Structural characterization of core-bradavidin in complex with biotin

RESEARCH ARTICLE Structural characterization of core-bradavidin in complex with biotin Nitin Agrawal 1 , Juha A. E. Ma ¨a ¨tta ¨ 2,3 , Markku S. Kulomaa 2 , Vesa P. Hyto ¨nen 2,3 , Mark S. Johnson 1 , Tomi T. Airenne 1 * 1Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland, 2Faculty of Medicine and Life Sciences and BioMediTech, University of Tampere, Tampere, Finland, 3Fimlab Laboratories, Tampere, Finland *[email protected] Abstract Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Åresolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidinlike protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E.coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin– Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin. Introduction Avidins (Avds) are proteins produced in oviducts of birds, reptiles and amphibians, and in several different bacteria [1,2]. In nature, Avds are most stable in their tetrameric [1,3–5] and dimeric [6] forms. They have a high affinity for D-biotin (K d = ~10 −15 M for chicken Avd) [1,4,7], which makes them attractive proteins for numerous biotechnological applications [7– 10]. The best studied Avds to date are the eukaryotic chicken Avd [1–3,11] and the bacterial streptavidin from Streptomyces avidinii [5,12,13]. As a secreted protein, chicken Avd is posttranslationally modified by cleavage of the 24 amino acid N-terminal signal peptide and by glycosylation at Asn17; the resulting mature protein has 128 amino acids [1,14]. The 159 amino acid gene product of the full-length streptavidin, in turn, is naturally trimmed at both the N PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 1 / 21 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Agrawal N, Ma¨a¨tta¨JAE, Kulomaa MS, Hyto¨nen VP, Johnson MS, Airenne TT (2017) Structural characterization of core-bradavidin in complex with biotin. PLoS ONE 12(4): e0176086. https://doi.org/10.1371/journal.pone.0176086 Editor: Eugene A. Permyakov, Russian Academy of Medical Sciences, RUSSIAN FEDERATION Received: February 9, 2017 Accepted: April 5, 2017 Published: April 20, 2017 Copyright: ©2017 Agrawal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. The coordinates and structure factors for the X-ray structure of core-bradavidin have been deposited in the Protein Data Bank with entry code 4BBO. Funding: This work was supported by grant funding from the Academy of Finland (257814 (MSJ), 272283 (MSJ), 290506 (VPH), 261285 (MSK), and 272288 (MSK)); Sigrid Juselius Foundation; Joe Pentti, and Tor Borg Memorial Fund; Academy of Finland FIRI program (141398) and C termini: the most studied form has 127 amino acids, only containing residues 13–139 of the full-length, and is referred to as core-streptavidin [5]. This naturally occurring truncated form of streptavidin has been shown to have low aggregate formation and high solubility, while retaining high affinity for biotin [15]. Moreover, the crystal structure of full-length streptavidin revealed that the 20-residue C-terminal extension (residues 139–159) binds on the surface of the protein and that residues 150–153 occupy the ligand-binding site of the same subunit—acting as an intrasubunit intrinsic ligand [16]. In addition to chicken Avd and streptavidin, other Avds have been characterized. They include the natural eukaryotic Avds from, for example, zebrafish (Danio rario) [17], frog (Xenopus tropicalis) [18], and mushroom (Pleurotus cornucopiae) [19], and the bacterial Avds, such as the dimeric rhizavidin (Rhizobium etli) [6], shwanavidin (Shewanella denitrificans) [20] and hoefavidin (Hoeflea phototrophica) [21]; the thermostable tetrameric burkavidin (Burkholderia pseudomallei) [22]; as well as bradavidin II (Bradyrhizobium diazoefficiens), which has a highly dynamic oligomeric structure [23]. Apart from the naturally occurring Avds, a number of genetically engineered Avds [7] have been produced, too. These include the dualchain Avd (dcAvd) [24] and single-chain Avd (scAvd) [25], respectively with two and four simultaneously modifiable ligand-binding sites, the monomeric streptavidin [26–28], the steroid-binding Avd (sbAvd) [29,30] and an extremely thermostable and protease resistant chimeric Avd [31]. Bradavidin is a tetrameric Avd from a nitrogen-fixing bacterium (B.diazoefficiens) found in root nodules of soy beans [32]. Wild-type (wt) bradavidin, after cleavage of the 25-residue signal peptide, has 138 amino acid residues, of which the last ten C-terminal residues are known as the Brad-tag [33]. The Brad-tag ( 129 GSEKLSNTKK) binds to the ligand-binding site of a neighboring subunit and hence serves as an intrinsic, intersubunit ligand, dissimilar both in mode of interaction and sequence to the C-terminal sequence found in full-length streptavidin [16] that acts as an intrinsic intrasubunit ligand. The key residues of Brad-tag interacting with the ligand-binding site are Glu131, Lys132 and Leu133, whereas in full-length streptavidin the key residues are Asn150, Gly151, Asn152, and Pro153. In the case of streptavidin, several peptide tags have been developed, including strep-tag I [34], strep-tag II [34,35], Nano-tag [36] and SBP-tag [37]; all of which have a different binding mode in comparison to the Brad-tag. Here, we report the tetrameric X-ray structure of core-bradavidin in complex with biotin at 1.60 Åresolution [PDB:4BBO]. In comparison to the X-ray structure of wt bradavidin (tetramer; 138 amino acids/14 kDa per subunit), core-bradavidin is artificially truncated at the Cterminus containing only residues 1–118 (12 kDa per subunit) and hence missing the Brad-tag [32]. In addition to these bradavidin structures, three structures of bradavidin II (each 115 amino acids/13 kDa per subunit) are known: two different crystalline forms of the apo protein, the monomeric Form-A [PDB:4GGR] and the dimeric Form-B [PDB:4GGT], as well as a tetrameric structure in complex with biotin [PDB:4GGZ] [23]. All the known X-ray structures of bradavidin and bradavidin II are from the same bacterium (B.diazoefficiens sp. nov; this strain was earlier known as the strain USDA 110 of Bradyrhizobium japonicum [38]). The core-bradavidin structure not only gives insight into the detailed biotin-binding mode of bradavidin but also helps us to further engineer core-bradavidin as a receptor with tighter binding towards ligands such as the Brad-tag itself. We also report homology models for the avidin-like proteins from Rhodopseudomonas palustris (rhodavidin [39]) and Bradyrhizobium sp. Ai1a-2 (referred to here as bradavidin A2); both of these proteins have the Brad-tag sequence at their C-terminus. Our better understanding about bradavidin binding to different ligands may aid in the development of novel constructs providing additional, improved tools for biotechnological purposes. Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 2 / 21 and Åbo Akademi Center of Excellence in Cell Stress and Aging. The funders had no influence in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Fimlab Laboratories (Tampere, Finland) provided support in the form of salaries for author VPH and in the form of research materials for the group lead by VPH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: The authors have declared that no competing interests exist. VPH is affiliated to Fimlab Laboratories; Fimlab Laboratories has supported financially the research conducted in the research group led by VPH. This does not alter our adherence to PLoS ONE policies on sharing data and materials. Results Overall structure of core-bradavidin The 3D structure of core-bradavidin in complex with biotin was solved at 1.60 Åresolution. This structure represents an artificially truncated form of bradavidin and lacks the C-terminal residues Gly114-Lys138 (residues Gly129-Lys138 correspond to Brad-tag) [32]. The sequence of the solved structure shares 35% identity with chicken Avd [PDB:1AVD] [3], and the overall tetrameric structure and the folds of the individual subunits I-IV (numbering according to [4]) of core-bradavidin are typical for Avds, including the wt bradavidin structure reported in [33] (Fig 1). We have also tried to crystallize wt bradavidin in complex with biotin without success, which may have been due to the presence of the biotin-competing C-terminal Brad-tag sequence in the wt bradavidin. It is also possible that reconfiguration of the C-terminus of wt bradavidin occurred due to biotin binding and that this may have altered crystal contacts and affected crystal formation. Biotin-binding mode of core-bradavidin—Conserved features Despite the low sequence identity between core-bradavidin and chicken Avd, the deeply buried residues involved in biotin binding and the mode of binding are highly conserved (Fig 2). Like in chicken Avd [PDB:1AVD], the core-bradavidin–biotin interaction is stabilized by several hydrogen bonds (H-bonds) (Fig 2). In more detail, 1) Asn9 Nδ(Asn12 in Avd), Ser13 Oγ (Ser16) and Tyr31 Oη(Tyr33) all form H-bonds with the 2´ oxygen atom of the ureido ring of biotin; 2) Asp107 Oδ(Asn118) forms a H-bond interaction with the 1´ nitrogen atom of the ureido ring; 3) Asn33 Oδ(Thr35) forms a H-bond to the 3´ N atom of the ureido ring; 4) Thr77 Oγ(Thr77) has polar interactions with the sulfur atom of the tetrahydrothiophene ring; and 5) Ser75 Oγ(Ser75) forms a H-bond to one oxygen atom of the carboxylate group of the valeric acid moiety (bradavidin numbering according to [40]). Three structural water molecules (HOH73, HOH145 and HOH189) are also located close to the carboxylate end of biotin. Moreover, several conserved hydrophobic interactions typical for Avds are also seen in corebradavidin and include the interaction of biotin with Trp89 (Trp97 in Avd) and Trp99 (Trp110; from another subunit); these residues are respectively 3.7 Åand 4.3 Ådistant from biotin. Biotin-binding mode of core-bradavidin—Unique features Asp107 and Asn33 of core-bradavidin are equivalent to Asn118 and Thr35 in Avd [PDB: 1AVD]. The side-chain oxygen atom of each of these residues interacts with the 1´ and 2´ N atoms of biotin but the overall H-bonding network of the side chains of these residues with the surrounding residues varies between core-bradavidin and Avd (Fig 3). Asp107 in core-bradavidin is within H-bonding distance of a structural water molecule (HOH2014), Asn9, Gln10, Trp75 (equivalent to Phe79 in Avd), Trp89 and Ala106, whereas in Avd only Asn12, Asp13, Trp97 and Ile117 are sufficiently close to the corresponding residue Asn118. Both sets of residues make hydrophobic interactions with the ureido ring moiety of biotin in core-bradavidin and in Avd. In bradavidin Tyr11 may be of special importance for ligand binding, since in the structure of the biotin complex of core-bradavidin it has moved significantly in comparison to the location in the wt bradavidin structure (see below). This residue is also poorly conserved and, to our knowledge, a tyrosine residue at the equivalent position is only found in a few bacterial Avds, which includes the only other reported Brad-tag containing Avd, rhodavidin from Rhodopseudomonas palustris [39], and the novel Avd-like sequences that we have identified in the Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 3 / 21 genera Bradyrhozibium,Mesorhizobium,Burkholderia (Pseudomonas),Catenulispora and Actinocatenispora (data not shown). The Nδatom of Asn33 in core-bradavidin is H-bonded to Ser38 Oγand Cys39 O (Fig 4), whereas in chicken Avd the equivalent H-bonds are missing. Out of the known crystal structures of other Avds, similar H-bond interactions are only seen in rhizavidin [PDB:3EW2] and hoefavidin [PDB:4Z28]. In both structures, an asparagine residue equivalent to Asn33 of corebradavidin is stabilized by H-bonds to the side-chain oxygen atom of a threonine residue (Ser38 Oγin core-bradavidin) and to the main-chain oxygen atom of a glycine residue (Cys39 Fig 1. Superimposition of the Cαtraces of subunit I of core-bradavidin (orange) [PDB:4BBO], wt bradavidin (magenta) [PDB:2Y32] and chicken Avd (cyan) [PDB:1AVD]. For clarity, only the biotin bound to core-bradavidin is shown (stick model). The loop regions L1,2 to L7,8, and the N and C termini, are labelled. https://doi.org/10.1371/journal.pone.0176086.g001 Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 4 / 21 O in core-bradavidin). In wt bradavidin, Asn33 is not connected to these residues but instead H-bonds to Asn33 Nδand Asp40 Oδ, the orientation of the Asp40 side chain being flipped in the opposite direction in comparison to core-bradavidin due to a rearrangement of the L3,4 loop. Moreover, in core-bradavidin, the side chain of Asp40 is H-bonded to Leu67 N and Fig 2. Comparison (stereo view) of the biotin-binding residues (sticks) of core-bradavidin (orange; bold labels) [PDB:4BBO] and chicken Avd (cyan; labels in brackets) [PDB:1AVD]. The Cαtraces were superimposed. Trp99 (Trp110 in Avd) is shown from subunit III; other residues are from subunit I. The bound biotin ligands are drawn as thick sticks. Nitrogen atoms are shown in blue, oxygen atoms in red and sulfur atoms in yellow. Water molecules are drawn as red spheres and H-bonds for core-bradavidin as grey dashed lines (distances in Ångstro ¨ms; *= 2.8 Å). https://doi.org/10.1371/journal.pone.0176086.g002 Fig 3. Interactions of Asp107 of core-bradavidin (orange; bold labels) and the equivalent Asn118 of chicken Avd (cyan; labels in brackets). Seven H-bonds stabilize the side chain of Asp107 in core-bradavidin (a), whereas only five H-bonds stabilize the equivalent Asn118 in chicken Avd (b). Non-carbon atom colouring as in Fig 2. H-bonds are drawn as grey dashed lines for core-bradavidin (a) and blue dashed lines for chicken Avd (b); distances in Ångstro ¨ms. https://doi.org/10.1371/journal.pone.0176086.g003 Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 5 / 21 Gly68 N of the L5,6 loop, to Lys43 Nzof the L3,4 loop, and to Cys69 S (L5,6 loop) that forms the disulfide bridge with Cys39 (Fig 5). The side-chain orientation of Glu41 is also flipped to the opposite direction in the core-bradavidin structure (closed L3,4 loop) versus the wt bradavidin structure (open L3,4 loop), and stabilized by different interactions (Fig 6). Hence, Asp40 Fig 4. Interactions of Asn33 of core-bradavidin (orange; bold labels) and the equivalent Thr35 of chicken Avd (cyan; labels in brackets). Asn33 of core-bradavidin is H-bonded (grey dashed lines) to Ser38 and Cys39, whereas in chicken Avd the equivalent H-bonds cannot be formed. Biotin molecules for both proteins are shown as thin sticks. Noncarbon atoms are coloured as in Fig 2. Distances are shown in Ångstro ¨ms. https://doi.org/10.1371/journal.pone.0176086.g004 Fig 5. Comparison of Asp40 in core- (orange; bold labels) [PDB:4BBO] and wt bradavidin (magenta; labels in brackets) [PDB:2Y32]. In corebradavidin (a), the side chain of Asp40 is flipped to an opposite direction as compared to wt bradavidin (b). Biotin (a) and residues K132 and L133 (b) occupying the same space as biotin in core-bradavidin (see a) are shown as spheres. Non-carbon atoms are coloured as in Fig 2. H-bonds are shown as dashed lines; distances in Ångstro ¨ms. The weighted 2Fo-Fc electron density map around Asp40 (a, b) is shown as a blue mesh (contour level of 1.0 σ). https://doi.org/10.1371/journal.pone.0176086.g005 Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 6 / 21 and Glu41 may have a vital role for ligand binding and in stabilizing the unique L3,4 loop conformations of wt bradavidin (Brad-tag as ligand) and core-bradavidin (biotin as ligand), two variations of the same protein with quite different bound ligands. In comparison with Trp99 in wt bradavidin, in core-bradavidin the tryptophan residue moves by 2 Å, enabling the residue to better seal the ligand-binding pocket containing biotin (see below). An Ångstro¨m-scale shift in atomic position is also seen for Ser38 of core-bradavidin [PDB:4BBO] in comparison to wt bradavidin [PDB:2Y32]. The movement of Ser38 mimics the “pinching effect” reported for the equivalent threonine residue in hoefavidin [21] [PDB:4Z6J, 4Z28] and rhizavidin [41] [PDB: 3EW1, 3EW2]; both Thr55 of hoefavidin and Thr48 of rhizavidin of the L3,4-loop respectively more closely approach Leu113 and Leu104 (located on the β7-strand; Leu91 in bradavidin) as a result of biotin binding (a similar “pinching effect” can also be seen in shwanavidin [20] [PDB:3SZH, 3SZJ] and bradavidin II [23] [PDB:4GGT, 4GGZ]). In each of these structures, the leucine side chains occupy the same relative locations, suggesting that the pinching effect might not be restricted only to dimeric Avds and, in general, might reflect an adaptation of the L3,4-loop for ligand binding. The aromatic residues Trp70 and Phe79 of chicken Avd are respectively replaced by Phe66 and Trp75 in bradavidin. Even subtle differences such as these may have an effect on the flexibility of the L5,6 loop and the biotin-binding properties of core-bradavidin: in Avd, Trp70 Nη is H-bonded to Thr77 Oγ; whereas, in bradavidin, an equivalent H-bonding interaction is missing. To our knowledge, all of the bacterial Avds identified so far—including both dimeric and tetrameric proteins—have a tryptophan residue at the position equivalent to Trp75 of bradavidin, whereas all of the characterized eukaryotic Avds have a phenylalanine residue at this position. The disulfide bridge of bradavidin—Unusual configuration The L3,4 loop of tetrameric core-bradavidin and of wt bradavidin is stabilized by a disulfide bridge between residues Cys39 (L3,4 loop) and Cys69 (L5,6 loop), similarly to dimeric rhizavidin [PDB:3EW2] [41], shwanavidin [PDB:3SZJ] [20] and hoefavidin [PDB:4Z28] [21], as well as bradavidin II [PDB:4GGZ] with its highly dynamic oligomeric structure [23]. In bradavidin, Fig 6. Comparison (stereo view) of Glu41 in core-bradavidin (orange; bold labels) [PDB:4BBO] and wt bradavidin (magenta; labels in brackets) [PDB:2Y32]. In core-bradavidin, the side chain of Glu41 is Hbonded (grey dashed lines) to several neighboring residues and is oriented in the opposite direction as compared to wt bradavidin, where the side chain of Glu41 is facing the solvent and H-bonded (blue dashed line) only to Lys137. A part of the acyl moiety of the bound biotin (BTN) of the core-bradavidin structure is shown as light grey sticks. Non-carbon atom colouring as in Fig 2. Water molecules are drawn as small red spheres. Distances are shown in Ångstro ¨ms. https://doi.org/10.1371/journal.pone.0176086.g006 Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 7 / 21 however, cysteine Cys39 of the L3,4 loop is located one residue earlier in the sequence, as reported recently by Avraham et al. (2015) for hoefavidin [21]. Thus, Cys39 is not exactly structurally equivalent to Cys50 in rhizavidin, Cys45 in shwanavidin, Cys57 in hoefavidin and Cyss44 in bradavidin II, whereas the other cysteine of the disulfide bond within the L5,6 loop, Cys69 in bradavidin, is conserved despite the fact that the conformation of the L5,6 loop varies within these proteins. Moreover, in the case of the biotin-complex structure of core-bradavidin, the earlier position of the cysteine residue does not seem to clearly affect the conformation of the L3,4 loop, which is in a similar conformation in all of the biotin complex structures listed above. In comparison to the wt bradavidin structure, which lacks biotin but has the Brad-tag sequence within the ligand-binding pocket, the configuration of the disulfide bridge of corebradavidin is, however, altered and directly related to the position and conformation of Cys39 (Fig 7). As described above, the residue adjacent to Cys39 in bradavidin, Asp40, may have a special importance here, too, because the side chain is flipped in the opposite direction in core-bradavidin versus wt bradavidin. It is not yet known how the Brad-tag itself affects the conformation of the L3,4 loop when and if biotin were bound in the intact wt structure, since we have not been not able to crystallize wt bradavidin in complex with biotin. All in all, the configuration of the disulfide bond in both forms of bradavidin, corewith biotin and wt sans biotin, are unique, and likely do represent the unique structural features needed to enable the presence and recognition of two different ligands by the bradavidin structure. Fig 7. Superimposition of the residues 35–41 (L3,4 loop) of core-bradavidin (orange; bold labels) [PDB:4BBO] and wt bradavidin (magenta; labels in brackets) [PDB:2Y32]. https://doi.org/10.1371/journal.pone.0176086.g007 Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 8 / 21 Subunit interfaces Avds are stabile over a wide range of conditions, including temperature and pH. The core-bradavidin–biotin complex with T m = 97.9±0.2˚C is less stable than chicken Avd (T m with bound biotin 118˚C), streptavidin (T m with bound biotin 112˚C), [32] and even wt bradavidin (T m without biotin = 96.2±0.1˚C, and 101.7±0.1˚C with biotin) [33]. The key interactions responsible for stability are found at the subunit interfaces, which can be divided into three major categories: the interface between subunits I and II (IF1,2), between subunits I and III (IF1,3) and between subunits I and IV (IF1,4 interface); in the dimeric Avds, only the IF1,4 interface is present. All of the subunit-subunit interfaces of core-bradavidin and wt bradavidin are structurally highly similar. A key interfacial residue—Tyr90 from each of the four subunits—is located at the center of the tetramer in both forms of bradavidin, whereas in chicken Avd [PDB:1AVD] and streptavidin [PDB: 3YR2] the residues equivalent to Tyr90 are respectively Leu98 and Leu109. Since we have recently published the detailed analysis of the subunit interface of wt bradavidin [33], we will focus here only on regions unique to core-bradavidin as compared to wt bradavidin. At the IF1,2 interface of core-bradavidin, the position of Trp99 (subunit II; equivalent to Trp110 of chicken Avd) at the tip of the L7,8 loop and the spatial arrangement of Trp99 with respect to Tyr11 (subunit I) differ by over an Ångstro¨m in comparison to wt bradavidin (S1 Fig). These differences, together with the conformational adaptation of the L3,4 loop, are the major differences that help these bradavidin structures recognize two very different ligands, biotin and the Brad-tag. The C-terminal Brad-tag sequence enters the ligand-binding pocket of wt bradavidin between Tyr11 and Trp99, and Tyr11 Oη(subunit I) forms a H-bonding interaction with Ser130 N (3.4 Å; subunit III) of the Brad-tag sequence and with one structural water molecule (HOH2027). In core-bradavidin, Tyr11 OηH-bonds to three water molecules (HOH2014, HOH2015 and HOH2017). The coreof the IF1,3 interface in the coreand wt bradavidin structures is formed by residues Gln86, Leu88, Tyr90, Ala104 and Ala106, and is structurally highly similar. The IF1,4 interface is also very similar in both structures: it is clearly the largest interface in terms of contact area and the number of residues involved—47 in wt bradavidin [33]—and both the coreand wt bradavidin structures are stabilized by various non-covalent interactions as listed in [33]. The L7,8 loops, however, have different conformations since the residues Gly57-Tyr63 are in contact with and adapt to the binding of biotin to core-bradavidin and the Brad-tag to wt bradavidin. Effect of the C-terminal Brad-tag sequence for the fold of bradavidin In wt bradavidin, the open conformation of the L3,4 loop accommodates the amino acids of the Brad-tag sequence [42], whereas in the biotin-complex structure of core-bradavidin the L3,4 loop adopts a closed conformation (Fig 7). The most dramatic differences are found in the coordinates of residues Ala35-Glu41 of the L3,4 loop and the conformation of the side chain of Tyr31 is also different in these structures. As mentioned above, the conformation of Cys39, and the position of its Cαatom, varies also between these two bradavidin structures even though Cys69, which pairs with Cys39, has the same conformation in both structures. The importance of the equivalent disulfide bridge for biotin binding to shwanavidin and rhizavidin has been recently demonstrated using mutagenesis analysis [20]. This disulfide bridge has also been suggested to be important for biotin binding in hoefavidin and, in general, for all dimeric Avds [21], where the disulfide bridge is considered to maintain the L3,4-loop in the closed conformation. Interestingly, several different crystal structures of hoefavidin were recently determined by Avraham et al. (2015), including intact hoefavidin with its C-terminal, Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 9 / 21 of the proteins were analyzed using SDS-PAGE (15%) in reducing conditions. In addition, the protein concentration was determined with a UV/Vis spectrophotometer (NanoDrop 1000 Spectrophotometer, Thermo Scientific, Wilmington, DE, USA) by measuring the absorbance at 280 nm and using an extinction coefficient of 43555 M –1 cm –1 and 43430 for core-bradavidin V1 and CC mutant, accordingly. Biophysical analysis of core-bradavidin V1 & CC mutant The unfolding temperature of core-bradavidin V1 was analyzed using the VP-Capillary DSC instrument (GE Healthcare, MicroCal, Northampton, MA, USA) in 50 mM sodium phosphate buffer (150 mM NaCl, pH 7.2) with protein concentration of 0.2 mg/ml. Solutions were degassed prior to measurements. Samples were heated from 20˚C to 130˚C at a scanning rate of 2˚C/min. Feedback mode was set to ‘low’ and the filter period was 5 s. The temperature transition midpoint (T m ) was obtained from the midpoint of the curve that was fitted to the data after first subtracting the baseline from the measurement data and then using the Levenberg-Marquardt non-linear least-squares method to fit the curve using the MicroCal Origin 7.0 software (MicroCal, Malvern Instrument Ltd). Similar analysis was not possible with the CC mutant due to lack of protein for proper analysis. The dissociation rate constant (k diss ) of fluorescently labelled biotin was determined by fluorescence spectrometry using the biotin-labelled fluorescent probe ArcDia™BF560 as described in [69]. In practice, 50 nM dye in a buffer containing 50 mM sodium phosphate, 650 mM NaCl and 0.1 mg/ml BSA (pH 7) was mixed with 100 nM core-bradavidin V1 (or CC mutant) and the change in fluorescence intensity was measured over time. A 100-fold molar excess of free biotin (D-biotin, Sigma-Aldrich Co. LLC., St. Louis, MO, USA) was used to monitor the dissociation of this complex. The assay was performed at 50˚C using a QuantaMaster™Spectrofluorometer (Photon Technology International, Inc., Lawrenceville, NJ, USA). Biotinylated BF560 was excited at 560 nm, and emission was measured at 578 nm. The affinity of core-bradavidin V1 towards Brad-tag (peptide SEKLSNTK; GenScript, Piscataway, NJ, USA) was measured by ITC. The purified core-bradavidin V1 was dialyzed against 50 mM sodium phosphate (pH 7.0) buffer containing 100 mM NaCl, Brad-tag was dissolved in the same buffer and the samples were degassed using MicroCal™ThermoVac. The analysis was performed at 40˚C using an isothermal titration calorimetry VP-ITC MicroCalorimeter (GE Healthcare, MicroCal, Northampton, MA, USA) with 10 μl titration aliquots of Brad-tag in 30 repeated additions at intervals of 200 s using constant stirring speed of 440 rpm. The data were analyzed with Microcal Origin 7.0 (MicroCal LLC, Northampton, MA, USA) software. The observed reaction heats were corrected by subtracting the heat of dilution caused by the titration of the ligand alone into buffer. K a ,ΔH and n (stoichiometry per subunit) were obtained through non-linear least-squares fit of the corrected reaction heats for each titration step. Miscellaneous methods PyMOL [60,61] and Bodil [62] were used for analyzing structures, visualization and for creating figures. The structure-based sequence alignment was done by Malign [67] of the Bodil software package for biomolecular visualization and modeling [62]. A cut-off distance of 3.5 Å between non-hydrogen atoms was used for hydrogen bonds. Subunit one was used to create all figures unless not otherwise specified in the figure legends. Supporting information S1 Fig. Comparison of the subunit IF1,2 interface residues Trp99, Leu91 and Tyr11 of core-bradavidin (orange; bold labels) [PDB:4BBO] and wt bradavidin (magenta; labels in Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 16 / 21 brackets) [PDB:2Y32] based on superimposition of the Cαtraces of the proteins. The biotin molecule (BTN) of the core-bradavidin structure and the side chains of Trp99, Leu91 and Tyr11 are shown as sticks. Nitrogen atoms are coloured blue, oxygen atoms red and sulphur atoms yellow. (TIF) S2 Fig. Comparison of the tertiary structure of wt bradavidin [PDB:2Y32] to the homology models of rhodavidin (Uniprot: Q218I6; Genbank: WP_011472104.1) and bradavidin A2 (Genbank: WP_051334960.1). Superimposition of the Cαtraces of subunit I and subunit III of wt bradavidin (magenta), rhodavidin (blue) and bradavidin A2 (grey) are shown. The loop regions L1,2 to L7,8, and the N and C termini, are labelled. (TIF) S3 Fig. Structure-based sequence alignment of core-bradavidin, chicken Avd, rhizavidin, shwanavidin, bradavidin II, hoefavidin, wt bradavidin, rhodavidin and bradavidin A2. The biotin-binding residues (top six structures) are marked with black squared boxes; the blue squared boxes indicate cysteine residues forming disulphide bridges in non-tetrameric Avds; the green ‘1’ indicates the cysteine residues forming disulphide bridges in bradavidins and rhodavidin; the black triangle indicates the tryptophan residue in equivalent position to Trp99 of the core-bradavidin structure that is present only in tetrameric Avds; and the ‘Brad-tag’ residues are highlighted with yellow background. The beta-strands 1–8 of core-bradavidin are labeled and indicated by arrows. The conserved residues are coloured by the default scheme of the ESPript 3 program (http://espript.ibcp.fr/ESPript/ESPript/). (TIF) S1 Table. Yields of core-bradavidin, core-bradavidin V1 and CC mutant produced in E. coli BL21-AI. (DOCX) Acknowledgments We thank the bioinformatics infrastructure support (J.V. Lehtonen) from Biocenter Finland, and CSC IT Center for Science for laboratory and computational infrastructure support. Docent Tiina Salminen is acknowledged for the excellent facilities at the Structural Bioinformatics Laboratory and Dr. Heidi Kidron for the initial crystallization trials of core-bradavidin. We acknowledge the MAXLAB, Sweden, for provision of synchrotron radiation facilities, and we would like to thank the local contacts for assistance in using the beamline. We also acknowledge the infrastructure support from Biocenter Finland to the University of Tampere Protein Technologies core facility and valuable technical support from Niklas Ka¨hko¨nen. We also acknowledge Dr. Tiina Riihima¨ki and Dr. Jenni Leppiniemi for their valuable help in protein expression and analysis. Finally, acknowledgments to Dr. Henri Nordlund, who had a vital role for this work in the beginning of this study but, unfortunately, passed away in 2008. Author Contributions Conceptualization: NA MSJ MSK VPH TTA. Data curation: NA TTA. Formal analysis: NA TTA VPH JAEM. Funding acquisition: VPH MSJ MSK TTA. Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 17 / 21 Investigation: NA TTA JAEM. Methodology: NA TTA VPH. Project administration: TTA VPH. Resources: MSJ MSK VPH. Supervision: TTA MSK MSJ VPH. Validation: NA JAEM MSJ VPH TTA MSK. Visualization: NA JAEM TTA VPH. Writing – original draft: NA TTA. Writing – review & editing: NA TTA JAEM VPH MSJ MSK. References 1. Green NM. Avidin. Advances in protein chemistry. Elsevier; 1975; 29: 85–133. 2. Korpela J, Kulomaa M, Elo H, Tuohimaa P. Biotin-binding proteins in eggs of oviparous vertebrates. Experientia. Springer; 1981; 37: 1065–1066. 3. Pugliese L, Coda A, Malcovati M, Bolognesi M. Three-dimensional structure of the tetragonal crystal form of egg-white avidin in its functional complex with biotin at 2.7 Åresolution. Journal of molecular biology. Elsevier; 1993; 231: 698–710. 4. Livnah O, Bayer EA, Wilchek M, Sussman JL. Three-dimensional structures of avidin and the avidinbiotin complex. Proceedings of the National Academy of Sciences. National Acad Sciences; 1993; 90: 5076–5080. 5. Pa ¨hler A, Hendrickson W, Kolks M, Argarana C, Cantor C. Characterization and crystallization of core streptavidin. Journal of Biological Chemistry. ASBMB; 1987; 262: 13933–13937. 6. Helppolainen S, Nurminen K, Ma ¨a ¨tta ¨J, Halling K, Slotte J, Huhtala T, et al. Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family. Biochem J. Portland Press Limited; 2007; 405: 397–405. 7. Laitinen O, Hytӧnen V, Nordlund H, Kulomaa M. Genetically engineered avidins and streptavidins. Cellular and Molecular Life Sciences CMLS. Springer; 2006; 63: 2992–3017. 8. Barnard G, Bayer E, Wilchek M, Amir-Zaltsman Y, Kohen F. Amplified bioluminescence assay using avidin-biotin technology. Methods in enzymology. Elsevier; 1986; 133: 284–288. 9. Laitinen OH, Nordlund HR, Hytӧnen VP, Kulomaa MS. Brave new (strept) avidins in biotechnology. Trends in biotechnology. Elsevier; 2007; 25: 269–277. 10. Diamandis EP, Christopoulos TK. The biotin-(strept) avidin system: principles and applications in biotechnology. Clinical chemistry. Am Assoc Clin Chem; 1991; 37: 625–636. 11. Tuohimaa P, Joensuu T, Isola J, Keina¨nen R, Kunnas T, Niemela¨A, et al. Development of progestinspecific response in the chicken oviduct. Int J Dev Biol. 1989; 33: 125–134. PMID: 2485692 12. Rosebrough SF, Hartley DF. Biochemical modification of streptavidin and avidin: in vitro and in vivo analysis. Journal of nuclear medicine: official publication, Society of Nuclear Medicine. 1996; 37: 1380– 1384. 13. Bayer EA, Ben-Hur H, Hiller Y, Wilchek M. Postsecretory modifications of streptavidin. Biochemical Journal. Portland Press Limited; 1989; 259: 369–376. 14. Gope ML, Keina ¨nen RA, Kristo PA, Conneely OM, Beattie WG, Zarucki-Schulz T, et al. Molecular cloning of the chicken avidin cDNA. Nucleic acids research. Oxford Univ Press; 1987; 15: 3595–3606. 15. Sano T, Pandori MW, Chen X, Smith CL, Cantor CR. Recombinant Core Streptavidins A Minimumsized Core Streptavidin has Enhanced Structural Stability and Higher Accessibility to Biotinylated Macromolecules. Journal of Biological Chemistry. ASBMB; 1995; 270: 28204–28209. 16. Le Trong I, Humbert N, Ward TR, Stenkamp RE. Crystallographic analysis of a full-length streptavidin with its C-terminal polypeptide bound in the biotin binding site. Journal of molecular biology. Elsevier; 2006; 356: 738–745. 17. Taskinen B, Zmurko J, Ojanen M, Kukkurainen S, Parthiban M, Ma ¨a ¨tta ¨JA, et al. Zebavidin-An AvidinLike Protein from Zebrafish. PloS one. Public Library of Science; 2013; 8: e77207. Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 18 / 21 18. Ma ¨a ¨tta ¨JA, Helppolainen SH, Hytӧnen VP, Johnson MS, Kulomaa MS, Airenne TT, et al. Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis. BMC structural biology. BioMed Central; 2009; 9: 63. 19. Takakura Y, Tsunashima M, Suzuki J, Usami S, Kakuta Y, Okino N, et al. Tamavidins-novel avidin-like biotin-binding proteins from the Tamogitake mushroom. FEBS journal. Wiley Online Library; 2009; 276: 1383–1397. 20. Meir A, Bayer EA, Livnah O. Structural adaptation of a thermostable biotin-binding protein in a psychrophilic environment. Journal of Biological Chemistry. ASBMB; 2012; 287: 17951–17962. 21. Avraham O, Meir A, Fish A, Bayer EA, Livnah O. Hoefavidin: A dimeric bacterial avidin with a C-terminal binding tail. Journal of structural biology. Elsevier; 2015; 191: 139–148. 22. Sardo A, Wohlschlager T, Lo C, Zoller H, Ward TR, Creus M. Burkavidin: A novel secreted biotin-binding protein from the human pathogen Burkholderia pseudomallei. Protein expression and purification. Elsevier; 2011; 77: 131–139. 23. Leppiniemi J, Meir A, Ka ¨hkӧnen N, Kukkurainen S, Ma ¨a ¨tta ¨JA, Ojanen M, et al. The highly dynamic oligomeric structure of bradavidin II is unique among avidin proteins. Protein Science. Wiley Online Library; 2013; 22: 980–994. 24. Nordlund HR, Laitinen OH, Hytӧnen VP, Uotila STH, Porkka E, Kulomaa MS. Construction of a dual chain pseudotetrameric chicken avidin by combining two circularly permuted avidins. Journal of Biological Chemistry. ASBMB; 2004; 279: 36715–36719. 25. Nordlund HR, Hytӧnen VP, Hӧrha ¨J, Ma ¨a ¨tta ¨JAE, White DJ, Halling K, et al. Tetravalent single-chain avidin: from subunits to protein domains via circularly permuted avidins. Biochemical Journal. Portland Press Ltd; 2005; 392: 485. 26. Laitinen OH, Nordlund HR, Hytӧnen VP, Uotila ST, Marttila AT, Savolainen J, et al. Rational design of an active avidin monomer. Journal of Biological Chemistry. ASBMB; 2003; 278: 4010–4014. 27. Wu S-C, Wong S-L. Engineering soluble monomeric streptavidin with reversible biotin binding capability. J Biol Chem. 2005; 280: 23225–31. https://doi.org/10.1074/jbc.M501733200 PMID: 15840576 28. DeMonte D, Drake EJ, Lim KH, Gulick AM, Park S. Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate. Proteins: Structure, Function, and Bioinformatics. Wiley Online Library; 2013; 81: 1621–1633. 29. Riihima ¨ki TA, Hiltunen S, Rangl M, Nordlund HR, Ma ¨a ¨tta ¨JAE, Ebner A, et al. Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein. BMC Biotechnol. 2011; 11: 64. https://doi.org/10.1186/1472-6750-11-64 PMID: 21658230 30. Lehtonen S, Tullila A, Agrawal N, Kukkurainen S, Ka ¨hkӧnen N, Koskinen M, et al. Artificial AvidinBased Receptors for a Panel of Small Molecules. ACS chemical biology. 2016; 11: 211. https://doi.org/ 10.1021/acschembio.5b00906 PMID: 26550684 31. Hytӧnen VP, Ma¨a¨tta¨JA, Nyholm TK, Livnah O, Eisenberg-Domovich Y, Hyre D, et al. Design and construction of highly stable, protease-resistant chimeric avidins. Journal of Biological Chemistry. ASBMB; 2005; 280: 10228–10233. 32. Nordlund HR, Hytӧnen VP, Laitinen OH, Kulomaa MS. Novel avidin-like protein from a root nodule symbiotic bacterium, Bradyrhizobium japonicum. Journal of Biological Chemistry. ASBMB; 2005; 280: 13250–13255. 33. Leppiniemi J, Gro ¨nroos T, Ma ¨a ¨tta ¨JA, Johnson MS, Kulomaa MS, Hyto ¨nen VP, et al. Structure of Bradavidin-C-Terminal Residues Act as Intrinsic Ligands. PloS one. Public Library of Science; 2012; 7: e35962. 34. Schmidt TG, Koepke J, Frank R, Skerra A. Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. Journal of molecular biology. Elsevier; 1996; 255: 753–766. 35. Korndӧrfer IP, Skerra A. Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site. Protein science. Wiley Online Library; 2002; 11: 883–893. 36. Perbandt M, Bruns O, Vallazza M, Lamla T, Betzel C, Erdmann V. High resolution structure of streptavidin in complex with a novel high affinity peptide tag mimicking the biotin binding motif. Proteins: Structure, Function, and Bioinformatics. Wiley Online Library; 2007; 67: 1147–1153. 37. Keefe AD, Wilson DS, Seelig B, Szostak JW. One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. Protein expression and purification. Elsevier; 2001; 23: 440–446. 38. Delamuta JRM, Ribeiro RA, Ormeño-Orrillo E, Melo IS, Mart’\inez-Romero E, Hungria M. Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobiumdiazoefficiens sp. nov. International journal of systematic and evolutionary microbiology. Microbiology Society; 2013; 63: 3342–3351. Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 19 / 21 39. Larimer FW, Chain P, Hauser L, Lamerdin J, Malfatti S, Do L, et al. Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris. Nature biotechnology. Nature Publishing Group; 2004; 22: 55–61. 40. DeTitta GT, Edmonds JW, Stallings W, Donohue J. Molecular structure of biotin. Results of two independent crystal structure investigations. Journal of the American Chemical Society. ACS Publications; 1976; 98: 1920–1926. 41. Meir A, Helppolainen SH, Podoly E, Nordlund HR, Hytӧnen VP, Ma ¨a ¨tta ¨JA, et al. Crystal structure of rhizavidin: insights into the enigmatic high-affinity interaction of an innate biotin-binding protein dimer. Journal of molecular biology. Elsevier; 2009; 386: 379–390. 42. Repo S, Paldanius TA, Hyto ¨nen VP, Nyholm TK, Halling KK, Huuskonen J, et al. Binding properties of HABA-type azo derivatives to avidin and avidin-related protein 4. Chemistry & biology. Elsevier; 2006; 13: 1029–1039. 43. Helppolainen SH, Ma ¨a ¨tta ¨JA, Halling KK, Slotte JP, Hytӧnen VP, Ja ¨nis J, et al. Bradavidin II from Bradyrhizobium japonicum: A new avidin-like biotin-binding protein. Biochimica et Biophysica Acta (BBA)- Proteins and Proteomics. Elsevier; 2008; 1784: 1002–1010. 44. Dakshinamurti K, Chalifour L, Bhullar RP. Requirement for Biotin and the Function of Biotin in Cells in Cultureaa. Annals of the New York Academy of Sciences. Wiley Online Library; 1985; 447: 38–55. 45. Siqueira A, Ormeño-Orrillo E, Souza R, Rodrigues E, Almeida L, Barcellos F, et al. Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean. BMC genomics. 2014; 15: 420–420. https://doi.org/10.1186/1471-2164-15-420 PMID: 24888481 46. Kabsch W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. Journal of applied crystallography. International Union of Crystallography; 1993; 26: 795–800. 47. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. Journal of applied crystallography. International Union of Crystallography; 2007; 40: 658–674. 48. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta crystallographica Section D, Biological crystallography. 1994; 50: 760. https://doi.org/10.1107/ S0907444994003112 PMID: 15299374 49. Potterton E, Briggs P, Turkenburg M, Dodson E. A graphical user interface to the CCP4 program suite. Acta Crystallographica Section D: Biological Crystallography. International Union of Crystallography; 2003; 59: 1131–1137. 50. Murshudov GN, Skuba ´k P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallographica Section D: Biological Crystallography. International Union of Crystallography; 2011; 67: 355–367. 51. Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallographica Section D: Biological Crystallography. International Union of Crystallography; 2004; 60: 2126–2132. 52. Lamzin VS, Wilson KS. Automated refinement of protein models. Acta Crystallographica Section D: Biological Crystallography. International Union of Crystallography; 1993; 49: 129–147. 53. Langer G, Cohen SX, Lamzin VS, Perrakis A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nature protocols. Nature Publishing Group; 2008; 3: 1171– 1179. 54. Morris RJ, Perrakis A, Lamzin VS. ARP/wARP and Automatic Interpretation of Protein Electron Density Maps. Methods in enzymology. Elsevier; 2003; 374: 229–244. 55. Perrakis A, Morris R, Lamzin VS. Automated protein model building combined with iterative structure refinement. Nature Structural & Molecular Biology. Nature Publishing Group; 1999; 6: 458–463. 56. Adams PD, Grosse-Kunstleve RW, Hung L-W, Ioerger TR, McCoy AJ, Moriarty NW, et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallographica Section D: Biological Crystallography. International Union of Crystallography; 2002; 58: 1948–1954. 57. Adams PD, Afonine PV, Bunko ´czi G, Chen VB, Davis IW, Echols N, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D: Biological Crystallography. International Union of Crystallography; 2010; 66: 213–221. 58. Zwart PH, Afonine PV, Grosse-Kunstleve RW, Hung L-W, Ioerger TR, McCoy AJ, et al. Automated structure solution with the PHENIX suite. Structural Proteomics. Springer; 2008. pp. 419–435. 59. Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic acids research. Oxford Univ Press; 2007; 35: W375–W383. 60. DeLano WL. The PyMOL molecular graphics system. 2002; Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 20 / 21 61. Schrӧdinger L. The PyMOL Molecular Graphics System, Version~1.3r1. 2010; 62. Lehtonen JV, Still D-J, Ekholm J, Bjӧrklund D, Iftikhar Z, Huhtala M, et al. BODIL: a molecular modeling environment for structure-function analysis and drug design. Journal of computer-aided molecular design. Springer; 2004; 18: 401–419. 63. Berman HM, Battistuz T, Bhat T, Bluhm WF, Bourne PE, Burkhardt K, et al. The protein data bank. Acta Crystallographica Section D: Biological Crystallography. International Union of Crystallography; 2002; 58: 899–907. 64. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat T, Weissig H, et al. The protein data bank. Nucleic acids research. Oxford Univ Press; 2000; 28: 235–242. 65. Consortium U, others. The universal protein resource (UniProt). Nucleic acids research. Oxford Univ Press; 2008; 36: D190–D195. 66. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of molecular biology. Elsevier; 1990; 215: 403–410. 67. Johnson MS, Overington JP. A structural basis for sequence comparisons: an evaluation of scoring methodologies. Journal of molecular biology. Elsevier; 1993; 233: 716–738. 68. Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. Journal of molecular biology. Elsevier; 1993; 234: 779–815. 69. Hytӧnen VP, Laitinen OH, Airenne TT, Kidron H, Meltola NJ, Porkka EJ, et al. Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli. Biochemical Journal. Portland Press Ltd; 2004; 384: 385. Core-bradavidin PLOS ONE | https://doi.org/10.1371/journal.pone.0176086 April 20, 2017 21 / 21