Novel insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F

Accepted Manuscript Novel insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F

Yew Kwang Toh, Asha Manikkoth Balakrishna, Malathy Sony Subramanian Manimekalai, Boon Bin Chionh, Ramya Ramaswamy Chettiyan Seetharaman, Frank Eisenhaber, Birgit Eisenhaber, Gerhard Grüber PII: DOI: Reference:

S0304-4165(17)30295-7 doi: 10.1016/j.bbagen.2017.09.011 BBAGEN 28944

To appear in: Received date: Revised date: Accepted date:

30 June 2017 11 September 2017 15 September 2017

Please cite this article as: Yew Kwang Toh, Asha Manikkoth Balakrishna, Malathy Sony Subramanian Manimekalai, Boon Bin Chionh, Ramya Ramaswamy Chettiyan Seetharaman, Frank Eisenhaber, Birgit Eisenhaber, Gerhard Grüber , Novel insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F, (2017), doi: 10.1016/j.bbagen.2017.09.011

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ACCEPTED MANUSCRIPT Novel insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F Yew Kwang Toh1#, Asha Manikkoth Balakrishna2#, Malathy Sony Subramanian Manimekalai2, Boon Bin Chionh2, Ramya Ramaswamy Chettiyan Seetharaman2, Frank

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Eisenhaber1,3, Birgit Eisenhaber1 and Gerhard Grüber1,2,*

Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), 30

Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive,

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Biopolis Street, #07-01 Matrix, Singapore 138671, Republic of Singapore

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Singapore 637551, Republic of Singapore

School of Computer Engineering, Nanyang Technological University (NTU), 50 Nanyang

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Drive, Singapore 637553, Republic of Singapore

Authors contributed equally to this work

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To whom correspondence should be addressed: Prof. Dr. Gerhard Grüber, Tel.: + 65 - 6316

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2989; Fax: + 65 - 6791 3856, E-mail: [email protected]

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Keywords: Protein disulfide oxidoreductases, hydrogen peroxide, alkylhydroperoxide reductase, Enterococcus faecalis, AhpF, thioredoxin-like domain

Abbreviations: H2O2, hydrogen peroxide; AhpR, alkyl hydroperoxide reductase; AhpF, alkyl hydroperoxide reductase subunit F; AhpC, alkyl hydroperoxide reductase subunit C; CP, peroxidatic cysteine; CR, resolving cysteine; NADH, nicotinamide adenine nucleotide; CTD:Pyr_redox_2, pyridine nucleotide-disulfide oxidoreductase domain; NTD_N/C, thioredoxin-like domain; FAD, flavin adenine nucleotide; EfAhpF, E. faecalis AhpF; 1

ACCEPTED MANUSCRIPT EcAhpF, E. coli AhpF; StAhpF, S. typhimurium AhpF; SAXS, small-angle x-ray scattering; PDO, protein disulfide oxidoreductase; PfPDO, Pyrococcus furiosus protein disulfide

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oxidoreductase.

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ACCEPTED MANUSCRIPT Abstract The ability of the vancomycin-resistant Enterococcus faecalis (V583) to restore redox homeostasis via antioxidant defense mechanism is of importance, and knowledge into this defense is essential to understand its antibiotic-resistance and survival in hosts. The flavoprotein disulfide reductase AhpR, composed of the subunits AhpC and AhpF, represents

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one such vital part. Circular permutation was found to be a feature of the AhpF protein family. E. faecalis (V583) AhpF (EfAhpF) appears to be a representative of a minor subclass

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of this family, the typically N-terminal two-fold thioredoxin-like domain (NTD_N/C) is

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located at the C-terminus, whereas the pyridine nucleotide-disulfide oxidoreductase domain

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is encoded in the N-terminal part of its sequence. In EfAhpF, these two domains are connected via an unusually long linker region providing optimal communication between

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both domains. EfAhpF forms a dimer in solution similar to Escherichia coli AhpF. The crystallographic 2.3 Å resolution structure of the NTD_N/C domain reveals a unique loop-

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helix stretch (409ILKDTEPAKELLYGIEKM426) not present in homologue domains of other 415PAKELLY421-helix

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prokaryotic AhpFs. Deletion of the unique

or of

415PAKELL420

affects

protein stability or attenuates peroxidase activity. Furthermore, mutation of Y421 is described

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to be essential for E. faecalis AhpF’s optimal NADH-oxidative activity.

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ACCEPTED MANUSCRIPT 1. Introduction In pathogenic bacteria, multiple antioxidant mechanisms to counteract the deleterious effect of reactive oxygen species such as hydrogen peroxide (H2O2), are of importance as the macrophages in the mammalian immune system produce an oxidative burst of diffusible H2O2 molecules to kill the pathogen [1-3]. One such bacterial defense mechanism is the alkyl

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hydroperoxide reductase (AhpR) system, which consists of a 2-Cys peroxiredoxin, AhpC and a thiol-dependent peroxiredoxin reductase, AhpF. In most prokaryotes, expression of both

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subunits is regulated by the transcription factor OxyR which becomes activated by an

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oxidative-stress induced disulfide bond formation with the subsequent up-regulation of AhpC

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and AhpF in the cell [4, 5]. Other than protecting the host cells, the AhpR system also seeks to modulate the signaling effect of the intracellular H2O2 molecules [6].

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In most bacteria, AhpC exists as a basic dimeric unit when it is oxidized and forms a decamer- or dodecamer ring under reduced conditions [7-10]. A peroxidatic cysteine (CP)

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residue in one unit of the dimeric AhpC reacts with H2O2 to form sulfenic acid. A resolving

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cysteine (CR) residue in the other unit of the dimer then attacks the sulfenic acid to release water. As a result, a disulfide bond is formed between the peroxidatic and resolving cysteine

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residues. Finally, the oxidized AhpC becomes regenerated by the NADH-dependent oxidoreductase AhpF [11]. The latter is a homodimer protein characterized by two distinct

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domains connected by a flexible linker region [12]. The pyridine nucleotide-disulfide oxidoreductase domain (CTD:Pyr_redox_2) contains the FAD and NADH-binding sites and a CXXC-motif, while a two-fold thioredoxin-like domain (NTD_N/C) harbors a second CXXC-motif (Fig. 1). The catalytic mechanism reveals a cascade of electron transfers from NADH via FAD, a prosthetic molecule, to the first CXXC-redox center within the CTD:Pyr_redox_2 domain and then onto a second CXXC-redox center at the NTD_N/C. Small angle X-ray scattering (SAXS) studies showed that E. coli AhpF (EcAhpF) and AhpC (EcAhpC) form a complex with a stoichiometry of 10:2 (EcAhpC:AhpF) in solution [13]. 4

ACCEPTED MANUSCRIPT Electrons from two CXXC-catalytic sites of AhpF can then reduce two oxidized AhpCs, thus regenerating them for H2O2 reduction [11, 13]. E. faecalis is a gram-positive, commensal bacterium of the gastrointestinal tract of humans and other mammals. However, they can become opportunistic pathogens and as a result of broad misuse in antibiotic application, multi-drug resistant strains such as

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vancomycin-resistant E. faecalis (V583) have emerged as leading cause of nosocomial infection [14]. It has been shown that antibiotics often kill bacteria by a common mechanism

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of action which is oxidative stress [15-17]. Vancomycin-induced superoxide production has

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also been linked to nephrotoxicity, which is a common side effect of vancomycin therapy

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[18]. Thus it is important for antibiotic-resistant bacterial strains to upregulate antioxidant mechanisms in order to protect against the bactericidal effect of the antibiotic. Indeed,

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superoxide dismutase responsible for detoxifying superoxide radicals is found to be important for E. faecalis resistance to vancomycin and penicillin [19]. Furthermore, studies have shown

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that E. faecalis is able to survive inside the oxidative stress environment of macrophages and

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even to increase its virulence [20, 21]. It is worth noting that E. faecalis generally lacks an active catalase in the absence of heme [22]. In this context, it means that other players of the

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E. faecalis antioxidant mechanism system have to upregulate their activity to compensate for the inactive catalase. Interestingly, the E. faecalis genome has more genes encoding for

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antioxidant enzymes than phylogenetically closely related bacteria [23] and the E. faecalis AhpC and AhpF genes are under the control of an OxyR homologue, HypR. [24]. To date, no studies on the E. faecalis AhpR system’s peroxidatic activity have been described. In this paper, we uncover the phenomenon of domain swapping and unique structural features in the EfAhpF. Despite these striking structural characteristics, EfAhpF forms a dimeric protein, whose flexible traits and conformations were studied by SAXS. Truncations or swapping of these unique elements as well as single substitutions of EfAhpF

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ACCEPTED MANUSCRIPT mutants provide insights into their enzymatic role and into the evolutionary divergence of the

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vancomycin-resistant, gram-positive E. faecalis (V583) and other gram-negative bacteria.

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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Cloning of wild-type EfAhpF and mutant genes Wild-type (WT) E. faecalis (V583) AhpF (Gene ID: EF2738) was cloned from E. faecalis (V583) genomic DNA (kindly provided by Prof. Gregory Cook, Department of Microbiology and Immunology, Otago School of Medical, Sciences, University of Otago, New Zealand) by

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polymerase chain reaction (PCR) using the forward and reverse primers with NcoI and SacI restriction site (bold) respectively, (Supplementary Table 1) and ligated to a modified pET9-

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d1-His6 vector [25].

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EfAhpF354-560 was cloned using the forward and reverse primers with NcoI and SacI

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restriction site (bold) respectively, (Supplementary Table 1) and ligated to the pET9-d1-His6 vector.

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Cloning of EfAhpF (residues 1-313 and 354-560) with the E. coli AhpF (EcAhpF) linker (residues 195-215) mutant (where the native linker in EfAhpF was switched with the EcAhpF

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linker) was performed using Inverse Fusion PCR method [26] with primer A (Supplementary

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Table 1) (bold indicates EfAhpF residues 310-313, while underlined indicates EcAhpF linker residues 195-200), primer B (Supplementary Table 1) (corresponding to the EcAhpF linker

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residues 206-215) and primer C (Supplementary Table 1) (corresponding to EfAhpF residues 354-363). First, the EcAhpF linker region was amplified by conventional PCR using primer

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A and B. Inverse fusion PCR was completed by mixing primer B and C, EfAhpF template vector and EcAhpF linker PCR product (with a molar ratio of vector to linker PCR product, 1:100) and using KAPA HiFi DNA polymerase under conditions as described in the paper [26]. The gene product was ligated to the pET9-d1-His6 vector. The EfAhpF ∆415-421 mutant was cloned by Inverse Fusion PCR with primer A (Supplementary Table 1) (corresponding to EfAhpF residues 354-363), primer B (Supplementary Table 1) (corresponding to EfAhpF residues 405-414) and primer C (Supplementary Table 1) (corresponding to EfAhpF residues 422-430) as described above 7

ACCEPTED MANUSCRIPT and ligated into pET9-d1-His6. The EfAhpF ∆415-420 mutant was cloned using primer A and primer B as for the EfAhpF ∆415-421 mutant and primer C (Supplementary Table 1) (corresponding to EfAhpF residues 421-430) by Inverse Fusion PCR, followed by ligation into pET9-d1-His6. The single EfAhpF mutant L420P was cloned using primers (Supplementary Table 1)

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(underlined indicates single amino acid substitution), while the EfAhpF Y421A mutant was cloned using primers (Supplementary Table 1) by non-overlapping mutagenesis and ligation

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into the pET9-d1-His6 vector. Fidelity of all cloning products was confirmed by DNA

2.2 Cloning of the WT E. faecalis AhpC gene

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sequencing.

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The gene encoding WT EfAhpC (Gene ID: EF2739) was cloned from E. faecalis (V583) genomic DNA by PCR using forward primer (Supplementary Table 1) with NcoI restriction

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site (bold) and the reverse primer (Supplementary Table 1) with SacI restriction site (bold).

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The PCR product was ligated into pET9-d1-His6. Fidelity of the correct clone was confirmed

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by DNA sequencing.

2.3 Purification of WT and mutant proteins of E. faecalis

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WT E. faecalis AhpR subunits AhpC and F as well as its mutants were transformed into E. coli BL21 (DE3) cells (Stratagene, USA) and induced with 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) at 20 °C (EfAhpF) or 37 °C (EfAhpC) for protein production. Cells were lysed on ice by sonication for 3 × 1 min in buffer A (50 mM Tris/HCl, pH 7.5, 200 mM NaCl, 0.8 mM DTT and 2 mM PefablocSC (BIOMOL)). EfAhpF ∆415-421 mutant cells were lysed via French press at 15 000 psi. The cell lysate was centrifuged at 10 000 × g for 35 min before the supernatant was filtered (0.45 μm; Millipore) and incubated with NiNTA Agarose (Qiagen) for one hour at 4 °C. His6-tagged protein was then eluted with an 8

ACCEPTED MANUSCRIPT imidazole gradient (20 mM to 500 mM) in Buffer A. For EfAhpC, fractions were pooled and applied onto a gel filtration column (SuperdexTM 200 HR 10/300 column, GE Healthcare) in buffer B (50 mM Tris/HCl, pH 7.5 and 200 mM NaCl). In the case of EfAhpF, fractions containing the recombinant protein were pooled and applied onto an anion chromatography column ResourceQ (6 ml). Fractions containing recombinant protein were pooled and applied

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onto a gel filtration column (SuperdexTM 200 HR 10/300 column, GE Healthcare) in buffer B (50 mM Tris/HCl, pH 7.5 and 200 mM NaCl), and the purified recombinant protein was

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pooled and concentrated. Recombinant EcAhpF and EcAhpC were produced and purified as

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described [10].

2.4 Crystallization of EfAhpF354-560

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Recombinant EfAhpF354-560 (NTD_N/C) was concentrated to 5 mg/ml in a buffer composed of 50 mM Tris/HCl, pH 7.5 and 200 mM NaCl. Hexagonal-shaped crystals were

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grown in precipitant solution of 0.1 M Bis-Tris (pH 6.5), 1.5 M ammonium sulfate and 0.1 M

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sodium chloride by hanging-drop vapor-diffusion method. Initial crystals were cryo-frozen in 20% – 30% glycerol and diffracted to about 4.5 Å. Further optimization of the cryo-buffer

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was done using different concentrations of Proline. The final optimized EfAhpF354-560 crystals were flash-frozen in liquid nitrogen at 100 K in crystallization buffer containing 0.1 M Bis-

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Tris (pH 6.5), 2.0 M ammonium sulfate, 0.1 M sodium chloride and 2.0 M Proline.

2.5 Data collection and structure determination of EfAhpF354-560 X-ray diffraction data for the EfAhpF354-560 were collected at 100 K at beamline 13B1 of the National Synchrotron Radiation Research Centre (NSRRC, Hsinchu, Taiwan) using the ADSC Quantum 315 CCD detector. Data were collected as a series of 0.5° oscillation images covering a crystal rotation range of 100° on cryo-cooled crystals. A complete 2.3 Å data set

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ACCEPTED MANUSCRIPT was collected and the diffraction data were indexed, integrated and scaled using HKL2000 suite [27]. The results of data processing and data statistics are summarized in Table 1. The crystallographic structure was solved by molecular replacement method using the Salmonella typhimurium AhpF (StAhpF) structure (PBD ID: 1ZYN_B chain) [28] as model in PHASER [29]. The amino acid identity between EfAhpF and StAhpF is 31.8% as

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determined by Clustal Omega program [30]. The starting model was improved by iterative cycles of model building manually using Coot [31] and refinement by REFMAC5 [32] of

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CCP4 suite. Refinement was done until convergence and the geometry of the final model was

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validated with MolProbity [33]. This analysis indicated that the overall geometry of the final model ranked in the 100th percentile (MolProbity score of 1.36). The clash score for all atoms

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was 2.39 corresponding to a 100th percentile ranking of structures of comparable resolution.

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The data were cut off at 2.3 Å based on the CC1/2 statistics [34] (correlation coefficient between two random halves of the data set where CC1/2 > 10%) to determine the high-

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resolution cut-off for our data. Phenix [35] was used to compute CC1/2 (88.9% for the highest

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resolution shell and 99.9% for the entire data set), supporting our high-resolution cut-off determination. The figures were drawn using PyMOL [36] and structural comparison analysis

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was carried out using SUPERPOSE [37] from CCP4 suite [38, 39]. The coordinates and

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structure factors of EfAhpF354-560 at 2.3 Å have been deposited in the Protein Data Bank with accession code 5H29.

2.6 NADH-dependent peroxidase assay NADH-dependent peroxidase assay was monitored at 340 nm following the decrease in NADH absorbance using a stopped flow spectrophotometer Applied Photophysics SX20. The assay was carried out at 25 °C containing a 50 mM phosphate buffer at pH 7.0, 100 mM ammonium sulfate and 0.5 mM EDTA. One syringe arm contained a final concentration of

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ACCEPTED MANUSCRIPT 300 µM of NADH, 1 mM of H2O2, 1 µM of AhpC, and the other arm contained a final concentration of 1 µM AhpF. The assay was done in triplicates.

2.7 SAXS studies of EfAhpF SAXS data of EfAhpF were collected with the BRUKER NANOSTAR SAXS instrument,

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equipped with a Metal-jet X-ray source and VANTEC 2000-detector system according to

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[10, 40, 41]. The X-ray radiation was generated from a liquid gallium alloy with microfocus electron source (K = 1.3414 Å with a potential of 70 kV and a current of 2.857 mA). The X-

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rays were filtered through Montel mirrors and collimated by a two pinhole system. The

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sample to detector distance was set at 67 cm and the sample chamber and X-ray paths were evacuated. This setup covers the momentum transfer range 0.016 < q < 0.4 Å-1 (q = 4π

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sin(θ)/λ, where 2θ is the scattering angle). SAXS measurement for a standard compact globular protein, lysozyme, at a concentration of 5 mg/ml in buffer containing 100 mM acetic

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acid, pH 4.5 was also done, employing the same strategy as above for comparison. The data

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were flood-field, spatial corrected and processed using the build-in SAXS software. The data were tested for possible radiation damage by comparing the six data frames, which indicated

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no change. The scattering of the corresponding buffer was subtracted and the difference

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curves normalized for the concentration as well as initial intensity. All the data processing steps were performed using the program package PRIMUS [42]. The experimental data obtained was analyzed for aggregation using the Guinier region. The forward scattering I(0) and the radius of gyration, Rg were computed using the Guinier approximation assuming that at very small angles (q < 1.3/Rg) the intensity is represented as I(q) = I(0)·exp(-(qRg)2/3). These parameters were also computed from the extended scattering patterns using the indirect transform package GNOM [43], which provides the distance distribution function P(r) hence the maximal particle dimension, Dmax, and Rg. The normalized Kratky plot was generated by normalizing the scattering intensity I(q) to the forward scattering I(0), and the scattering 11

ACCEPTED MANUSCRIPT vector q to the radius of gyration Rg. The normalized Kratky-plot is used to assess the conformational behaviour of the polypeptide chain. In order to determine the oligomeric state of the protein, the molecular mass (MM) of the protein can be estimated in several ways from the SAXS data and cross-validation between them is very important. The MM was determined from the forward scattering of the sample

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I(0), by comparison with a reference protein, lysozyme [44]. This method is limited by the accurate measurement of protein concentration and partial specific volume, which leads to

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error in the MM determination by about 10-15% [44]. Alternatively, the MM was estimated

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from the scattering data based on the excluded (i.e. hydrated) particle volume, Vp [45, 46].

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The MM determination using the volume of correlation, Vc (defined as the ratio of I(0), to its total scattered intensity) is contrast and concentration independent and shown to have 5%

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error [47]. The MM estimated for EfAhpF (Table 2) confirm the dimeric state of the protein. Comparison of the experimental scattering curve with the theoretical scattering curves

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calculated from available high resolution crystal structures were performed with CRYSOL

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[48]. The compact and extended crystal structures were then used in OLIGOMER [42] to find the best fit to a multi-component mixture of proteins. Rigid body modeling was performed

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using the software CORAL [49] with the solved crystal structure of EfAhpF354-560 and a homology model of the CTD:Pyr_redox_2 (residues 1-313) that was generated by the

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RaptorX web server [50], that allows the flexibility of the loop region (residues 314-353), which connects the NTD_N/C and CTD:Pyr_redox_2.

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ACCEPTED MANUSCRIPT 3. Results 3.1 Sequence analysis of E. faecalis AhpF E. faecalis AhpF (EfAhpF) is a 560-amino acid protein (Fig. 1A). A striking feature of this protein is the so-called swapped domain phenomenon [51], where the pyridine nucleotide-disulfide oxidoreductase domain (CTD:Pyr_redox_2), called E. faecalis AhpF1-313,

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is found at the N-terminus (This domain is located at the C-terminus for E. coli and S. typhimurium). It contains the FAD- and NADH-binding sites and a CXXC-motif. The two-

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fold thioredoxin-like domain (NTD_N/C), called E. faecalis AhpF354-560, responsible for

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reducing AhpC, resides at the C-terminus (This domain is found at the N-terminus of EcAhpF

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and StAhpF) (Fig. 1A and 1B). Another notable feature is that EfAhpF has a purported linker region of forty residues (314-353) compared to twenty residues found in the well-studied

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EcAhpF [9, 10].

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3.2 Crystallographic structure of EfAhpF354-560

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To understand better about the structural features of EfAhpF, we have crystallized EfAhpF354-560, as it shows differences in amino acid composition and length to other related

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bacterial AhpF domains (Fig. 2A). The structure of EfAhpF354-560 was crystallized in the cubic space group P4132 and was refined to 2.3 Å resolution. The final R-factor and R-free

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(calculated with 5% of reflections that were not included in the refinement) were 19.7% and 23.3%, respectively (Table 1). There is one molecule of EfAhpF354-560 in the asymmetric unit with a solvent content of 71.62%. The EfAhpF354-560 monomer contained 207 amino acid residues with almost all main chain residues fitting well into the electron density. The EfAhpF354-560 monomer also contained one molecule each of SO4, Tris and proline and 106 molecules of water (Table 1). The overall structure of EfAhpF354-560 is shown in Fig. 2B. The side chain density for all the amino acids is very well resolved. The average B-factors was calculated to be 63.5 Å2. Analysis of the stereochemical quality of the final model by 13

ACCEPTED MANUSCRIPT PROCHECK has identified that 92.5% of all the residues are within the core regions of the Ramachandran plot, 7.5% are within the allowed regions. There were no residues in the disallowed region. Overall, EfAhpF354-560 consists of two “fused” so-called N- and C-terminal thioredoxinlike folds. The structure of EfAhpF354-560 reveals an α/β structure consisting of eight α-helices

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and eight β-strands (Fig. 2B). The β-sheets are arranged in the order β2, β1, β3, β4, β6, β5, β7 and β8, in which the orientation of β3 and β7 are antiparallel with respect to others. The eight

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β-strands form an intrinsic β-sheet that constitutes the core of the protein. The eight α-helices

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are distributed asymmetrically on either side of the central β-sheet. Five of the helices are located on one side and three of them are located on the other one. The EfAhpF354-560 chain

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starts with helix 1 and terminates with helix 8. Helix 2, 3, 6 and 7 are sandwiched between β-

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strands. Helix 4 and 5 are connected by a loop of residues 464SEGQPLEA471, with a sequence which is different to the one of the related NTD_N/C of EcAhpF (Fig. 2A). A unique stretch of residues

409ILKDTEPAKELLYGIEKM426

is present in-between β2 and β3 of EfAhpF,

The catalytic

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which encloses a short helix α3 composed of residues 415PAKELLY421 (Fig. 2C). 493CXXC496-motif

forms an intra-chain disulphide bond (Fig. 2D). In order

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to exclude any radiation effect, we investigated carefully the initial frames, which confirmed

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the disulphide formation. The dihedral angle for the disulfide bond is χ1 = 161.6°, χ2 = -138.4° for C493, χ1ꞌ = -76.1°, χ2ꞌ = 72.2° for C496, and χ3 for the S-S bond is determined to be 94.5°. The Sγ-atom of C493 in the disulfide is more exposed on the protein surface, while that of C496 is buried. The sequence alignment in figure 2A shows that the

493CXXC496-motif

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very well conserved and in majority of the organisms, the second cysteine is preceded by an asparagine, but in case of EfAhpF, the asparagine is substituted by an aromatic residue phenylalanine (Fig. 2D). This might in part explain the increased conformational strain in the 493CXXC496-motif.

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ACCEPTED MANUSCRIPT Along with the

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disulphide in EfAhpF354-560, there is another cysteine at

position 503, which is missing in both E. coli and S. typhimurium AhpF and is replaced by a leucine residue in the sequence. The extra cysteine C503 is located in helix α6 and present between β5 and β6 (Fig. 2A). Sequence alignments of the assemble partner EfAhpC reveals two additional cysteines (C13 and C66) when compared to other AhpCs (data not shown).

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However, whether the additional C503 of EcAhpF and C16, as well as C66 of EfAhpC are

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3.3 Structural comparison with other AhpF proteins

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mechanistically important is not known yet.

When EfAhpF354-560 was overlaid with full-length EcAhpF, it aligned well with the

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NTD_N/C of EcAhpF (residues 1-196), with an r.m.s.d of 2.7 Å for 182 Cα atoms (Fig. 2E). 409ILKDTEPAKELLYGIEKM426

loop-helix

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The major difference was seen in the unique

region present between β2 and β3. A conserved cis-proline, P427 is present between the loop-helix segment and β3, situated on the opposite side of

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409ILKDTEPAKELLYGIEKM426

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the catalytic 493CXXC496 motif. Previous studies have shown that the cis-conformation of the proline exposes the main chain oxygen of the preceding residue on the surface of the protein

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[52], which in case of EfAhpF is a methionine, M426 (Fig. 2F). This can then facilitate the formation of hydrogen bonds between the loop and stabilize the interaction [52]. In

2A).

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comparison, the preceding residue of the cis-proline in EcAhpF and StAhpF is a lysine (Fig.

3.4 SAXS analysis of EfAhpF SAXS experiments were performed to determine whether the oligomeric state and behavior of EfAhpF in solution would be similar or different from the recently described EcAhpF [12]. The Guinier plot at low angles is linear and revealed good data quality with no indication of protein aggregation (Fig. 3A, inset). The radius of gyration (Rg) value from the 15

ACCEPTED MANUSCRIPT Guinier approximation was 41.9 ± 0.99 Å and SAXS data of EfAhpF gave a calculated molecular weight (MWVc) of 126 kDa which is double its theoretical molecular weight of 61 kDa (Table 2). This shows that EfAhpF is dimeric in solution, and is further corroborated by the program CRYSOL, where the theoretical scattering curves of monomeric and dimeric form of the extended EcAhpF (PDB ID: 4O5Q) and compact StAhpF (PDB ID: 1HYU)

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respectively, were fitted against the experimental solution X-ray scattering data of EfAhpF [48]. A ribbon representation of the extended EcAhpF and compact StAhpF model is shown

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in supplementary Fig. 1. The analysis showed that the dimeric form of the extended EcAhpF

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(χ2 = 1.23) and compact StAhpF (χ2 = 1.37) had a better fit than the monomeric extended

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EcAhpF (χ2 = 7.68) or compact StAhpF (χ2 = 8.68) conformations (Fig. 3A). The distance distribution function (P(r)) revealed that the maximum particle dimension (Dmax) of EfAhpF

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is 153 ± 15 Å. As in the case of EcAhpF [12], the dimeric EfAhpF protein exists in open and closed conformations in solution. To analyze this possibility, the program OLIGOMER [42] was used to fit the experimental scattering curve against the dimeric extended and compact

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structures of EcAhpF and StAhpF, respectively. A fit with a χ2-value of 1.35 (Fig. 3B) was achieved, where a mixture of extended- and closed-dimeric conformations of EfAhpF were

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present in the ratio of 43% : 57%.

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Next we wanted to examine whether the longer linker of EfAhpF is more flexible and thus could result in faster closed-open conformation leading to more optimal electron transfer rate between the CTD:Pyr_redox_2 and NTD_N/C. To test this, the normalized Kratky-plot (q4 I(q) vs. q4) of EfAhpF were plotted along with EcAhpF and lysozyme (Fig. 3C). Peak maxima for both EcAhpF and EfAhpF shifted to the right of the peak maximum for lysozyme, indicating that both AhpF proteins exhibited increase in flexibility as compared to lysozyme. However, it also showed that both EcAhpF and EfAhpF peak maxima overlapped, indicating that EfAhpF has similar flexibility as EcAhpF. 16

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3.5 Flexibility of EfAhpF in solution The SAXS-data described above showed that like EcAhpF, EfAhpF is a flexible, dimeric protein in solution which adopts a closed-open conformation. To analyse the flexibility of EfAhpF in solution in more depth, we performed rigid-body modelling of the dimeric

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EfAhpF in the program CORAL, using the SAXS data obtained earlier [46], and allowing

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flexibility for the loop region (residues 314-353) between CTD:Pyr_redox_2 (residues 1-313)

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and NTD_N/C (residues 354-560). The determined crystal structure of EfAhpF354-560 was used as a model for NTD_N/C, while the model for CTD:Pyr_redox_2 was derived from

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homology modelling using the RaptorX web server [50]. The program CORAL performs a SAXS-based rigid modelling of complexes using a combined rigid body for the atomic

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structures of the domains and the ab initio modeling of the missing linker region. As shown in figures 4A and 4B, the CORAL model revealed that both a closed and an open

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conformation are present in the same dimeric protein and the experimental data had a good fit

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to the model with a χ2-value of 0.39.

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3.6 NADH- dependent peroxidase assay of WT EfAhpF and EfAhpC

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Given the interesting features described above, we seek to understand whether these differences have any direct impact on the EfAhpF enzymatic ability to reduce EfAhpC for hydrogen peroxide detoxification. Full-length EfAhpF was cloned and purified. The size exclusion chromatogram with gel inset (Supplementary Fig. 2A) showed that recombinant EfAhpF was purified with monodispersity and a good purity yield. It has been described that the activity of Amphibacillus xylanus NADH oxidase in the presence of peroxiredoxins was same in aerobic condition as was in anaerobic condition [53]. NADH oxidase with a 51.2% identity to StAhpF, was shown to act as a functional reductase 17

ACCEPTED MANUSCRIPT for StAhpC [54]. Thus in our study, NADH oxidation by EfAhpC-AhpF complex to detoxify hydrogen peroxide was measured in an aerobic condition. Figure 5A shows that the wild-type EfAhpC-AhpF complex is enzymatically active and NADH oxidation to detoxify hydrogen peroxide, which was measured by the decrease in absorbance for NADH at 340 nm, reached completion at about 50 seconds.

415PAKELLY421

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To determine whether the presence of the additional α3-helix with the amino acids of EfAhpF354-560 is critical for its reducing ability, a deletion mutant EfAhpF

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∆415-421 was generated (Supplementary Fig. 2B). As revealed in the SDS-gel of

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supplementary figure 2B, this deletion mutant undergoes degradation and indicates the

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importance of the unique helix in protein stability. Since the EfAhpF ∆415-421 cannot be used in the peroxidase assay and to understand more in depth the importance of critical 415PAKELLY421

segment in protein stability, the deletion mutant EfAhpF

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residues of the

∆415-420 was engineered. Supplementary Figure 2C reveals its elution profile and the stable and pure EfAhpF ∆415-420 protein observed in the gel. The NADH-dependent peroxidase

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assay showed that NADH oxidation for EfAhpF ∆415-420 does not reach completion before 150 seconds as compared to WT EfAhpF (Fig. 5A), indicating the importance of the short

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helix α3 for EfAhpF activity.

The purification of the EfAhpF ∆415-421 mutant demonstrates the importance of amino

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acid Y421 in protein stabilization. Furthermore, the crystallographic structure reveals that Y421 is stabilized by a hydrogen bond network (Fig. 5B), including the hydrogen bond between the main chain carbonyl oxygen of Y421 and the epsilon amino group of K442 located on β4. In addition, the hydroxyl group of Y421 interacts with the main chain carbonyl oxygen of G436 via a water molecule. Interestingly, this G436 is highly conserved among all AhpFs. These hydrogen bonding interactions as well as the aromatic interaction between the side chains of Y421 and Y438, located on the loop between β3 and β4, may play a significant role in stabilizing the α3-helix (Fig. 5B). To ascertain whether Y421 is essential for the 18

ACCEPTED MANUSCRIPT optimal activity, it was mutated to an alanine (Y421A), resulting in the EfAhpF Y421A mutant, which was purified according to the protocol of WT EfAhpF. As shown in figure 5A, the EfAhpF Y421A mutant did not reach completion of NADH oxidation after 150 seconds with a similar profile as observed for the EfAhpF ∆415-420 mutant which were much slower than the WT enzyme (50 seconds). Taken together, Y421 is important for optimal protein

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stability and enzymatic catalysis of EfAhpF. To accentuate the critical role of Y421, the neighboring residue L420, which has no

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further contact with other intra-residues, was mutated to proline and the EfAhpF L420P

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mutant was expressed. Since the rate of NADH oxidation by the L420P mutant is similar to

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that of the WT enzyme (Fig. 5A), one can conclude that L420 is not critical to NADH oxidation and confirms further the importance of amino acid Y421.

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One additional notable difference between EfAhpF and EcAhpF is the length of the linker connecting the catalytic domains of EfAhpF1-313 and EfAhpF354-560 (Fig. 1). To investigate the

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relationship between the linker-length and the peroxidase activity, the linker of EfAhpF was

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replaced with the linker of EcAhpF to create the chimeric mutant termed EfAhpF linker mutant as demonstrated in supplementary figure 3A. The SDS-gel in supplementary figure

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3B confirmed the high purity of the mutant. Its peak elution profile has shifted to the left as compared to the WT, implying that switching for its non-native shorter linker has resulted in

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slight conformational effect. The time-dependent NADH-oxidation profile of the EfAhpF linker mutant demonstrates that shortening the native linker by insertion of the E. coli one, affects the activity when compared to WT EfAhpF as NADH oxidation was completed later (Fig. 6). This indicates that the length of the linker is fundamental for optimal catalysis.

19

ACCEPTED MANUSCRIPT 4. Discussion 4.1 Phenomena of the AhpR-system in E. faecalis Flavoprotein disulfide reductases, which are important for antioxidant mechanisms, are grouped into three subgroups: namely the disulfide reductases (glutathione reductases and mycothione reductases), alkyl hydroperoxide reductases (thioredoxin reductases and AhpF)

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and peroxidase-oxidase-reductases (NADH oxidases and NADH peroxidases) [55]. Although extensive data about these antioxidant proteins are published from gram-negative bacteria

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such as E. coli [9, 10, 12, 13, 56] and S. typhimurium [28, 57-60], there is still a knowledge

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gap of these antioxidant proteins of the gram-positive bacterium E. faecalis, particularly the

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vancomycin-resistant strain V583. It is known that the E. faecalis catalase, an important hydrogen peroxide scavenger, is not active in the absence of heme [22]. E. faecalis has also

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been shown to contain more genes encoding for H2O2 detoxification enzymes in its genome compared to its phylogenetically closely related bacteria such as Lactobacillus lactis IL1403

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and Streptococcus pyogenes MGAS10394 [23]. However, in the context of the AhpC-AhpF

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ensemble, it is not known whether it upregulates its peroxidase activity in E. faecalis as compared to other organisms and/or whether tolerance to H2O2 may in part be a regulator.

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As presented here, the E. faecalis (V583) AhpF gene reveals a swap of the usually Nterminal domain to the C-terminus (NTD_N/C domain) and the CTD:Pyr_redox_2 domain to

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the N-terminus, which is in striking contrast to the well-known E. coli and S. typhimurium AhpFs. This swapped domain phenomenon might be explained by the concept of circular permutation, whereby proteins have a reorganized order of amino acids in their sequences due to gene rearrangement, resulting in improved catalytic activity and altered substrate or ligand binding affinity [51]. Domain swapping is a means of generating very strong intermolecular interactions, but to what extent is still to be addressed. Apparently, E. faecalis AhpF is not the only AhpF protein that displayed a swapped domain feature. As documented in PFAM (release 28) [61], a HMMER search with both Pyr_redox_2 (PF07992) and 20

ACCEPTED MANUSCRIPT Thioredoxin_3 (PF13192) domains against the UniProt protein database reports 10532 sequences with the ‘E. coli-like’ AhpF sequence architecture while only 600 sequences were found to have their domain architecture swapped like E. faecalis (V583) AhpF (Supplementary Fig. 4A), With regard to species, the EcAhpF-like architecture was found in 762 different species, the Ef(V583)AhpF-like form only in 104. Interestingly, there are 4

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species with both types of sequence architecture for different strains, including E. faecalis (Supplementary Fig. 4B). Other species possessing an Ef(V583)AhpFs architecture include

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numerous Bifidobacterium strains, Clostridium stercorarium and Lactobacillus reuteri, etc. A

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recent minimum evolution tree of protein disulfide oxidoreductases (PDOs) and the

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thioredoxin-like domain of AhpFs showed a swapped orientation of domains in AhpFs of some bacteria, including Lactobacillus, Fusobacteria and Clostridia [62]. Despite the domain

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swapping and longer linker characteristics, EfAhpF behaves closely similar to EcAhpF [12] in solution according to the SAXS analysis (Fig. 3 and 4). The data showed EfAhpF exists as

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a dimeric protein in solution which adopts a dynamic closed-open conformation with a

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degree of flexibility similar to EcAhpF.

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4.2 The importance of the extra short α3-helix in E. faecalis AhpF354-560 As shown for the EcAhpF-AhpC formation, its interaction via the two-fold thioredoxin-

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like domain (NTD_N/C) of EcAhpF, EcAhpF1-196, is specific and long-lived as a result of a well-defined core binding epitope of EcAhpC, including the peroxidatic cysteine (CP) residue inside helix α2’ in one unit of the dimeric AhpC and the resolving cysteine (CR) inside helix α6 of the second unit, and the packing of the AhpC C-terminus via a groove on the backside of EcAhpF1-196 (Fig. 7A, left). The binding of the C-terminus of AhpC onto the back surface of EcAhpF1-196 is proposed to slow the dissociation of the AhpC-AhpF1-196 complex in E. coli and to enable efficient transfer of electrons between both proteins [56]. In comparison, the atomic structure of EfAhpF354-560 shows a similar groove wrapping around the protein and 21

ACCEPTED MANUSCRIPT providing the area for the packing of the EfAhpC C-terminus (Fig. 7A, right). Interestingly, while the C-terminus of EfAhpC would bind to the backside of EfAhpF354-560, it would interact with the unique helix

415PAKELLY421

(Fig. 7B) and could induce a sequential

interaction with the helix residue Y421, which makes interactions with the neighbouring residues G436 via a water molecule and Y438 and K442, with the later residues bridging the

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connection to helix α4. This helix is involved in the redox process with the catalytic disulphide (CPS-SCR) of bound EfAhpC, and thereby providing rapid electron transfer. The

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EfAhpF ∆415-421 and EfAhpF ∆415-420 mutants presented, confirm the importance of this

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unique helix for protein stability and catalytic activity. Furthermore, the NADH-dependent

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peroxidase data of the EfAhpF Y421A mutant underline the critical role of residue Y421 for a higher enzymatic activity and support the structural interconnection described above.

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A structural similarity search (DALI server) revealed similarity of EfAhpF354-560 with a glutaredoxin-like protein (pdb code: 2AYT [63] and 1J08) and protein disulphide

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oxidoreductase (PDO) (pdb code: 1A8L [52]) with a Z-score of 28 and an r.m.s.d. of ~1.7 Å,

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followed by EcAhpF and StAhpF. The high similarity of EfAhpF354-560 with the glutaredoxinlike protein and PDOs, like the Pyrococcus furiosus enzyme (PfPDO) [64]), reflects the

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presence of the extra loop-helix stretch of EfAhpF354-560, being also present in these classes of enzymes but absent in the gram-negative bacteria EcAhpF and StAhpF. Overall the structure

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of EfAhpF354-560 is quite similar to PfPDO, including the extension of the loop and the presence of a short helix at the N-terminus (Fig. 8). However, in PfPDO, each thioredoxin fold consists of one active disulphide center, whereas EfAhpF354-560 comprises only one redox active disulphide center, located in the second thioredoxin fold with a distinct CXXC active site motif (Fig. 8). This may depict EfAhpF as an in-between or hybrid protein between PDOs and other bacterial AhpFs.

22

ACCEPTED MANUSCRIPT 4.3 Linker region of E. faecalis AhpF The linker regions of EcAhpF and EfAhpF are diverse in sequence and length. Studies on the four serotypes of dengue virus non-structural protein 5 (NS5) have shown that specific amino acid residues in the linker region are crucial for the interaction between two linked enzymatic domains and the flexibility of the NS5 proteins [65, 66]. SAXS analysis showed

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that there is no significant difference in the flexibility of both EcAhpF and EfAhpF protein despite the heterogeneity in linker characteristics. However, when the longer linker region of

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EfAhpF was replaced with the shorter linker of EcAhpF, the linker mutant suffered a slight

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slowdown in its NADH oxidation with EfAhpC. This implies that the linker length is specific

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to each AhpF species, whereby the two domains of AhpF could come within an optimal spatial arrangement for efficient electron transfer. An interesting review by Ma et al (2011)

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presented that flexible linkers could perform an allosteric role encoded by its diverse sequence and length that is often found within the same protein family [67]. Once

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allosterically activated, the linker can undergo a series of conformational changes leading to

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the protein domains populating a more favorable state for faster interaction and cellular response. It was further suggested that this specificity of the diverse linker sequence and/or

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length could be exploited for drug discovery. Although this study highlights the fact that distinctive features found in E. faecalis AhpF

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are important to its catalytic activity observed in the assay, it must not be neglected that AhpF is only one part of the AhpR-system. Contributory factor(s) from 2-Cys peroxiredoxin AhpC is/are also important. As described above, E. faecalis AhpC (EfAhpC) has the two extra cysteine residues C13 and C66. Given the role of cysteine residues in the redox reaction, these extra cysteines could be important for the peroxidatic activity and is for future investigation.

23

ACCEPTED MANUSCRIPT Acknowledgement We are grateful to Prof. Gregory Cook, Department of Microbiology and Immunology, Otago School of Medical, Sciences, University of Otago, New Zealand, for providing E. faecalis (V583) genomic DNA. We thank the staff of beamline 13B1 at the National Synchrotron Radiation Research Centre (NSRRC), a national user facility supported by the

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National Science Council of Taiwan, ROC for expert help with data collection. The Synchrotron Radiation Protein Crystallography Facility at NSRRC is supported by the

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National Research Program for Genomic Medicine. This study was supported by the

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Singapore Ministry of Education Academic Research Fund Tier 1 (RG140/16) to G.G.. B.E.

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and F.E. kindly note the support from the Genome Informatics IAF311010 grant. Ramya Seetharaman thanks the Nanyang Technological University, Singapore, for awarding her a

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PhD Scholarship during her studies.

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Author contributions

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YKT designed and performed most of the experiments, analyzed the data and drafted the manuscript. AMB collected and analyzed the crystallography data and drafted the

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manuscript. MSSM collected and analyzed the SAXS data. BBC helped with the cloning and purification of one of AhpF mutant protein. RRCS helped with the cloning and purification of

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AhpF WT protein. FE and BE helped perform the computational analysis and editing of the manuscript. GG conceived the study and analyzed the data and wrote the manuscript. All authors read and approved the manuscript.

Conflict of Interest The authors declare that they have no conflict of interest.

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solution scattering of biological macromolecules from atomic coordinates, Journal of applied crystallography, 28 (1995) 768-773.

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[49] M.V. Petoukhov, D. Franke, A.V. Shkumatov, G. Tria, A.G. Kikhney, M. Gajda, C. Gorba, H.D.T. Mertens, P.V. Konarev, D.I. Svergun, New developments in the ATSAS

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protein structure modeling using the RaptorX web server, Nat Protoc, 7 (2012) 1511-1522. [51] Y. Yu, S. Lutz, Circular permutation: a different way to engineer enzyme structure and

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function, Trends Biotechnol, 29 (2011) 18-25. [52] B. Ren, G. Tibbelin, D. de Pascale, M. Rossi, S. Bartolucci, R. Ladenstein, A protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units, Nat Struct Biol, 5 (1998) 602-611. [53] T. Arai, S. Kimata, D. Mochizuki, K. Hara, T. Zako, M. Odaka, M. Yohda, F. Arisaka, S. Kanamaru, T. Matsumoto, S. Yajima, J. Sato, S. Kawasaki, Y. Niimura, NADH oxidase and alkyl hydroperoxide reductase subunit C (peroxiredoxin) from Amphibacillus xylanus form an oligomeric assembly, FEBS open bio, 5 (2015) 124-131. 30

ACCEPTED MANUSCRIPT [54] Y. Niimura, L.B. Poole, V. Massey, Amphibacillus xylanus NADH oxidase and Salmonella typhimurium alkyl-hydroperoxide reductase flavoprotein components show extremely high scavenging activity for both alkyl hydroperoxide and hydrogen peroxide in the presence of S. typhimurium alkyl-hydroperoxide reductase 22-kDa protein component, The Journal of biological chemistry, 270 (1995) 25645-25650.

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Eisenhaber, F. Eisenhaber, G. Grüber, NMR studies reveal a novel grab and release

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mechanism for efficient catalysis of the bacterial 2-Cys peroxiredoxin machinery, The FEBS journal, 282 (2015) 4620-4638.

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[57] L.B. Poole, H.R. Ellis, Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 1. Purification and enzymatic activities of overexpressed AhpF and AhpC

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proteins, Biochemistry, 35 (1996) 56-64.

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typhimurium. 2. Cystine disulfides involved in catalysis of peroxide reduction, Biochemistry,

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peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium, Biochemistry, 36 (1997) 13349-13356. [60] S. Nirudodhi, D. Parsonage, P.A. Karplus, L.B. Poole, C.S. Maier, Conformational studies of the robust 2-Cys peroxiredoxin Salmonella typhimurium AhpC by solution phase hydrogen/deuterium

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spectrometry, International journal of mass spectrometry, 302 (2011) 93-100. [61] R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangrador-Vegas, G.A. Salazar, J. Tate, A. Bateman, The Pfam 31

ACCEPTED MANUSCRIPT protein families database: towards a more sustainable future, Nucleic acids research, 44 (2016) D279-285. [62] A. Becerra, L. Delaye, A. Lazcano, L.E. Orgel, Protein disulfide oxidoreductases and the evolution of thermophily: was the last common ancestor a heat-loving microbe?, J Mol Evol, 65 (2007) 296-303.

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oxidoreductase from the bacterium Aquifex aeolicus, J Mol Biol, 356 (2006) 155-164.

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[64] Z.A. Wood, L.B. Poole, P.A. Karplus, Structure of intact AhpF reveals a mirrored

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thioredoxin-like active site and implies large domain rotations during catalysis, Biochemistry, 40 (2001) 3900-3911.

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[65] W.G. Saw, G. Tria, A. Grüber, M.S. Subramanian Manimekalai, Y. Zhao, A. Chandramohan, G. Srinivasan Anand, T. Matsui, T.M. Weiss, S.G. Vasudevan, G. Grüber,

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virus in solution, Acta crystallographica. Section D, Biological crystallography, 71 (2015) 2309-2327.

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ACCEPTED MANUSCRIPT Table 1: Data Collection and Refinement Statistics

Wavelength (Å) Space group Unit cell parameters (Å)

1.00 P4132 a = b = c = 134.28 α = β = γ = 90° 30-2.3 71.62 217 226 19 025 22.1(3.0) 99.8 (100) 5.1(66.2) 11.4 (11.7)

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Resolution range (Å) Solvent content (%) Total number of reflections Number of unique reflections I/ σ(I)a Completeness (%) Rmergeb (%) Redundancy

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Refinement Statistics R factorc (%) R freed (%)

19.7 23.3

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Number of amino acid residues

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Ramachandran statistics Most favored (%) Additionally allowed (%) Generously (%) Disallowed (%)

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63.5 56.58

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Mean atomic B values Overall Wilson B Factor

99.9 (88.9) 100 (97.0)

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CC1/2 CC*

92.5 7.5 0.0 0.0 0.005 0.979

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R.M.S. Deviations Bond lengths (Å) Bond angles (º)

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Clash score Molprobity score:

2.39 1.36

Values in parentheses refer to the corresponding values of the highest resolution shell (2.38 – 2.3 Å). Rmerge = ΣΣi|Ih - Ihi|/ ΣΣi Ih, where Ih is the mean intensity for reflection h. c R-factor = Σ||FO| -|FC||/ Σ|FO|, where FO and FC are measured and calculated structure factors, respectively. d R-free = Σ||FO| - |FC|/ Σ|FO|, calculated from 5% of the reflections selected randomly and omitted during refinement. a

b

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ACCEPTED MANUSCRIPT Table 2: SAXS data collection and derived parameters for EfAhpF Data collection parameters Instrument (source & detector)

Bruker NanoStar equipped with MetalJet eXcillum X-ray source and VÅNTEC-2000 detector

Beam geometry

100 m slit

Wavelength (nm)

0.134 0.016 – 0.4

q range (Å-1) Exposure time (min)

30 (6 frames x 5 min)

Protein sample

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EfAhpF

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Concentration range (mg ml )

3.5

288.15

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Temperature (K) Structural parameters†

87.66 ± 0.95

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I(0) (arbitrary units) [from P(r)] Rg (Å) [from P(r)]

43.73 ± 0.52 86.16 ± 1.51

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I(0) (arbitrary units) (from Guinier) Rg (Å) (from Guinier) Porod volume estimate (Vp) (Å3) DAMMIF excluded volume (Vex) (Å3) Dry volume from sequence (Å3) ‡

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Molecular mass determination†

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Calculated monomeric MM (kDa) [from sequence*]

41.91 ± 0.99 153 ± 15 ~190,991 ~263,737 ~74,059

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~61 119 ± 12

MM from excluded volume (Vex/2) (kDa)

131 ± 13

MM from volume of correlation ( Vc) (kDa)

126 ± 13

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Software employed

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MM from Porod invariant (Vp/1.6) (kDa)

Primary data reduction

BRUKER SAS

Data processing

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Ab initio analysis

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Validation and averaging

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Computation of model intensities

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Rigid body modeling

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3D graphics representations

PyMOL

* http://web.expasy.org/compute_pi/ ‡ http://www.basic.northwestern.edu/biotools/proteincalc.html

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ACCEPTED MANUSCRIPT Figure Legends Figure 1. The domain architecture of (A) EfAhpF and (B) EcAhpF. The two-fold thioredoxin-like domain (NTD_N/C, cd02974/cd03026) and the pyridine nucleotide-disulfide oxidoreductase domain (CTD:Pyr_redox_2, PF07992) are in a swapped orientation. EfAhpF contains a longer linker between NTD_N/C and CTD:Pyr_redox_2. Two cysteine redox

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centers are also illustrated within. The figure was created with DOG 1.0 [68].

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Figure 2. (A) Sequence alignment of the NTD_N/C domain of subunit AhpF from S.

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typhimurium, E. coli and E. faecalis (EfAhpF354-560). The secondary structure elements of

blue. The

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EcAhpF (orange) and EfAhpF354-560 (blue) are shown. Conserved residues are highlighted in 409ILKDTEPAKELLYGIEKM426-loop-helix

stretch of EfAhpF354-560 is outlined

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within a red box. The CXXC conserved motif is highlighted in yellow. The extra cysteine, C503 in EfAhpF354-560 is marked in green. The conserved cis-Proline is highlighted in red. (B) Cartoon representation of one monomer of EfAhpF354-560 shown in blue, the α-helices and

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β-strands are labeled. All the cysteines including the disulphide bond are shown in yellow spheres. (C) The FO-FC map contoured at 3 σ for the EfAhpF unique loop-helix sequence

493CHFC496-motif,

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is shown. (D) The FO-FC map at the EfAhpF active site

which is contoured at 3 σ. (E) The EfAhpF354-560, blue is overlapped with

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full-length EcAhpF (PDB ID: 4O5Q). The EfAhpF354-560 domain aligns well with the Nterminal domain of EcAhpF (residues 1-196, orange). The structure of EcAhpF is completed by the linker (green), the FAD-domain (yellow) and the NADH-domain (brown). The unique loop with the short helix

409ILKDTEPAKELLYGIEKM426

is marked with an asterisk. (F)

EfAhpF354-560., blue is overlapped with the NTD_N/C domain of S. typhimurium AhpF (PDB ID: 1ZYN; deep salmon). The

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stretch is colored red. The

residues M426 and P427 of EfAhpF354-560 as well as the equivalent residues in StAhpF are shown as sticks. 35

ACCEPTED MANUSCRIPT Figure 3. (A) SAXS pattern (○) of EfAhpF is displayed in logarithmic units for clarity. (Inset) Guinier plot show linearity indicating no aggregation. CRYSOL program is used to compare the theoretical scattering curves for monomeric (blue line) and dimeric (orange line) forms of extended EcAhpF (PDB ID: 4O5Q) and monomeric (red line) and dimeric (olive green line) forms of closed StAhpF (PDB ID: 1HYU) to the experimental scattering curve

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from EfAhpF. (B) Oligomer fits between experimental scattering data (○) of EfAhpF and theoretical scattering pattern (red line) calculated using open and closed dimers of EcAhpF

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structures. (C) Normalized Kratky plot of EcAhpF (green) and EfAhpF (magenta) forms

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compared to the compact globular lysozyme (grey) with it exhibiting a bell-shaped profile

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and the expected peak (dashed line) representing the theoretical peak assuming an ideal

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Guinier region of a globular particle.

Figure 4. (A) Fit of the scattering profile of the CORAL model (red line) to the experimental

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scattering pattern (○) of EfAhpF. (B) The CORAL-model generated for EfAhpF. The dimeric

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CTD:Pyr_redox_2 domain are colored yellow and pale yellow and the dimeric NTD_N/C

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domain are colored orange and light orange. The linker region (dots) is colored green.

Figure 5. (A) NADH-dependent peroxidase assay of WT EfAhpF (blue line), EfAhpF ∆415-

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420 (magenta line), EfAhpF Y421A (olive green line) and EfAhpF L420P (orange line) with E. faecalis AhpC. Data shown are the mean of triplicates. (B) The molecular network and hydrogen bonding of Y421, red with the neighboring residues in EfAhpF354-560, blue. All the residues involved are presented as sticks and are labeled.

Figure 6. NADH-dependent peroxidase assay of EfAhpF linker mutant with EfAhpC (violet line) and WT EfAhpF is shown for comparison (blue line). Data shown are the mean of triplicates. 36

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Figure 7. (A) (Left) A dimeric EcAhpC, brown and sand: PDB ID; 4QL9 has been manually docked onto the NTD_N/C of EcAhpF, wheat: PDB ID; 4O5Q where the C-terminal tail of EcAhpC, violet runs along the back groove of EcAhpF1-196, which is shaded pale green. The CP and CR of EcAhpC are represented by yellow and green lime spheres. The catalytic

560

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cysteine residues of EcAhpF1-196 are labeled. (Right) A surface representation of EfAhpF354where the back groove is colored pale green and the additional α3-helix segment is

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colored red. The model is rotated about 45° around the y-axis to show the α3-helix segment.

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(B) Hinging of Y421 with the catalytic area in EfAhpF354-560, blue white. The α3-helix is colored red. Residues G436, Y438, K442 interacting with Y421 are colored orange. Residues

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443-459 in EfAhpF354-560 helix α4 (blue) and residues 483-495 in the β5 sheet-loop bordering

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the catalytic sites (slate) form a contiguous patch that is purported to interact with AhpC. The

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cysteine catalytic center is represented by yellow spheres.

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Figure 8. Comparison of PfPDO (PDB: 1A8L, wheat, left) with EfAhpF354-560 (slate, right).

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Cysteine redox centers are represented by yellow spheres and α3-helix is colored red.

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ACCEPTED MANUSCRIPT Highlights

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Circular permutation identified in the Enterococcus faecalis AhpF (EfAhpF) First low-resolution solution structure and flexibility studies of EfAhpF Crystallographic structure of the catalytic N-terminal domain of EfAhpF EfAhpF residues identified to be essential for protein stability and activity Unravelling of a unique linker length of EfAhpF important for enzyme activity The data form a platform for structure-guided drug design

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     

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