance in understanding the pathogenesis of mycobacterium. This information shall provide a gui-dance for the development of anti-mycobacteria regimen.

1. Introduction

Tuberculosis, an airborne disease caused by Mycobacterium tu-berculosis (M. tuberculosis; termed Mtb), remains a major global healththreat and the emergence of multidrug resistant tuberculosis poses agreat challenge in disease control [1–3]. In order to design more ef-fective therapeutic regimens to counteract Mtb infection, understanding

the molecular pathogenesis of Mtb is of pivotal importance. Adhesion isthe precursor step in Mtb infection which is mediated by a group of cell-surface associated or secreted components called adhesins. Adhesinsfacilitate cell-to-cell or cell-to-extracellular matrix (ECM) interactionsand mediate the entry of bacteria cells. These molecules also relate tobacterial aggregation, biofilm production, and immune evasion, con-tributing to virulence and pathogenesis [4,5]. Accordingly,

https://doi.org/10.1016/j.bbagen.2019.06.003Received 18 April 2019; Received in revised form 26 May 2019; Accepted 3 June 2019

⁎ Corresponding authors at: State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China.

E-mail addresses: xjy51@hotmail.com (X. Yu), guoreyting@gmail.com (R.-T. Guo), ccckate0722@gmail.com (C.-C. Chen).

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mycobacterial adhesins have been considered as attractive targets fordevelopment of novel anti-mycobacterial agents. Thus, investigation ofmolecular mechanism of mycobacterial adhesin is of great interests.

Fibronectin (Fn) is an ECM component which has numerous func-tions including cell adhesion, growth, migration, and differentiation[6,7]. Fn comprises multiple copies of three types of homologousmodules (FnI, FnII, and FnIII). These modules further assemble intoseveral functional domains: an N-terminal domain (NTD), a gelatin-binding domain (GBD), a cell-binding domain (CBD), a 40-kDa domainthat contains heparin-binding domain II (Hep-2) and FnIII-15, and afibrin-binding domain II [8]. Fn is a common target for bacterial ad-hesins and a large number of Fn-binding bacterial adhesins have beenidentified [9]. For mycobacteria, a group of secreted protein antigen 85complex (Ag85) including Ag85A, Ag85B, and Ag85C that are encodedby three independent genes are the most well-known Fn-binding ad-hesins [10]. Functional and structural analyses demonstrated that Ag85also acts as mycolyltransferases and plays a role in cell wall assembly[11]. Ag85D, a member of the Ag85 family that shares 40% sequenceidentity to Ag85A but lacks catalytic residues functions purely as anadhesin [12]. A consensus Fn-binding motif of Ag85 complex has beenreported [13], which is located on protein surface and presumed toform electrostatic and hydrophobic interactions to Fn [11,14]. Re-cently, we mapped the Ag85-binding motif of Fn to 17SLLVSWQPPR26

of FnIII-14 module located within the Hep-2 domain, providing the firstdetailed analysis of Fn-binding mycobacterial adhesin [15].

Another major mycobacterial Fn-binding adhesin is fibronectin at-tachment protein (FAP), also known as Apa, ModD, Rv1860, or MPT-32. Apa is an alanine-proline-rich immunodominat antigen, which hasbeen identified in several mycobacterial species such as M. tuberculosis,M. leprae,M. vaccae,M. bovis,M. kansasii,M. smegmatis,M. avium subsp.paratuberculosis, and M. bovis BCG (Fig. S1) and mediates the attach-ment and internalization of these bacteria [16–21]. It has been shownthat four Thr residues in Pro-rich domains near the N- and C-terminusof Apa are O-glycosylated with mannose via α-1,2 glycosidic linkages(Fig. 1A) [22], which plays indispensable roles in eliciting T-cell re-sponses [23]. Moreover, blocking Fn-Apa interaction significantlyabolishes bacterial binding and invasion to host cells, implying that Apais crucial to mycobacterial pathogenesis [19,24]. In a previous report, aseries of overlapping peptides covering Apa of M. avium (Apa-A) weresynthesized and examined for their binding affinity to Fn [20]. Peptide177–201 and 269–292 of Apa-A exhibited specific Fn-binding, with thelatter capable of blocking the full-length protein interactions. A follow-up study further narrowed down the Fn-binding fragment to 269–280,where the motif 273RWFV276 within this fragment was strictly con-served across various species and thus considered to be essential for Fnbinding [16]. In contrast to Ag85, the structure of Apa remains un-known and the binding characteristics of Apa-Fn has not been studiedyet.

In the present study, we reported the crystal structure of Mtb Apaand its Fn-binding features. The Fn modules that contributed to Apabinding were also identified. Since Apa has been demonstrated topossess essential immunological properties for eliciting T- and B-cellresponses during infection [25] and sequences of Apa among myco-bacterial species are highly homologous, the structural and Fn-bindingcharacteristics we reported here shall provide insights into the multi-functional protein Apa of mycobacterium.

2. Material and methods

2.1. Bacterial strains, reagents and antibodies

Escherichia coli strains were cultured in Luria-Bertani broth (LB)with appropriate antibiotics (Table S1). Fibronectin (extracted fromhuman plasma), the N-terminal domain of Fn (NTD), the gelatin-binding domain of Fn (GBD), ethylenediaminetetraacetic acid (EDTA),sodium chloride, sodium phosphate monobasic, sodium phosphate

dibasic, Tris, magnesium chloride, manganese chloride, zinc chloride,copper chloride, and calcium chloride were purchased from Sigma-Aldrich (Milwaukee, WI). The Fn cell-binding domain (CBD), the Fn40 kDa domain, and mouse anti-fibronectin were ordered fromMillipore (Billerica, MA). Mouse anti-α-actin antibody, mouse anti-histidine tag, mouse anti-GST antibody, and HRP-conjugated goat anti-mouse antibody were purchased from Invitrogen (Carlsbad, CA). HRPconjugated goat anti-mouse IgG antibody and TMB peroxidase substratewere purchased from KPL (Gaithersburg, MD). DNA sequencing ser-vices were order from Mission Biotech (Taipei, Taiwan).

2.2. Plasmid construction and site-directed mutagenesis

The oligonucleotides used in this study are listed in Table S2. Thegene encoding M. tuberculosis Apa without signal peptide(WP_003911690.1, Apa hereinafter) (Fig. 1A) was cloned from thegenomic DNA of M. tuberculosis strain H37Rv using polymerase chainreaction (PCR) and constructed into pET-16b vector (Merck, Brookfield,WI). Truncated Apa (ΔApa) avoiding putative glycosylated region onboth N- (residue 40–69) and C-termini (residue 311–325) was amplifiedby using primers ΔApa-F and ΔApa-R and cloned into pET46Ek/LICvector. Recombinant glutathione S-transferase (GST)-tagged Fn CBDconstructs (FnIII-1–11 modules) were generated using the pGEX-6P-1vector (GE Healthcare, Pittsburgh, PA). Apa mutants (A160C, R164A,R258A, W259A, F260A, and V261A) were prepared by using a Quick-Change site-directed mutagenesis kit (Agilent Technologies, SantaClara, CA, USA) following the manufacturer's instruction withpET46Ek/LIC-ΔApa as a template and mutagenesis oligonucleotides.Mutant constructs were confirmed by DNA sequencing.

2.3. Recombinant protein expression and purification

For expression and purification of Apa, ΔApa, and variants (ΔApa-A160C, Apa-R164A, Apa-R258A, Apa-W259A, Apa-F260A, and Apa-V261A), plasmids were transformed into E. coli BL21(DE3) cells andprotein expression were induced by 0.8 mM isopropyl β-D-1-thioga-lactopyranoside (IPTG) at 16 °C for 24 h. Cells were harvested by cen-trifugation at 5000×g for 15min and resuspended in buffer containing25mM Tris-Cl, (pH 7.5), 150mM NaCl and 20mM imidazole followedby disruption with a French Press. Cell debris was removed by cen-trifugation at 17,000×g for 1 h. The supernatant was then applied to anickel-nitrilotriacetic acid (Ni-NTA) column FPLC system (GEHealthcare). The target proteins eluted at ~100mM imidazole whenusing a 20–500mM imidazole gradient. Proteins were dialyzed againstbuffer containing 25mM Tris-Cl (pH 7.5) and loaded onto a DEAESepharose column. Target proteins were eluted at ~300mM NaCl whenusing a 0–500mM NaCl gradient. The purified proteins were furtherconcentrated to 100mg/ml. Finally, all recombinant proteins (exceptΔApa-A160C) were in buffer containing 25mM Tris-Cl (pH 7.5),150mM NaCl. ΔApa-A160C was in 25mM Tris-Cl (pH 7.5) with 0.1M(NH4)2SO4 for crystallization purpose. Similar procedures were appliedto recombinant Fn CBD modules preparation. Recombinant plasmidswere transformed into E. coli BL21(DE3) cells and induced with 1mMIPTG at 20 °C for 16 h. The cells were suspended in phosphate-bufferedsaline (PBS) and disrupted as mentioned above. The protein-containingsupernatant was collected and purified by using a GST column whichwas equilibrated with PBS as described previously [15,26]. Proteinpurity was verified by SDS-PAGE analysis.

2.4. Gel filtration determination of protein form

The molecular species of the Apa was determined on a pre-packedSuperose 12 10/300 GL column (GE Healthcare) by comparing theelution volume of protein with the molecular mass standards (GelFiltration LMW Calibration Kit, GE Healthcare). PBS was used as run-ning buffer at a flow rate of 0.5ml/min.

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2.5. Crystallization and data collection

ΔApa crystals were initially obtained by using a Wizard IV kit(Emerald BioSysterms) (No. 41: 20% PEG 8000, 0.1M HEPES pH 7.5,0.2 M (NH4)2SO4, 10% isopropanol) with the sitting-drop vapor diffu-sion method. ΔApa-A160C crystals were initially obtained by using aProPlex II kit (Molecular Dimensions) (No. 37: 0.1M sodium citrate,pH 6.0, 2M NaCl) with the sitting-drop vapor diffusion method. Ingeneral, 1 μl protein (100mg/ml) was mixed with 1 μl of reservoir so-lution in 24-well Cryschem Plates, and equilibrated against 100 μl of thereservoir at 25 °C. Within one month, crystals reached the size suitablefor X-ray diffraction. For heavy-atom derivatives, the Hg-containingreagents of Heavy Atom Screen Hg (Hampton Research) were used.Cryoprotectant solutions (0.15M sodium citrate, pH 6.0, 2.5M NaCl,30% glycerol) containing 2mM Hg derivatives were used in soaking theΔApa-A160C crystals for 3 to 5 h prior to data collection. All datasetswere collected at 100 K.

2.6. Data collection, structure determination and refinement

Data sets were collected at beam lines BL15A1 and TPS-5A of theNational Synchrotron Radiation Research Center (NSRRC, Hsinchu,Taiwan) and processed by using the HKL2000 program [27]. Prior tostructure refinement, 5% randomly selected reflections were set asidefor calculating Rfree as a monitor of model quality [28]. The single-wavelength anomalous diffraction (SAD) datasets of mercury-con-taining derivatives [29] of ΔApa-A160C were collected at a wavelengthof 1.0093 Å. Using SHELXC/D/E [30] from CCP4i program suit [31],datasets from different Hg-derivative crystals were tested, the best re-sults were obtained using the derivative of ethylmercurithiosalicylicacid, the figure of merit (FOM) values were over 0.9, a better modelwith most side chains was built by ARP/wARP [32]. The model andmap were further improved by refinement using Refmac5 [33] and Coot[34]. Structures of the ΔApa crystals were solved by molecular re-placement (MR) using SAD solved model by using program Phaser [35]from CCP4i suite. Water molecules were modeled at the 1.0 σ maplevel. All of the following structural refinements were carried out using

Fig. 1. Characteristic analysis of recombinant Mtb Apa. (A) Schematic diagram showing the sequence of full-length Apa. Four of the O-glycosylated Thr residues(T49, T57, T66, and T316) were boxed. Signal peptide (residue 1–39) and gray colored sequence (residue 40–69 and 311–325) harboring the O-glycosylated residueswere truncated to form the ΔApa (residue 70–310) used for crystallization. (B) SDS-PAGE analysis of the recombinant Apa. M, molecular mass markers. Lanes 1 is theHis-tagged Apa devoid of signal sequence (Apa, residue 40–325). The molecular weight of Apa was ~47 kDa. Lane 2 is ΔApa with the molecular weight of ~35 kDa.(C) The elution profile of the Apa (0.6 mg/ml) by gel filtration chromatography for the determination of the oligomeric form. The standard curve retention timeversus log MW graph was plotted by using protein standards of known molecular masses (inset). The estimated molecular mass of Apa calculated from the equation ofthe best-fit line was approximately 30.2 kDa.

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the programs Refmac5 and Coot. All data collection and refinementstatistics are summarized in Table 1, and all structure figures wereprepared by using PyMOL (http://pymol.sourcefore.net/).

2.7. Protein binding assays by ELISA

To map the Apa binding domain of Fn, 0.5 μM of full-length Fn, fourFn proteolytic domains (including NTD, GBD, CBD, and 40-kDa do-main) or bovine serum albumin (BSA, negative control) was coated onmicrotiter plate wells using 0.1 M NaHCO3 (pH 9.3) coating buffer at4 °C overnight and blocked by PBS plus 3% BSA at 37 °C for 1 h.Subsequently, Apa (0.2 μM) was added to each well and incubated at37 °C for 1 h. After washing by PBST (PBS plus 0.05% Tween-20) bythree times, the bound Apa was hybridized and detected consecutivelyby mouse anti-histidine (1000-fold diluted) and HRP-conjugated goatanti-mouse IgG (1000-fold diluted) [26]. After washing the plates thricewith PBST, 100 μl of TMB substrate (KPL) was added to each well, al-lowed to react for 5min, and quenched with the addition of 100 μl of0.5% hydrofluoric acid. Microtiter plates were read at 460 nm using anELISA plate reader (Bioteck EL-312). For mapping the Apa bindingmodules in the CBD, individual GST-tagged FnIII-1~11 modules, glu-tathione S-transferase (GST, negative control), or BSA (negative con-trol) (0.5 μg) was coated on microtiter plate and Apa (0.2 μM) wasadded to each well. Binding detection was performed using the sameprocedures described previously. To determine the dissociation con-stant (KD), GST-tagged FnIII-5 (0.5 μg) was coated on microtiter platewells and various concentrations (0.2, 0.5, 1, 2, 5, 10, and 20 μM) ofHis-tagged Apa proteins (wild type or mutants) were added to each welland incubated as mentioned previously. The data were fitted as shownin Eq. 1 by using KaleidaGraph software (version 2.1.3 Abelbeck soft-ware, Reading, PA). Each value represents the mean ± SEM of threetrials.

=

+KOD

OD [rApa][rApa]D

460460 max

(1)

2.8. Binding kinetics study by surface plasmon resonance (SPR)

The interactions of recombinant Apa to Fn and Fn CBDs were ana-lyzed by an SPR technique using a Biacore 2000 instrument (GEHealthcare, Pittsburgh, PA). To determine the Fn binding activity ofApa, full-length Fn or CBD (25 μg) was immobilized on a CM5 chip (GEHealthcare, Pittsburgh, PA). Then 10 μl Apa or BSA (negative control)of indicated concentration was injected into the flow cell at 10 μl/minat 25 °C. The chip surface was regenerated by removal of analyte with aregeneration buffer (10mM Glycine-HCl at pH 2.0). All sensorgramdata were subtracted from the negative control flow cell. To obtain thekinetic parameters of the interaction, the data of the sensograms werefitted by BIA evaluation software version 3.0 using the one step bio-molecular association reaction model (1:1 L model), which resulted inoptimum mathematical fits with the lowest χ values.

3. Results

3.1. Purification of recombinant Mtb Apa

The Mtb Apa was shown to be glycosylated on T49 and T57 withmannobiose (α-D-Manp(1→ 2) α-D-Manp), on T66 with a single Man(α-D-Manp), and on T316 with a mannose, a mannobiose, or a man-notriose (α-D-Manp(1→ 2) α-D-Manp(1→ 2) α-D-Manp) (Fig. 1A) [22].The His-tagged Apa (removal of signal sequences, named Apa in thisstudy) and a truncated form (residue 70–309, named ΔApa) lacking theglycosylation regions were over-expressed in E. coli BL21 (DE3) andpurified by Ni-NTA affinity chromatography. SDS-PAGE analysis of thepurified proteins gave bands with molecular weights of 47 and 35 kDafor Apa and ΔApa, respectively (Fig. 1B). In gel filtration chromato-graphy, Apa was eluted at an apparent molecular weight of 30.2 kDa,corresponding to the monomeric form (Fig. 1C). The inconsistent mo-lecular weight observed in SDS-PAGE and gel filtration might be a re-sult of a high content of proline, which may retard the migration rate ofApa on SDS-PAGE.

3.2. Crystal structure of Mtb Apa

The attempt to obtain crystals of full length Apa failed. We hy-pothesized that high degree of glycosylation might interrupt crystalpacking and thus tested ΔApa which contains no putative O-glycosy-lated regions for crystallization (Fig. 1A). ΔApa was successfully crys-tallized in the rhombohedral space group R32. The structure was solvedby SAD using a mercury derivative of the ΔApa A160C mutant crystal.The ΔApa crystal contained one polypeptide chain (residue110th–287th) in an asymmetric unit. The protein folds into a largeseven-stranded anti-parallel β-sheet (β3-β9) flanked by three α-helices(α1, α2, α3) and a smaller two-stranded β-sheet (β1 and β2; Fig. 2A).Strands β4 and β7 are twisted and the adjacent strands β5 and β6 eachcontains a large bulge. The N- and C-terminal segments, with 16 and 10prolines, as well as 10 and 9 alanines, in 39 and 23 amino acid residues,were not observed presumably due to disorder. Neither were portions ofthe β5-α2 and β8-β9 loops. By crystallographic symmetry operations,three molecules of ΔApa appear to assemble into a trimer, which thenpacked to another triad to form a hexamer (Fig. S2). Nevertheless, thesize exclusion chromatography analysis suggested that the protein is inmonomeric form. Thus the observed hexamer, despite involving in largecontact interfaces (630 and 700 Å2 surface areas on the triad and two-triad interfaces), is a result of crystal packing.

A search with the DALI server showed> 35 similar structures withZ-scores of 8.8–12.6 and RMSD of 2.7–3.7 Å (Fig. S3). The values weremeasured from superposing 110 to 146 Cα matching pairs of Apa andeach homologous structures (Fig. S3). Among the top of the list are theyeast Ran-binding Mog1P and the plant PSII-stabilizing PsbP (Fig. 2B).Despite the similar β-sheet topology, there are significant structuralvariations, especially in the connecting loops, and the sequence identity

Table 1Data collection and refinement statistics for A160C and wild type ΔApa.

ΔApa-A160C-Hg ΔApa

Data collectiona

Space group R32 R32Unit-cell

a, b, c (Å) 118.0, 118.0, 118.9 118.7, 118.7, 117.9α, β, γ (°) 90, 90, 120 90, 90, 120

Resolution (Å) 25–1.77 (1.83–1.77) 25–1.54 (1.60–1.54)Unique reflections 31,151 (3101) 46,832 (4645)Redundancy 6.2 (5.9) 16.8 (13.4)Completeness (%) 99.9 (99.9) 99.9 (100.0)Average I/σ (I) 25.7 (3.0) 38.2 (3.0)Rmerge (%) 5.9 (42.0) 6.3 (99.9)CC1/2 0.974 (0.903) 0.950 (0.824)

RefinementNo. of reflections 31,149 (3091) 46,822 (4594)Rwork (%) 16.3 (23.8) 15.7 (21.9)Rfree (%) 18.1 (23.2) 17.0 (25.7)Bond length rmsd (Å) 0.018 0.016Bond angle rmsd (°) 1.50 1.46Ramachandran plot

Favored (%) 99.4 98.7Outliers (%) 0 0

Average B (Å2)/atomsProtein 31.0/1220 32.3/1341Ligand 49.9/7 44.6/6Water 45.7/178 49.7/269

PDB ID code 5ZXA 5ZX9

a Numbers in parentheses are for the outermost resolution shell.

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is only about 10%. Also, the high content of proline and alanine is notobserved in these structures. The location of proline and alanine re-sidues in ΔApa are analyzed as the unusual high ratio of these residuesis a unique feature of Apa family. As shown in Fig. 2C, proline residuesare distributed in several extensive loop regions, while alanine residuesare widespread in the overall structure. Many prolines are found in themissing fragments, suggesting they should be located in unstructuredloops and are not visible in the structure.

3.3. Mtb Apa binds to Fn CBD

To map the Apa-binding region of Fn, the interaction between Apaand four principal domains of Fn, including NTD, GBD, CBD, and the40 kDa domain (Fig. 3A), were examined by ELISA. As shown in Fig. 3B,CBD was the only domain that showed significant binding to Apa,whereas no interaction between Apa and the other three domains wasdetected. Furthermore, the binding affinity (KD) of Apa to the fulllength Fn and CBD measured by surface plasmon resonance (SPR) were0.54 ± 0.06 μM (Fig. 3C, left panel) and 0.83 ± 0.04 μM (Fig. 3C,right panel), respectively. ΔApa that was used for crystallizationshowed comparable Fn- and CBD-binding affinity (data not shown).

3.4. FnIII-4 and FnIII-5 modules of CBD bound to Mtb Apa

Further studies were performed to identify the Apa-binding moduleswithin CBD. Eleven recombinant FnIII modules of CBD (FnIII-1~11,Fig. 4A) were individually purified and then subjected to Apa bindingmeasurement by using ELISA. The data showed that FnIII-4 and FnIII-5strongly bind to Apa. No significant interaction between Apa and theother FnIII modules was detected (Fig. 4B). Similar results were ob-served when using ΔApa in the experiments (data not shown).

3.5. RWFV motif of Mtb Apa is not involved in Fn binding

Previous study has indicated that 273RWFV276 motif ofM. avium Apacontributed for Fn binding [16]. However, the crystal structure of ΔApaindicates that the 273RWFV276 motif is buried within the protein and isnot accessible to the counterpart of binding (Fig. 5A). In order to va-lidate our observations, each of the four residues of RWFV motif weremutated to alanine and their binding affinity towards FnIII-5 were ex-amined. As shown in Fig. 5B, no significant reduction in the FnIII-5binding was observed between wild type protein and mutant proteins(dissociation constant: wild type, 1.72 ± 0.56 μM; R258A,1.95 ± 0.48 μM; W259A, 2.57 ± 0.52 μM; F260A, 2.46 ± 0.31 μM;V261A, 1.82 ± 0.49 μM).

4. Discussion

Bacterial infections are initiated by molecular interactions betweenthe pathogen and molecules on host cells, resulting in microbial ad-hesion and the potential for subsequent internalization [4]. Moreover,when bacteria adhere to surfaces, they become more resistant to hostantimicrobial defenses [5]. The ECM of the cells is a complex macro-molecular mixture that includes collagens, Fn, fibrinogen, vitronectin,

Fig. 2. Overall structural analysis of ΔApa. (A)The structure of monomericΔApa is shown as cartoon representation. (B) Cartoon representations of yeastRan-binding Mog1P (left panel, PDB ID: 1EQ6) and the plant PSII-stabilizingPsbP (right panel, PDB ID: 4RTI). (C) Proline and alanine residues in ΔApastructure are shown in blue and magenta stick models, respectively. (For in-terpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

Fig. 3. Apa binds to Fn CBD. (A) Schematic representation of modular struc-ture of Fn. (B) Relative binding affinities of Apa to immobilized full length andindividual domains of Fn or BSA measured by ELISA. The assay was performedin triplicate and the readings of OD460 nm absorbance are averaged and pre-sented as mean ± SEM. (C) The KD determination of Fn (left panel) and CBD(right panel) to Apa by SPR analysis. The measured KD were 0.58 ± 0.06 μMfor Apa and Fn, and 0.83 ± 0.04 μM for Apa and CBD.

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laminin, and heparan sulfate [5], all of which provide opportunities fordirect and specific bacterial adhesion. Pathogenic bacteria producespecific ECM attachment molecules that are often referred to as mi-crobial surface components recognizing adhesive matrix molecules(MSCRAMMs). MSCRAMMs, a group of virulence factors located in theouter surface of bacteria that mediate adhesion of a wide variety ofpathogenic bacteria to ECM components [5,15,26,36–42].

Adhesion of M. tuberculosis to the alveolar macrophage and epi-thelial cells is a determining step in infection, which enables subsequentinvasion and colonization of host cells [4]. Adhesins are surface ex-posed molecules that facilitate cell-to-cell or cell-to ECM adherence.These molecules also relate to bacterial aggregation, biofilm produc-tion, and immune evasion, contributing to virulence and pathogenesis[5]. Several M. tuberculosis adhesins have been reported. The mostcharacterized M. tuberculosis adhesin is heparan-binding hemagglutininadhesion (HBHA, Rv0475), which binds to heparan sulfate-containingreceptors on epithelial cells via C-terminal lysine-rich domain [43]. A19-kDa lipoprotein mediates macrophage binding through binding tomannose receptor [44]. Mycobacterial pili comprising protein Rv3312Ahas been found to bind to epithelial cells, implying its role as an adhesin[45]. Some intracellular proteins including chaperone-like protein andmalate synthase have been found to locate on bacterial surface and

mediate adhesion [46,47].Bacterial cell-wall-associated fibronectin binding proteins (FnBPs)

are important multifunctional virulence factors that facilitate host at-tachment. Recently, application of protein vaccines using FnBP-basedderivatives has been used as a promising strategy against staphylo-coccal infections [48]. Binding to Fn, likely a significant factor in thevirulence of mycobacteria, suggests a potential first step in the at-tachment and entry of mycobacteria into host cells. In addition to Apa, anumber of mycobacterial proteins were also identified to serve as FnBPssuch as Ag85 complex [13,15,49] and MPT51 (also called Ag85D orFbpD) [50]. Apa glycoprotein was found to be restricted to the myco-bacterium complex and not found in other mycobacterial species in-vestigated (i.e. M. avium, M. marinum, and M. smegmatis). Moreover,transfected M. smegmatis expressing Mtb Apa glycoprotein gains in-creased binding with human pulmonary surfactant protein A (hPSP-A)in comparison to the wild strain [51]. Formerly considered secreted,Apa has also been shown to be associated with cell wall for long enoughto aid attachment of PSP-A [51]. It is also important to note that MtbApa and M. bovis MPB83 are glycosylated. Mtb Apa has α-1,2-manno-oligosaccharides, whereas MPB83 has been shown to be substituted byα-1,3-linked manno-oligosaccharides [51]. Mtb Apa has also beenshown to be involved in various other activities like CD4+ and CD8+ T

Fig. 4. Mapping the binding fragments of Apa on CBD. (A) Sequence alignment of CBD modules 1–11. The conserved residues are highlighted (identical-red;similar-boxed). (B) Binding of Apa to immobilized recombinant GST-tagged CBD modules. Individual truncated CBD modules were coated on microtiter plate wells(0.5 μg), incubated with his-tagged Mtb Apa (0.2 μM), and detected by ELISA. Each value represents the mean ± S.D. of three trials performed in triplicate samples.

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cell proliferation [52], induction of the delayed type hypersensitivityreaction [25], and eliciting protection against Mtb challenge in theguinea pig [53]. It may also have role in host selectivity.

From current results, Mtb Apa which plays a role in the initial stepof bacterial infection could specifically bind to FnIII4/FnIII5. Blockingthe interaction between Mtb Apa and Fn via small molecules or poly-peptides, or designing vaccine to induce Apa-specific immune responsesmay hold potentials in developing new anti-mycobacterial therapeutics[54]. To achieve this purpose, three-dimensional structure of Mtb Apais essential information. In this study, the crystal structure of Mtb Apa isreported. Despite Mtb Apa shares very low sequence identity to knownstructures, several proteins with homologous overall fold were identi-fied through a search in DALI server (Fig. S3). In addition to the mostsimilar structures yeast Ran-binding Mog1P and plant PSII-stabilizingPsbP (Fig. 2), the search also hit a human Ran-binding Mog1 (PDB ID,5YFG), a trans-kingdom toxin TplEi (PDB ID, 5H7Z) and a periplasm-

localized immunity protein Tsi3 from Pseudomonas aeruginosa (PDB ID,4N7S), and a PSII-stabilizing PsbP from cyanobacteria (PDB ID, 2XB3).One should pay attention to the human Mog1 in the context of de-signing Apa-based anti-mycobacterial agents. Mog1 stimulates GDP/GTP release from small GTPase Ran and forms tight complex to apo-form Ran [55]. As the Ran cycling between nucleotide-bound and freestatus is critical in chromatin-driven spindle assembly and chromosomesegregation, the homeostatic control of Ran-Mog1 binding is consideredimportant during mitosis [56]. Though Mtb Apa shares very low se-quence identity to human Mog1 (< 14%), the effects of human Mog1and its related function upon applying any Apa inhibitor/competitorshould be taken into consideration.

Previously, Zhao et al., identified the Fn binding motif of M. aviumApa as 273RWFV276 using peptide binding assay [16]. However, basedon the protein structure that we reported in this study, RWFV motifmight not contribute to Fn-binding since the side chains of R258, W259,

Fig. 5. Binding of Apa mutants to FnIII-5 module. (A) Location of RWFV motif in ΔApa structure. Two images on the right panel are related by 180°. (B) Variousconcentrations (0.2, 0.5, 1, 2, 5, 10, and 20 μM) of GST-tagged FnIII-5 were coated on microtiter plate wells and incubated with 0.2 μM His-tagged wild type andmutant Apa proteins (wild type, R258A, W259A, F260A, and V261A). The measured KD values were 1.71 ± 0.54 μM, 1.83 ± 0.52 μM, 2.37 ± 0.56 μM,2.34 ± 0.43 μM, and 1.81 ± 0.49 μM, for wild type, R258A, W259A, F260A, and V261A, respectively. Each value represents the mean ± S.D. of three trialsperformed in triplicate samples.

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F260, and V261 (corresponding to R273, W274, F275, and V276 in M.avium Apa) are buried in the interior of the protein (Fig. 5A). Consistentresults were obtained in mutagenesis experiments. Ala substituents ofRWFV motif showed minimal effect on Fn-binding compared to wildtype protein (Fig. 5B). The discrepancy between present and previousreports might result from the experimental design. In previous studies,peptides were utilized in the binding assay while peptide fragment maynot fold into the real protein structure. On the other hand, the re-combinant protein with single mutations used in our study is the goldstandard to validate protein-protein interaction. In conclusion, this isthe first study to report the structure and Fn-binding characteristics ofApa. FnIII-4 and FnIII-5 within CBD are the only two modules con-tributing to the Apa-Fn interaction. Since Apa plays a role in stimulatingimmune responses and host cells adhesion, the data we reported heremay contribute to in-depth antimycobacterial studies.

Acknowledgements

This work was supported by Grant MOST 104-2311-B-005-015 fromthe Ministry of Science and Technology of the Republic of China;Taiwan Protein Project (grant no. AS-KPQ-105-TPP); the NationalNatural Science Foundation of China (grants 31600638, 31870790 and31570130); KFZD-SW-215-01 from CAS; CAS InterdisciplinaryInnovation Team; Youth Innovation Promotion Association, CAS;Taiwan Young Visiting Scholar Funding, CAS, USDA National Instituteof Food and Agriculture (grant no. 2019-67015-29846); NationalNatural Science Fundation of China (grant 31700057). The synchrotrondata collection was conducted at beam line TPS-05A and BL15A1 ofNSRRC (National Synchrotron Radiation Research Center, Taiwan).

Declaration of competing interests

The authors claim no conflict of interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbagen.2019.06.003.

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