SMYD1 promoter, participates in various cellular processes and is involved in human cancers. We report the first crystal structures of the human HDGF PWWP domain (residues 1–100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four β-strands and two α-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable α-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions.The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the  Previous studies have demonstrated that HDGF participates in various cellular processes, such as the formation of vascular lesion4, astrocyte proliferation5 and cardiovascular differentiation1. Over-expressed HDGF can be found in several type of human cancers, including hepatocellular carcinoma6, pancreatic cancer7, oral cancer8, breast cancer9 and non-small cell lung cancer9. Human HDGF has also been shown to be involved in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is related to breast cancer and hepatocellular carcinoma. This pathway regulates post-translational histone modification11. Moreover, HDGF is involved in cardiovascular development and transcriptional regulation, resulting in its function as a transcriptional repressor12.Human hepatoma-derived growth factor (HDGF) is highly expressed in the developing heart, tumour cell lines and normal tissue ubiquitously with mitogenic and angiogenic activities13. These two domains play distinct roles in physiology. The dominant nuclear location signal 2 (NLS2) located in the C-terminus in HDGF might assist the translocation of HDGF into the nucleus and subsequently regulates its mitogenic activity15. The PWWP motif of the N-terminal domain (PWWP domain) is named after the Pro-Trp-Trp-Pro motif, but only the third (Trp) and fourth (Pro) residues are highly conserved among various PWWP domain-containing proteins, whereas the first and second residues are variable16. The sequence of the PWWP motif of human HDGF is 24Pro-His-Trp-Pro27, of which the second residue is variable. The highly conserved PWWP domains are discovered in numerous eukaryotes, from unicellular organisms to humans. For instance, bromo and plant homeodomain finger–containing protein 1 (brpf1) that promotes histone acetylation utilizes its C-terminal PWWP domain to recognize histone modifications18. The mammalian DNA methyltransferase, DNMT3B, which contains the PWWP domain, is not only linked to human immunodeficiency, centromere instability and facial anomalies syndrome but also to distinct cancers21. Another PWWP-containing protein, Lens epithelium-derived growth factor (LEDGF/p75), plays an essential role in the integration of HIV-1 cDNA into human chromosomes25. To date, more than twenty PWWP domain-containing proteins from human genomes have been discovered, most of which are chromatin-associated26.HDGF generally comprises two portions, an N-terminal domain with a highly conserved PWWP motif (residues 1–100 for human HDGF) and a variable C-terminal domain (residues 101–240)27. The PWWP domain of HDGF may first bind to heparin through its positively charged residues and then be internalized into a cell28. In addition, nucleolin (NCL) located on the plasma membrane functions as a receptor for HDGF through interactions with of the HDGF PWWP domain6. Because of its chromatin-associated properties, the PWWP domain exhibits a methylated histone and DNA-binding capability30. The PWWP domain of DNA methyltransferase DNMT3B has been demonstrated to show no preference for double-stranded DNA (dsDNA), based on DNA binding assays31. Previous work has shown that the PWWP domain has greater binding affinities towards various types of dsDNA, instead of single-stranded DNA, indicating that the PWWP domain can interact with dsDNA nonspecifically32. In addition to the nonspecific DNA binding, the HDGF PWWP domain is capable of recognizing the SMYD1 promoter (5′-CAGGCTGGTCTTGAACTCCTGACCTCAGATGATCCATG TGCCTCGGCCTCCCAAGGTGGGGATTACAGGCGTGAGCCACC-3′)12. HDGF has been demonstrated to bind to the SMYD1 promoter containing a HDGF core-binding sequence with a shortest length of 37 bp from human chromatin and functions as a transcriptional repressor12. HDGF interacts with the SMYD1 promoter through its N-terminal PWWP domain; however, the precise interaction of the PWWP domain with SMYD1 remains unclear12.The PWWP domain can interact with heparin, a linear polymer consisting of repeating units of uronic acid and glucosamine residues on the cell surfaceapo form or complexes with histone-related peptides30. The NMR structures of a monomeric form of the apo soluble HDGF PWWP domain and one domain-swapped dimeric form of the apo refolded PWWP domain have been determined, respectively 28. Despite the preceding work, the mechanisms of DNA binding and interactions with the PWWP domain remain puzzling because of a lack of essential knowledge regarding the intact structure in complex with DNA molecules. Here, we report the first crystal structures of the human HDGF PWWP domain in complex with a 10-bp SMYD1 and its unbound apo form. Our structural studies, with physiological assays, provide new insights into the PWWP-DNA interaction pattern, which might facilitate research into the role of the PWWP domain in nucleosomal functions.Several structures of the PWWP domains of various proteins from different species have been reported using NMR and X-ray approaches, but they represent only either the apo PWWP domain after several crystallization attempts, and determined the crystal structure at 3.3 Å resolution  : the length and amounts of β-strands as well as the locations of the α-helices. One additional short β-strand (Leu63 – Phe64) exists in the solution structure. The positions of the defined first α-helix (αA) in the two structures are also distinct  Fig. . The 24Pain Fig. . The othain Fig. . The X-rnct Fig. .Table 1Capo PWWP domain reveals that the positively charged region and the slightly negatively charged and neutral region are separated on two opposite sides of the molecular surface  based on the omit map  is elongated further after this newly formed αC helix and interacts tightly with the N-terminal region of the other molecule (chain B) to form a dimer, which undergoes 3D domain swapping33  from chain B and loop4 (77NKRK80) from chain A in the swapped C/N terminus  of the hydrogen-bonding residues for swapped dimerization of the PWWP-SMYD1 complex is lower than that (0.94) of the corresponding residues of the monomeric apo PWWP domain  Fig. , the norain Fig. , suggestres Fig. . The denain Fig. , whereasain Fig.  and S3B.orm Fig. . Finallyain Fig. . Taken t10. The colony formation assay, a well-used indication for cancer research34, was performed for confirming whether the recombinant HDGF and PWWP domain retains biological functions. The results showed that HDGF exhibited functional ability to promote colony formation better than the PWWP domain because of the dominant NLS2 in the C-terminus of HDGF  via the PWWP domainsay Fig. . Interesive Fig. . Togetheapo PWWP domain, and the dimeric apo PWWP domain can be readily obtained by increasing the protein concentration during purification  and loop4 (Asn77 – Lys80)  Figs  and S5A.80) Figs .SMYD1 complex and the apo PWWP domain reveals several significant conformational distinctions  of the extended main-chain structure of the PWWP-SMYD1 complex, suggesting that the PWWP domain may explore the methylated histone and the promoter with both swapped C/N and N/C termini35.Correspondingly, structural comparison of the HDGF PWWP-ons Fig. . The comSMYD1 complex is similar to that in internal interactions of the monomeric apo PWWP domain  with a larger bond distance in the apo PWWP domain may be just sufficient to maintain the globular structure of a monomer with larger temperature factors, and provide the possibility for domain swapping to form a much more stable dimer of the complex   Fig. . The refnus Fig. . The fleapo PWWP domain shows an interpretable electron density but with an overall B-factor that is larger than for the complex  shows that these PWWP domains comprise four or five stranded β-barrel and α-helices in their C-terminus, indicating that the main structural features of those PWWP domains are conserved, especially for the β-barrel region  indicated that the N-terminus (Lys8 – Tyr10), the loop region (Lys19 – Tyr23) and two α-helix regions , which contribute to the positively charged surface, were involved in DNA binding, especially with Glu9, Tyr10, Gly22, Lys61 and Arg7931. Based on our crystal structure of the PWWP-SMYD1 complex, the DNA-binding regions are loop1 (Lys19 – Gly22), which is before the highly conserved PWWP motif, and a partial loop4 (Asn77 – Lys80), differing from the prediction obtained with NMR chemical-shift perturbation. This result potentially suggests that the PWWP domain interacts with a longer region of DNA through larger regions  and loop4 (Asn77 – Lys80) with DNA in the PWWP domain , implying that these PWWP domains may specifically contact DNA through the identified residues at the two loop regions  to examine truncated HDGF with 80-bp ace Fig. . We foun237 Figs  and 3B. 40. Hence, HDGF might first interact with heparin on the cell surface with its dimeric and monomeric forms with distinct binding affinities27, or bind to NCL, a plasma membrane receptor of HDGF6. After internalizing into the cell, the HDGF recognizes the receptor on the nucleus pole by its specific sequence of NLS2 in the variable C-terminus to translocate itself into the nucleus14. The interactions of monomeric or dimeric HDGF with the receptor remain unclear. Subsequently, HDGF of dimers or monomers may gather together in the nucleus under physiological conditions. Next, HDGF may undergo domain swapping with the PWWP domain, and the swapped dimeric HDGF in the nucleus transforms its flexible hinge loop further into a rigid hinge α-helix when the protein approaches DNA fibres with loop1 and loop4. Once the PWWP domain is activated by methyl peptides of histone and recognizes the promoter, the synergistic binding mechanism may enhance the binding affinity with the corresponding histone and DNA and participate further in the regulation of gene expression through the C-terminus of HDGF30.Taken together, we propose a hypothesis for the mechanism of DNA binding and the functional process of HDGF in gene regulation Fig. . The groSMYD1 of 10 bp and its apo form, respectively. The PWWP domain undergoes domain swapping to markedly transform its secondary structures and to alter the overall conformation from monomeric globular folding into an extended dimeric structure upon binding to DNA. The PWWP-DNA complex becomes more rigid and stable than the apo form. The PWWP domain utilizes residues with variable characters at loop1 and loop4 from two chains of the swapped dimer to interact with the minor grooves of DNA. Together with physiological assays, these novel structural findings provide new insights into the mechanism of DNA binding and the functional process of HDGF.In summary, we have determined the first crystal structures of the HDGF PWWP domain in complex with 41. After the DNA sequencing analysis, the PCR-amplified PWWP domain was subcloned into NdeI and EcoRI sites of the pET28a vector and transformed into Escherichia coli (E. coli) BL21-Codon Plus®-RIL for expression and purification of the recombinant PWWP protein. All constructed plasmids were confirmed by DNA sequencing.The construct of human HDGF was amplified from the library of human fetal brain cDNA  utilizing the polymerase chain reaction (PCR) as described previously600 reached 0.4–0.6, isopropyl β-D-thiogalactopyranoside  was added to the culture for induction with rotary shaking at 37 °C for another 20 h. The cells were harvested by centrifugation  at 4 °C for 10 min.A single colony of the bacteria was selected and placed into Luria-Bertani (LB) broth (20 mL) with kanamycin (50 μg/mL) at 37 °C overnight. The culture was then transferred into fresh LB medium (2 L) with kanamycin (50 μg/mL). After the ODg) at 4 °C for 30 min; the supernatant was loaded onto a Ni2+-NTA agarose column  that had been pre-equilibrated with binding buffer . The unwanted proteins were washed out with binding buffer containing imidazole (20–50 mM); the desired target protein of the PWWP domain was eluted with binding buffer containing imidazole (300 mM). The protein was further purified by anion-exchange chromatography (Hitrap Q) with buffer A  and buffer B  while monitoring the ratio of A260/280 based on the markedly distinct pI values of the apo PWWP domain and DNA during purification. This purification step removed endogenous DNA from E. coli and contaminated proteins to ensure that there was no interference and contamination of endogenous DNA for further structural studies and physiological assays32. The homogeneity of the PWWP domain protein was confirmed by size-exclusion chromatography (Superdex-200) with buffer . The ratio of A260/280 was examined, and showed that the purified apo PWWP domain was not contaminated with endogenous DNA from E. coli. The PWWP protein was concentrated  before crystallization. The purity of the final PWWP domain was greater than 95% based on SDS-PAGE (15%) and staining (Coomassie Brilliant Blue R-250).The cells were resuspended in lysis buffer  and then sonicated by ultrasonication (SONICS VCX 750) using a pulsation cycle  for a total duration of 12 min of sonication at 40% energy on ice. The soluble PWWP protein extract was collected by centrifugation  was prepared for a Ni2+-NTA agarose column. The unwanted proteins were washed out with binding buffer containing imidazole (20–50 mM), and the desired target protein of HDGF was eluted with a binding buffer containing imidazole (150 mM). The subsequent step with anion-exchange chromatography (Hitrap Q) was similar to that for the apo PWWP domain. The homogeneity of the full-length HDGF protein was confirmed by size-exclusion chromatography (Superdex-200) with buffer . HDGF was concentrated  before colony-formation and Western-blot assays and structural studies. The purity of HDGF was greater than 95% based on SDS-PAGE (12%) and staining (Coomassie Brilliant Blue R-250).The expression and purification procedures for full-length HDGF were similar to that for the E. coli, if any, we incubated DNA of various lengths with the sequence of the SMYD1  and the PWWP domain at 4 °C overnight. The SMYD1 sequences were 5′-CAGGCTGGTCTTGAA-3′, 5′-CAGGCTGGTC-3′ and 5′-CAGGC-3, respectively. Complementary single-stranded DNA molecules ordered from the commercial company (Mission Biotech) were mixed together and then heated at 95 °C for 10 min. After heating, the DNA mixtures were left near 23 °C overnight to form the double-stranded DNA. The monomeric PWWP domain and SMYD1 were incubated at a molar ratio of 1:1. The complex of the PWWP domain and SMYD1 was purified again by size-exclusion chromatography (Superdex-200) in a buffer  to ensure that the PWWP-SMYD1 complex of high homogeneity was properly collected and that unbound DNA was removed to avoid a perturbing effect on the subsequent crystallization.After purification by the ion-exchange chromatography to exclude endogenous DNA from SMYD1 complex, the PWWP domain and the PWWP-15 bp SMYD1 complex to the nucleolin (NCL) protein. Human NCL cDNA was constructed using the pET15b vector and transformed into E. coli BL21 (DE3) cells. After IPTG (1 mM) induction, the protein was purified using the Ni2+-NTA agarose column. For the solid-phase binding assay, NCL coating was applied at 10 µg/mL at 4 °C overnight. Each well was blocked with BSA (3%) in PBS buffer containing Tween-20 (0.05%) at 25 °C for 1.5 h. Antigen proteins (HDGF and PWWP) were reacted at 25 °C for 1.5 h. The PWWP-specific antibody and the HRP-conjugated antibody were used to detect HDGF and PWWP domain proteins. Each well was reacted in TMB substrate at 25 °C, and H2SO4 (1 N) was applied as a stop solution. Finally, the optical density of each well was measured at 450 nm using an ELISA reader.The solid-phase binding assay was performed to detect the binding ability of HDGF, HDGF-15 bp ® 2000 reagent. The conditional medium was collected, and the HDGF-Fc protein was purified using a protein G Sepharose® column. In the competitive assay plate, NCL was coated at a concentration 10 µg/mL at 4 °C overnight. After BSA (3%) blocking, the HDGF-Fc fusion protein (0.01 μM) was reacted at ten times the molar concentration of the PWWP domain (0.1 μM) and PWWP-SMYD1 (0.1 μM) for competition. The goat anti-human-HRP antibody was used for detection of the HDGF-Fc fusion protein. Finally, the TMB substrate and H2SO4 (1 N) as a stop solution were used for the colour reaction.The competitive assay was performed to validate whether the PWWP domain was competitive for the HDGF-NCL interaction. For this assay, the human HDGF cDNA was constructed in the pcDNA3.1 expression vector using a fusion of the human antibody-Fc fragment. For protein production, the plasmid was transfected into human embryonic kidney 293 T cells using LipofectamineSMYD1 complex proteins were concentrated to 10–20 mg/mL in a storage buffer  containing protease inhibitor cocktail . Crystallization trials were performed in 96-well plates (JET Biofil) using several crystal-screening kits based on the hanging-drop vapour-diffusion method utilizing hanging drops (0.4 μL) containing equal volumes of protein solution (0.2 μL) and a reservoir solution (0.2 μL) against the bottom reservoir solution (100 μL) at 8 and 18 °C, respectively. The apo PWWP in storage buffer with spermine tetrahydrochloride (10 mM), which functioned as a kind of precipitant for DNA to remove soluble DNA, was crystallized under conditions containing sodium chloride (200 mM), Tris , and polyethylene glycol (PEG) 4000 . The PWWP-SMYD1 complex was crystallized under conditions containing sodium nitrate (90 mM), sodium phosphate dibasic (90 mM), Tris; Bicine , PEG 1000 (12.5%) and PEG 4000 .The purified PWWP domain and HDGF PWWP-apo PWWP domain at the best resolution, a total rotation of 120° with oscillation of 1.0° were measured at BL44XU of SPring-8 using an X-ray wavelength of 0.9 Å with an exposure duration of 1 s and distance of 500 mm from the crystal to the detector at 110 K under a nitrogen stream provided by a cryo-system . For complete data collection of the PWWP-SMYD1 complex, a total rotation of 200° with oscillation of 0.5° were measured at the Taiwan Photon Source (TPS) beamline 05A equipped with a CCD detector  using an X-ray wavelength of 1.0 Å, exposure duration of 0.1 s and distance of 350 mm from the crystal to the detector at 110 K under a nitrogen stream provided by a cryo-system. All data were indexed, integrated and scaled using the programme suite HKL-200042.The crystals were mounted on a synthetic nylon loop  and then flash-cooled in liquid nitrogen. The protein crystals were first tested with X-rays from synchrotrons at TLS beamlines BL13B1, BL13C1 and BL15A1 at the National Synchrotron Radiation Research Center (NSRRC) in Taiwan and BL12B2 at SPring-8 in Japan. For complete data collection of the apo PWWP domain belonged to the space group P6422, with unit cell dimensions a = b = 79.49 Å and c = 105.10 Å, and diffracted to 3.3 Å resolution. The crystal of the PWWP-SYMD1 complex belonged to the space group P31, with unit-cell dimensions a = b = 32.40 Å and c = 205.82 Å, and diffracted to 2.84 Å resolution. Structures of both the apo PWWP domain and the PWWP-SMYD1 complex were determined using the molecular replacement method with the Molrep programme43. The crystal structure of the apo PWWP domain was first determined with the X-ray structure of the PWWP domain of human HDGF2 (PDB entry: 3QBY) as a search model. The HDGF PWWP-SMYD1 complex was later determined with the solved structure of the HDGF apo PWWP domain as the protein model template and the structure of a 10-bp DNA as the DNA search model, respectively. All structures were built with COOT44. Refinement was performed with REFMAC5 and CCP445. The correctness of the stereochemistry of each structure was validated with MolProbity46. The calculations of root-mean-square deviation from angles, bonds and dihedral and improper angles showed the satisfactory stereochemistry. All details of the crystallographic data and refinement statistics are shown in Table PyMOL (http://www.pymol.org/).The crystal of the SYMD1 complex and the apo PWWP domain have been deposited in PDB under the accession codes 5XSK and 5XSL, respectively.Coordinates and structure factors of the HDGF PWWP-Supplementary Information


Xanthomonas oryzae have added a new twist to our understanding of this fascinating co-evolutionary arms race (Plants are constantly challenged by a wide range of pathogens and have therefore evolved an array of mechanisms to defend against them. In response to these defense systems, pathogens have evolved strategies to avoid recognition and suppress plant defenses . Three rrms race . They sh Xanthomonas plant-pathogenic bacteria that modulate host transcription by trans-activating host susceptibility genes . Direct interaction of TALEs with TFIIAγ5 from rice activates disease susceptibility genes (Transcription activator-like effectors (TALEs) are major virulence factors of ty genes . For thity genes . Due to entified . Over the manner . Additioe manner . The impty genes .Xanthomonas that can therefore be considered a microbial pattern (Xanthomonas oryzae pv. oryzae (Xoo) and X. axonopodis pv. vesicatoria isolates  because TALE-derived (truncTALES and iTALES) effectors can suppress this resistance suggesting they might act as effector mimics. In the case of Xo1 this suppression is independent from DNA binding at least for the Tal2h truncTALE . iTALEs may interfere with TALE recognition in a dominant-negative manner by occupying some binding sites of Xa1 for TALEs and by this outcompete TALEs for Xa1-binding without triggering receptor activation . This model is consistent with the finding of How TALEs are recognized by Xa1 and Xo1, directly or indirectly, and how truncTALEs/iTALEs interfere with recognition is unknown. However, for the case of Xa1, it is tempting to speculate that it might involve the BED-type zinc finger domain which is integrated in the N-terminal region of this NLR protein . In facttrans-activation? If yes, it is expected that mutants of these BED domain proteins are less susceptible to xanthomonads that rely on TALEs for virulence. Finally, a better molecular  understanding of how TALEs and iTALEs interact with Xa1 may allow engineering NLR receptors that recognize full-length TALEs but not iTALEs and thus would truly confer broad-spectrum resistance against Xoo and Xoc.The integrated decoy model for NLRs is a valuable concept to generate several hypotheses that can be challenged experimentally but that are entirely speculative at this point: Is the BED domain of Xa1 required for TALE recognition and iTALE-mediated suppression and if so, do these activities rely on direct binding between the BED domain and certain motifs in the TALE? Do TALEs interact with other Xa1 domains? Do TALEs interact directly or indirectly with other BED domain proteins and do such interactions contribute to target gene PZ, BS, RK, TK, and J-BM participated to the writing of this mini-review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rice stripe virus may promote virus systemic infection by interacting with the full length of fibrillarin from Nicotiana benthamiana (NbFib2) in the nucleolus and cajal body (CB). NbFib2 contains three functional domains. We used yeast two-hybrid, colocalization, and bimolecular fluorescence complementation (BiFC) assays to study the interactions between p2 and the three domains of NbFib2, namely, the N-terminal fragment containing a glycine and arginine-rich (GAR) domain, the central RNA-binding domain, and the C-terminal fragment containing an α-helical domain. The results show that the N-terminal domain is indispensable for NbFib2 to localize in the nucleolus and cajal body. p2 binds all three regions of NbFib2, and they target to the nucleus but fail to the nucleolus and cajal bodies (CBs).p2 of  Rice stripe virus (RSV), an economically significant pathogen of rice, is the member of the genus Tenuivirus. It is transmitted by the small brown planthopper (Laodelphax striatellus) in a persistent, circulative-propagative manner, affected by global warming [Nicotiana benthamiana (N. benthamiana) can be infected by RSV through mechanical sap inoculation [ warming , 2. Nicoculation .The genome of RSV comprises four single-stranded RNAs, denoted as RNA1, RNA2, RNA3, and RNA4 in the decreasing order of their molecular weights , 6. RNA2Nicotiana benthamiana (NbFib2) mediates assembly of Umbravirus ribonucleoprotein particles (RNPs), which are capable of long-distance movement and causing systemic viral infection [Influenza A H3N2 subtype virus binds to the fibrillarin via the C-terminal nuclear localization signal 2 (NLS2) [Potato virus A (PVA) and the 2b silencing suppressor protein in Cucumber mosaic virus (CMV), respectively [α-helical domain [Many different viruses bind to the nucleolus to manipulate host-cell functions and recruit nucleoprotein to aid in virus infection. Fibrillarin, an important nucleolus protein, was reported to interact with viral proteins and regulate virus replication, movement, and so on. Fibrillarin from nfection . Protein2 (NLS2) . There iectively , 19. In ectively . NbFib2 l domain , but howIn this study, the interactions between p2 and the three domains of NbFib2 are identified using yeast two-hybrid, colocalization, and BiFC methods. The results reveal that p2 binds to the three domains of NbFib2 in the nucleus but fails to target to the nucleolus and cajal bodies (CBs), and the GAR domain is necessary for NbFib2 to localize in the nucleolus and CBs.α-helical domain (NbFib2-3) , histidine (His), leucine (Leu), and tryptophan (Trp) , and the cotransformants of pGAD-T/pGBK-53 and pGAD-T/pGBK-Lam were individually used as positive control and negative control. However, the cotransformants of pGBK/pGAD, pGBK-NbFib2s/pGAD, pGBK/pGAD-NbFib2s, pGBK-p2/pGAD, or pGBK/pGAD-p2 failed to grow on SD/Leu−Trp−His−, although they grew well on SD/Trp−Leu−  domain (NbFib2-1), the central RNA-binding domain (NbFib2-2), and C-terminal fragment, containing an bFib2-3) . As showColocalization result shows that only NbFib2-1 (GAR domain) can form bright spots in the nucleolus and cajal body (CB) . The othIn BiFC assay, p2 binds NbFib2-1, NbFib2-2, and NbFib2-3 individually, and they almost localize in the nucleus but not in the nucleus and CB in the shape of spots .In summary, we found that (i) GAR domain was essential for NbFib2 to target to the nucleolus and CB, (ii) p2 interacted with the three functional regions of NbFib2, and (iii) these interactions occurred in the nucleus but failed to form bright spots targeting to the nucleolus and CBs.Arabidopsis and human cell to the nucleolus and CBs [NbFib2 is divided into three functional domains in our study; those domains localize in the nucleus, but only the N-terminal (GAR) domain targets to the nucleolus and CB as same as the full length of NbFib2. The GAR domain might be important for fibrillarin accumulation in the nucleolus . It was  and CBs , 23. Som and CBs . Howeverporcine reproductive and respiratory syndrome virus (PRRSV), and ORF3 of groundnut rosette virus (GRV) [Arabidopsis (AtFib2) has two RNA-binding regions, one located in the central region and the other located in the C-terminal region, while the GAR domain is incapable of RNA binding [In this study, p2 binds to the three functional regions of NbFib2 in the nucleus but not in the nucleolus, CBs, or cytoplasm. These results are consistent with our previous studies, which show that p2 interacts with the full length of NbFib2, and NbFib2 plays a role in both the nucleolar localization and the appropriate cytoplasmic distribution of p2 . The N-tus (GRV) –27. Our us (GRV) , 28. FibIn short, the results of this study are consistent with the previous study that the full length of NbFib2 is essential for p2 to target to the nucleolus and CBs. In addition, p2 interacts with the three functional regions of NbFib2, respectively; the mechanisms of these interactions will be studied in the future.N. benthamiana plants were grown and maintained in a greenhouse at 25°C.The N. benthamiana and RSV sequences (GenBank accession nos.: AM269909 and EF493228) downloaded from the GenBank. The three domains of NbFib2 were first inserted into the entry vector pDonr221 and then the destination vectors pEarleyGate101 (YFP), pEarleyGate201-YN (YN), and pEarleyGate201-YC (YC), using the Gateway recombination system [cDNAs encoding the three domains of NbFib2 and RSV-p2 were amplified, respectively, by PCR using primers in n system . pEarleyFor yeast two-hybrid experiments, PCR products of RSV-p2 and the three domains of NbFib2 were digested with suitable restriction enzymes individually and then ligated to the vector pGADT7 or pGBKT7 digested with the same enzymes.These constructs were confirmed by capillary sequencing conducted by Takara .α-gal . The cotransformation of pGADT7-NbFib2s and pGBKT7-p2 was also done the same way.pGBKT7-NbFib2s (three domains of NbFib2) were introduced together with pGADT7-p2 into the yeast strain AH109 by cotransformation. The cotransformants grew on different SD mediums: medium lacking tryptophan (Trp) and leucine (Leu) (SD/Trp−Leu−); medium lacking histidine (His), Trp, and Leu (SD/Trp−Leu−His−); and medium lacking adenine (Ade−), His, Trp, and Leu but containing X-Agrobacterium tumefaciens (A. tumefaciens) strain EHA105 were grown separately to OD600 = 0.8 at 28°C on the Luria–Bertani liquid medium supplemented with 50 μg/μL of rifampicin and 50 μg/μL of kanamycin. The resulting cultures were centrifuged at 12,000g for 1 min and then resuspended in induction media . In colocalization and BiFC assays, A. tumefaciens containing NbFib2s were separately mixed with p2 in equal volumes. The mixtures of the bacterial cultures were incubated at room temperature for 3 h and then infiltrated onto fully-grown upper leaves. Six-week-old N. benthamiana was used for the experiment.Subcellular localizations of proteins were monitored at 48 h after infiltration under a confocal microscope . The fluorophores in CFP and YFP were excited at 458 and 514 nm, and images were taken using BA480–495 and BA535–565 nm emission filters, respectively.


Arabidopsisthaliana, Glycine max, Cucumis sativus, Oryza sativa ssp. japonica and Oryza sativa ssp. indica). The genomes were screened for genes encoding lectin domains using a combination of Basic Local Alignment Search Tool (BLAST), hidden Markov models, and InterProScan analysis. Additionally, phylogenetic relationships were investigated by constructing maximum likelihood phylogenetic trees. The results demonstrate that the majority of the lectin families are present in each of the species under study. Domain organization analysis showed that most identified proteins are multi-domain proteins, owing to the modular rearrangement of protein domains during evolution. Most of these multi-domain proteins are widespread, while others display a lineage-specific distribution. Furthermore, the phylogenetic analyses reveal that some lectin families evolved to be similar to the phylogeny of the plant species, while others share a closer evolutionary history based on the corresponding protein domain architecture. Our results yield insights into the evolutionary relationships and functional divergence of plant lectins.Lectins are present throughout the plant kingdom and are reported to be involved in diverse biological processes. In this study, we provide a comparative analysis of the lectin families from model species in a phylogenetic framework. The analysis focuses on the different plant lectin domains identified in five representative core angiosperm genomes ( Arabidopsis thaliana was the first plant to have its genome completely sequenced in 2000 [ in 2000 . More th in 2000 . To dateProtein domains are structurally conserved and represent functionally independent components of proteins. The domains themselves have evolved from shorter structural units  or by the association of small folding motifs ,5. TheseAgaricus bisporus agglutinin family, the amaranthins, the homologs of class V chitinases (CRA), the cyanovirin family, the Euonymus europaeus lectin (EUL) family, the Galanthus nivalis agglutinin (GNA) family, the hevein family, the jacalin-related lectin (JRL) family, the legume lectin family, the lysin motif (LysM) family, the Nicotiana tabacum agglutinin (Nictaba) family and the ricin B lectin family [The plant lectin family encompasses all proteins that selectively recognize and bind to specific carbohydrate structures that occur in a free form or as part of glycoconjugates, such as glycoproteins and glycolipids. The binding of lectins to glycan structures is reversible and does not alter the structure of the glycan moiety . This prn family . Proteinn family ,19,20. An family . More ren family ,24,25,26Glycine max (soybean) [Cucumis sativus (cucumber) [Arabidopsis thaliana  [Morus notabilis (mulberry) [Oryza sativa ssp. japonica and Oryza sativa ssp. indica. The distribution of lectin domains was examined and the phylogenetic relationships of the different plant lectin motifs were investigated. Our comparative study revealed that additional protein domains have recurrently been integrated into lectins from multiple plant species. Using a phylogenetic approach, we demonstrated the differential modes of evolutionary innovation and observed compelling differences between the lectin families.Although lectins from plants and other species have been an important topic of research for years, most studies dealt with the identification and expansion of a particular lectin family in one species, or specifically focused on one type of lectin motif and its distribution in the plant kingdom ,34,35,36soybean) , Cucumisucumber) , Arabidoe cress)  and Moruulberry) . In thisArabidopsis thaliana, Glycine max, Cucumis sativus, Oryza sativa ssp. japonica and Oryza sativa ssp. indica were scanned for the presence of genes encoding plant lectin domains. All predicted lectin domains and any additional protein domains were identified using InterProScan. Altogether, 1337 lectin domain containing sequences were identified, belonging to 10 of the 12 plant lectin families known today  as well as in monocots  and in several dicot plants from different families ; demonstrating that this lectin domain is widespread, yet not omnipresent [To gain insight into the evolution and diversity of lectin protein architectures across plants, the genomes of wn today . Lectin ichardii . Similarlymorpha ,42. Membipresent ,44.Physcomitrella patens and Amborella trichopodia) to monocots and dicots.The total number of identified lectin genes is highly divergent 146-368) when comparing different species, but no correlation was found between the genome size and the number of lectin genes . Conside-368 whenA comparative analysis of the size of the different plant lectin families , demonstProtein domains are the building blocks of all proteins, and the combination of particular protein domains defines protein functionality . Using aPhyscomitrella patens and Selaginella moellendorffii, mainly monocots comprise EUL domains arrayed in tandem while the single-domain proteins are present in both monocot and dicot species [Protein sequences consisting of multiple lectin domains were found in most lectin families ; though no combinations of different lectin domains within one putative lectin sequence were retrieved. Furthermore, the lectin domains arrayed in tandem can also be linked to additional, unrelated protein domains. Examples are the amaranthin/amaranthin/aerolysin combination in cucumber and the LysM/LysM/protein kinase domain combination that is present in all the species under study. The EUL lectin domain is the only lectin domain that is not present in combination with other annotated protein domains, though proteins composed of two EUL domains arrayed in tandem were identified in rice. These results support previous studies which concluded that in addition to  species ,61. Our analysis revealed that almost all lectin domains can occur as single-domain proteins. However, it should be mentioned that in those proteins, the full-length protein sequence does not always consist of the lectin domain alone. The additional N- or C-terminal sequences are unrelated to the lectin domain, but do not correspond to any known protein domain. The majority of the identified domain architectures represent multi-domain proteins, consisting of one or more lectin domain(s), in combination with other annotated protein domains. Glycosyl hydrolase (GH) domains were found in combination with the hevein domain (GH19), the legume lectin domain (GH1 or GH17) and the ricin B lectin domain (GH5 or GH27). Similarly, the F-box domain was identified in combination with the jacalin, LysM and Nictaba domains in multiple species. Another reoccurring protein domain found in all species is the protein kinase (PK) domain. This protein domain is associated with the CRA, GNA, jacalin, Nictaba, LysM and legume lectin domains in various species. Furthermore, a lot of unique domain combinations were identified, comprising a lectin domain and one or more additional protein domain(s). The presence of these protein domain architectures is predominantly limited to a particular species, supporting previous observations that long multi-domain architectures tend to be more species-specific. However, a large majority of all plant domain arrangements was shown to be present in all linages; as illustrated in A selection of lectin domains and some reccurring combinations of lectin domains and particular non-lectin protein domains were investigated in more detail. Evolutionary relationships between the protein domains from the different species were investigated by building maximum likelihood trees with RAxML. The results of the phylogenetic analyses and the biological importance of the protein domain combinations are discussed below.In the genomes of Arabidopsis, soybean, cucumber and rice, the ricin B lectin domain is found to be part of multi-domain proteins; including the combination with a GH27 or a GH5 domain, a second ricin B domain, or in the RIP/ricin B/ricin B architecture. The maximum likelihood tree built using the protein sequences of all ricin B domains a, clearlOryza sativa ssp. indica. Similar to the other clades, the subclades reflect the origin of the retrieved sequences. Surprisingly, the GH1 sequence shows a close connection to two GH19 domain sequences from soybean even though they are not part of the same clan of GHs (available online: www.cazy.org). A separate branch (clade 1) only containing the GH17 sequence from Oryza sativa ssp. japonica completes the phylogenetic tree. In this context, several studies investigated the evolution of glycoside hydrolase subfamilies. However, these reports mainly focused on different GH subfamilies, or were confined to bacterial and/or fungal species. Aspeborg and coworkers analyzed the large GH5 subfamily in plants, and group the GH5/ricin B proteins from Arabidopsis, rice and soybean identified in our study, together in a special cluster (GH5_11) similar to our phylogenetic analysis [Phylogenetic analyses of the GH domains b supportanalysis .Ricinus communis and known for its high toxicity to mammalian and other eukaryotic cells [N-glycosidase domain (A chain or RIP domain) and a lectin domain (B chain or ricin B domain) [N-acetylgalactosamine. The toxicity of ricin is mainly attributed to the N-glycosidase domain, though interaction of the lectin domain with galactosylated receptors on the cell surface helps the toxin to enter the cell [Abrus precatorius, Sambucus nigra and Viscum album has been confirmed but it should be mentioned that not all proteins are as toxic as ricin [The ricin B domain refers to the lectin domain of ricin, a protein identified in ic cells . Ricin i domain) . Multi-dthe cell . The toxas ricin . Expressas ricin ,71,72,73In cucumber, some ricin B lectins composed of two ricin B lectin domains are found. One of these proteins is referred to as XSP30 and was proposed to represent a signaling molecule produced in the xylem parenchyma and pericycle cells of cucumber roots and controlled by gibberellic acid . Althougwww.cazy.org) [Although GH/ricin B combinations have been identified in several of the species under study, this type of multi-domain protein has not yet been investigated in plants. In bacteria, combinations of such domains have been reported previously, and were shown to be involved in the degradation of insoluble or complex polysaccharides. These GH/ricin B proteins comprise an unusual combination of a carbohydrate-degrading (GH) and a carbohydrate-binding (lectin) domain and in bacterial homologs, the latter has been acknowledged to assist the enzymatic activity ,76,77. Bazy.org) ,78.Proteins containing the Nictaba domain or combinations of an F-box and a Nictaba domain are widespread in plants  and wereClades 6–9 in the phylogenetic tree are mainly composed of Nictaba domains originating from F-box/Nictaba proteins. Within these clades, a few sequences correspond to single-domain proteins only containing the Nictaba domain, possibly due to loss of the F-box domain during evolution. These rearrangements are isolated events and did not always occur in the closest homologs, implying that they took place after the diversification of the different species and/or subspecies. One of the subclades of clade 8 also clusters the sequences derived from PK/Nictaba proteins, a protein domain combination that most probably evolved after the deletion of the F-box domain. Noteworthy, clade 2 with Nictaba sequences originating from the F-box/Nictaba proteins is more separated from the other clades (6–9) containing sequences with the same domain architecture. This clade can be further separated into four subclades: clades 2c and 2d only contain sequences from rice while clades 2a and 2b encompass different dicot sequences. More generally, this organization among subclades also applies to the other subclades of the phylogenetic tree, which also group sequences of the same origin.Overall, there is a clear separation between the Nictaba domain sequences originating from Nictaba and F-box/Nictaba proteins, indicating that the F-box/Nictaba protein domain combination originated early in a shared ancestor of plants, long before the divergence of monocot and dicot plants. These conclusions are in correspondence with a previous study, in which Nictaba homologs from 15 plant genomes were analyzed .Oryza sativa) clades of the cladogram  or closely related  are in two distinct clades of the phylogenetic tree, suggesting that they are the result of two independent domain fusion events which could also explain why these sequences are not found in the other species since they do not share a common ancestor in land plants. The F-box sequences from the F-box/LysM and F-box/F-box AD1/jacalin sequences from Arabidopsis and rice also clustered in distinct clades in studies including all F-box sequences present in these species, supporting our results ,80.Remarkably, the corresponding F-box domains of the Nictaba domain sequences from clade 2 in Pseudomonas syringae infection [PP2-A1, an Arabidopsis homolog, repressed phloem feeding of Myzus persicae and addition of the protein to an artificial diet significantly reduced the weight gain of aphids [O-GlcNAc-modified histones [Nictaba was originally discovered in tobacco leaves treated with methyl jasmonate . Later, nfection . Similarf aphids ,86. It ihistones .F-box/Nictaba genes from Arabidopsis and rice are stress inducible [PP2-B10 and PP2-B11 from Arabidopsis showed a higher tolerance to Pseudomonas syringae infections and high salinity, respectively [In Arabidopsis, some Nictaba domains are found in combination with the TIR or AIG1 (avirulence induced gene 1) G-type domain, which are known for their roles in pathogen detection and defense responses ,89, againducible ,95,96. Oectively ,98, confOryza sativa ssp. japonica and Oryza sativa ssp. indica)  . Phytophthora ssp. [Botrytis cinerea [AtLecRK-I.9 gene was identified as the first receptor for extracellular ATP [Legume lectins were originally discovered in leguminous plants. Though legume lectins have been considered as a group of lectins that were restricted to leguminous plants for many decades, compelling evidence now shows that the so-called legume lectin domain is also widespread in the plant kingdom. Legume receptor-like kinases (RLKs) or L-type RLKs are a specific group of legume lectin homologs with diverse functions. Functional characterization of many L-type RLKs reported on the involvement of this type of protein in symbiosis ,101,102,ora ssp. ,106,107, cinerea , enhance cinerea , bacteri cinerea ,111, sal cinerea ,112 and ular ATP  and recoular ATP .Oryza sativa ssp. indica is less pronounced compared to Oryza sativa ssp. japonica. This is probably the consequence of differential expansion among the two subspecies.Analysis of the evolutionary relationships of the GNA homologs  shows reAll clades contain GNA domain sequences from proteins with the GNA/SG/PAN/PK/SRK domain organization in combination with GNA sequences from other protein configurations. However, the latter, i.e., GNA domains from single-domain proteins, cluster mostly in separate subbranches. The presence of GNA single-domain proteins in clades that are predominantly composed of GNA domains from the GNA/SG/PAN/PK/SRK combinations can be explained by recent single-step events in which the PK domain and/or other non-lectin domains were lost. Thus, evidence is accumulating for a large GNA/SG/PAN/PK/SRK family in the common ancestor of flowering plants. Subsequently, this family has further expanded and diverged in the different lineages, resulting in diverse family sizes . NotablyReports on the evolution of the GNA family in literature are limited. One study dealing with GNA RLKs in Brassicaceae concluded that some gene fusion events in the common ancestor of land plants resulted in almost all GNA RLKs, which is in agreement with our hypothesis . SimilarBIR2, which plays a role in plant innate immunity [OsLecRK1-OsLecRK3) was shown to confer broad-spectrum and durable insect resistance [Magnaporthe grisea [GNA or G-type RLKs are best studied in Brassicaceae and also known as S-domain RLKs due to the presence of the S-locus glycoprotein domain which is involved in pollen self-incompatibility ,116. Othimmunity . OsSIK2,immunity . Anotherimmunity . A gene sistance . In a ste grisea .Other GNA domain architectures including the thaumatin, LRR and NB-ARC domain suggest that these proteins are stress-related given the involvement of these domains in disease resistance and stress adaptation ,129,130.Analysis of the phylogenetic relationships between the different jacalin domains reveals that lectin sequences derived from one species show a closer relationship . This isIn agreement with the phylogenetic analysis of the F-box protein domains (discussed in 2.3.2), the jacalin domains of the (F-box/)F-box AD1/jacalin proteins from Arabidopsis and the F-box/jacalin protein from soybean cluster in two different branches  of the phylogenetic tree. This observation again confirms that these proteins do not share a common history.In clades 2, 3, 6 and 9; only jacalin domains from the single-domain architecture are grouped . All othThe dirigent/jacalin proteins are restricted to rice and most probably evolved from a recombination of the dirigent and jacalin domain before the diversification of rice. This event was followed by multiple tandem duplication events, resulting in the presence of these jacalin domains in a separate branch of clade 4.Clade 4 also contains the jacalin domain from the exclusive no apical meristem (NAM)/jacalin protein and one of the peptidase C48/jacalin-related jacalin domains. The other jacalin domain originating from the same domain architecture is grouped in clade 8, suggesting that the recombination of the jacalin domain with the peptidase C48 domain occurred twice, independently of each other.JRLs can be classified into 2 subgroups: the vacuolar galactose-binding JRLs and the nucleocytoplasmic mannose-binding JRLs . While mO-GlcNAc signaling and intracellular motility [Several JRLs are involved in disease resistance, abiotic stress signaling, wounding and plant defense. This is reflected in the domain architecture of these lectins, where the jacalin domain is fused to domains related to stress response and defense , NB-ARC). The combination of a jacalin and dirigent domains has only been reported in Poaceae species  and thesmotility .Orysata are upregulated in response to several stresses  and hormonal treatments (jasmonic acid and abscisic acid) [Orysata, also known as SALT, in rice plants improves salinity tolerance [Magnaporthe grisea infection [Arabidopsis JAC1 homolog prevents systemic infection of the tobacco etch potyvirus, the plum pox virus, and the lettuce mosaic virus [Next to the chimeric JRLs, also the JRLs solely composed of jacalin domains were reported as stress-related proteins. Transcript levels for the rice gene ic acid) ,137,138.ic acid) . Overexpolerance  and enhanfection . The Aranfection . Similaric virus ,145,146.ic virus .Other domains associated with the jacalin domain are annotated to be related to signal transduction. The C1-like and DC1 domain found in cucumber can bind to diacylglycerol , a membrThe maximum likelihood tree with the LysM domain sequences  reveals All clades contain LysM domains from all different species, but lineage-specific subclades reveal the expansion through duplication events. Surprisingly, for proteins with two in tandem arrayed LysM domains, the two LysM domains are not clustered in the same clade . While tLysM RLKs are cell wall localized receptors that recognize GlcNAc moieties in various types of bacterial peptidoglycans, fungal chitins and rhizobacterial Nod factors. Some LysM RLKs play a role in plant defense as they are part of the plant innate immunity. Next to the classical LRR receptor kinases, LysM RLKs are now acknowledged as true pattern recognition receptors and are involved in the perception of microbial signals ,152. GlcLotus japonica: LjNFR1/LjNRF5, Glycine max: GmNRF1/GmNFR5, Medicago truncatula: LYK3/NFP9) [Next to their role in plant innate immunity, some LysM RLKs are involved in symbiosis. In legumes and other plants, symbiosis signaling involves recognition of the beneficial microorganisms such as rhizobacteria and mycorrhiza ,155,156.K3/NFP9) ,160,161.Oryza sativa ssp. japonica and indica sequences containing four hevein domains (clade 2), the corresponding hevein domains from both subspecies are clustered together  4 proteins . The barnfection ,167,168.nfection .AtEP3, an Arabidopsis homolog of this family, is also considered as a stress-responsive gene since its expression can be upregulated upon Xanthomonas campestris infection, UV light, SA treatment and wounding [Phytophthora sojae infection [CHIT17 and CHIT7) were significantly upregulated in a Magnaporthe oryzae resistant rice cultivar [Cht-2 or Cht-3 chitinase gene showed a higher resistance against Magnaporthe grisea [rcc2 or rcg3) in banana enhanced resistance towards black leaf streak disease [RC24) in wheat helped the plant to cope with strip rust infection [Class I and class IV chitinases consist an N-terminal hevein domain and a C-terminal GH19 catalytic domain . AtEP3, wounding ,171. Ananfection . RNA-seqcultivar . Transgee grisea . Moreove disease  and the nfection .https://phytozome.jgi.doe.gov/pz/portal.html). The Cucurbit Genomics Database (available online: http://www.icugi.org/cgi-bin/ICuGI/index.cgi) and MSU Rice Genome Annotation Project Database (RGAP release 7) [Oryza sativa ssp. indica (ASM465v1) were identified using Ensembl Plants (available online: http://plants.ensembl.org/index.html). Subsequently, the top hit was used for a second BLASTp search and all retrieved sequences were selected as candidate lectin genes. The corresponding protein sequences were downloaded and the presence of the lectin domains and any other annotated protein domains was identified using InterProScan5 (available online: http://www.ebi.ac.uk/Tools/webservices/services/pfa/iprscan5_soap) with default settings [Putative lectin genes from Arabidopsis, soybean, cucumber and rice were identified using a combination of BLAST, InterProScan5 and hidden Markov models. Protein sequences encoding a representative protein from each lectin family  were uslease 7)  and Phytsettings . Proteinsettings  and the settings , respecthttp://www.ebi.ac.uk/Tools/msa/muscle/) or locally using molecular evolutionary genetics analysis 7 (MEGA7) [http://tree.bio.ed.ac.uk/software/figtree/).Maximum likelihood trees were generated using the amino acid sequences of the protein domains. For protein sequences containing multiple domains of the same type, the domains were separated and all domain sequences were included in the analysis. The domain sequences were aligned with MUSCLE using default settings . The ali (MEGA7) . Subsequ (MEGA7) . Based o (MEGA7) . RAxML uGene expansion through segmental and/or tandem duplications was analyzed with PLAZA .Arabidopsis thaliana, Glycine max, Cucumis sativus, Oryza sativa ssp. japonica and Oryza sativa ssp. indica. Our data demonstrated that most plant lectin families are present across all surveyed genomes. Although these findings are consistent with previous analyses made in dicot genomes, we are not aware of genome wide studies investigating the lectin sequences from monocot species. Furthermore, PLAZA analysis confirmed that all plant lectins under study are also present in Amborella trichopodia and Physcomitrella patens, signifying ancient lectin domain integrations and pointing out the occurrence and importance of lectins throughout the whole plant kingdom. In this study we presented an inventory of the lectin sequences in five genomes of model plants, including Additionally, we performed a comparative and evolutionary genomics study of some particular plant lectin domains and some other protein domains that are found in combination with the lectin domains. A detailed analysis of the lectin sequences revealed a wide range of domain architectures, involving the fusions of lectin domains with other annotated protein domains. Parts of these reccurring additional protein domains have been reported to be involved in plant defense, signaling and/or development, which agrees with the idea that lectin domains play a role in plant growth and defense. Some of the multi-domain proteins are widespread, indicating the retention of ancient fusions in a common ancestor. In contrast, the many species-specific domain arrangements can be explained by lineage-specific retention of a certain domain rearrangement or by a recent event of protein domain fusion or domain loss in a particular species .For some lectin families, there is significant variation in the number of lectin genes across flowering plants. Phylogenetics confirmed that duplication events and speciation are the driving source for this diversification. This is also reflected in the organization of lectin sequences in the phylogenetic trees. The JRL family clearly demonstrates a lineage-specific evolution while the ricin B family experienced extension of the different domain architectures in most species. These findings support the dynamic evolution of the different plant lectin families in respect to the domain organization and duplication events. In the future, it will be important to continue studying lectin families across species to enrich our understanding of their evolutionary history, but this method also has some limitations. The proteins encoded by the lectin genes are part of a larger and complex interactome, and it will be difficult to relate the emergence of a new protein domain combination to existing pathways. Therefore, a combined analysis of these traditional approaches in combination with co-expression networks would be advantageous and could expand our knowledge since recent studies demonstrated that co-expression networks are conserved also across species ,186. At It is clear that plants have a large variety of plant lectins at their disposal. During evolution, new lectin domain combinations have been created and used to the benefit of the plant to allow rapid adaptation to changing environmental conditions. Although the functionality of the putative lectin domains needs to be confirmed by experimental evidence, these lectins will most probably exert complementary activities since they are present in different subcellular/tissue specific locations and recognize different carbohydrate structures, either present in the plant or part of pathogens. Further functional investigation of these lectins will substantially contribute to our knowledge of the lectin complement and their importance for the plant.


The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains—In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model. The traditional definition of a domain refers to the local compactness criterion ,2,3,4. ATwo databases—SCOP ,8 and CADomain-specific structural changes are often discussed in the context of amyloidogenesis where partial unfolding of domain fragments  is seen as a possible cause of amyloid plaque formation . AccordiVoronoi tessellation is a specialized model used in the study of inter-domain interfaces. ,14,15. SThe relation between interface structure and hydrophobicity density distribution is particularly relevant in the context of amyloidogenesis . SelectiA thorough discussion of protein complexation and interface conformation would not be possible without referring to the worldwide discussion forum offered by the CAPRI  project, originally launched in 2001 ,20. The A cursory review of techniques employed in the CAPRI challenge indicates one common characteristic: They all tend to focus on pairwise (atom-atom) interactions. Analysis of the protein-protein interface is usually restricted to the zone of contact, with the geometry of solids (monomers) often used as the sole criterion of structural compatibility.In our work, we propose an approach which regards complexation as an effect of global conformational characteristics produced by the fuzzy oil drop model (FOD) in each structural unit (domain or protein), as well as in multi-domain complexes and protein dimers . The presence of a shared hydrophobic core is seen as the primary factor responsible for quaternary structural stabilization. The paper presents a set of homodimers in which the status of the inter-domain (or inter-chain) interface varies. Some dimers exhibit a shared hydrophobic core while in others no such core is present. In some cases we can attribute this phenomenon to the specific biological function of a given homodimer. Special attention is given to two proteins: dystrophin ,23,24,25All homodimers presented in this work have been analyzed with the use of the fuzzy oil drop model —An extenThe innovation of the fuzzy oil drop approach rests in its ability to identify “hydrophobically ordered” domains in the protein body—Even when these domains are actually formed by fragments contributed by separate chains. Such emergent hydrophobic cores may play a key role in ensuring proper biological activity of protein complexes, particularly when contraction and stretching come into play . The preTo-date analyses of homodimers on the basis of the fuzzy oil drop model characterize protein-protein interfaces as areas exhibiting higher-than-expected hydrophobicity—A result of local exposure of hydrophobic residues on the protein surface ,40,41. AIn light of the above, we can divide homodimers into four distinct groups: (1) hydrophobic core present in each participating monomer as well as in the dimer; (2) hydrophobic core present in the dimer but not in either monomer; (3) hydrophobic core present in both monomers but not in the dimer; and (4) hydrophobic core present in one participating monomer as well as in the dimer. The main point of this paper is the recognition of a distinct interface which manifests itself as a quasi-domain comprising fragments contributed by both monomers and possessing its own hydrophobic core. Such quasi-domains have been found in dystrophin and utrophin.The analysis presented in this work acknowledges the FOD status of homodimers (as explained above), focusing on representative cases for each group.The presented homodimers have been selected in such a way as to cover the entire spectrum of relationships between individual units  :Methanothermus fervidus (a hyperthermophilic organism)—1BFM [(1) Hydrophobic core (according to FOD criterion) present in each participating monomer as well as in the dimer. This case is represented by the histone B dimer from sm)—1BFM .(2) Hydrophobic core present (according to FOD model) in the dimer but not present in either monomer, represented by protein 1Y7Q . 1Y7Q isStaphylococcus aureus in the Zn2-form (1R1V) is representative of this group [(3) Hydrophobic core present in both monomers but not in the dimer; the metal-sensing transcriptional repressor from is group .(4) Hydrophobic core present in one participating monomer as well as in the dimer. This category is represented by the 2CD0—Human lambda-6 light chain dimer, a variable domain from the lambda-6 type immunoglobulin light chain . This diThe main contribution of this paper is recognition of a distinct interface, which manifests itself as an independent domain, comprising fragments contributed by both monomers and possessing its own well-defined hydrophobic core. Such interfaces are found in dystrophin and utrophin.Dystrophin is one of the largest proteins found in humans. It is expressed in various tissues, including skeletal muscle, heart, nerves and liver, where it forms an important component of the cellular membrane. Dystrophin is a cytoplasmic protein with an elongated shape. It links the cytoskeleton of the muscle cell with the extracellular matrix across the plasma membrane . DystropUtrophin  is anothBoth utrophin and dystrophin interact with actin in a similar way. They both affect the mobility of actin—Specifically, its bending and twisting capabilities . FunctioAccording to some studies either dystrophin or utrophin may have formed via partial gene duplication . In biolThe set of proteins under consideration is presented in The fuzzy oil drop model is based on the oil drop introduced by Kauzmann assuming hydrophobic residues migrating to the center of the protein body with hydrophilic residues exposed on its surface ,40,41. TThe global discordance expressed by RD > 0.5 for complete protein (or domain) is interpreted as lack of ordered hydrophobicity core in particular molecule. Local discordance is also expressed by RD > 0.5 calculated for selected fragment of the profiles . In particular, the fragments of polypeptide chain representing well defined secondary structure can be taken as units to calculate RD to characterize their status versus the ordered hydrophobic core. It is also possible to calculate the RD value for the polypeptide chain with selected residues eliminated from calculation. This is to show the role of certain (selected) residues in the hydrophobic core construction. In particular, the resides engaged in protein-protein interaction can be used as objects to be eliminated. Their status depends on the internal construction of the domain (molecule) and on the external factors which is the second protein molecule. The residues located in the inter-protein interface are the object of the analysis presented here. The aim of this analysis is the definition of the role of residues responsible for dimers construction and its stability.To visualize the status of particular chain, domain or chain fragment the status of RD > 0.5 discordance versus the theoretical distribution expressed by the values of RD above 0.5 are given in bold.The fuzzy oil drop model is described in details in a paper recently published . This isIdentification of secondary structural folds and the composition of protein domains follows the CATH and PDBSum classifications. Inter-domain/inter-chain contacts have been identified on the basis of the PDBSum distance criterion .Methanothermus fervidus (a hyperthermophilic organism) labeled 1BFM [This category is represented by the histone B dimer from led 1BFM .Analysis of RD values listed in Elimination of fragments, which participate in the inter-chain interface, distorts the shared core (RD increases from 0.383 to 0.390). This suggests that the interface zone contributes to the common hydrophobic core and its elimination destabilizes the complex, distorting the alignment between the observed and ideal distributions .Methanothermus fervidus, an archaeon that grows optimally at 83 °C, making it a hyperthermophilic organism. It is, therefore, a very interesting study subject. Our experience indicates that well-ordered hydrophobic cores are particularly common in thermophilic proteins (publication currently in preparation). This is consistent with experimental studies which point to increased role of hydrophobic interactions under high temperature conditions [The presented protein comes from nditions .A highly simplified, one-dimensional visualization of the presented case is shown in This category is represented by 1Y7Q . Fuzzy oThe interface zone plays a significant role in shaping the shared hydrophobic core: elimination of its residues (row labeled “No P-P”) increases the value of RD, which implies substantial involvement in construction of the shared core. We may conclude that this portion of the chain is the main component of the shared core and should be characterized by high local hydrophobicity. Reductions in RD calculated for individual secondary folds support this conclusion, suggesting that those fragments are aligned with the shared core.Comparing RD values for both monomers and for the dimer reveals symmetry in the shared core, while individual assessment of each monomeric core points to certain differences—It seems that each monomer adapts to the shared core in a slightly different manner. We can conclude that most residues contribute to the shared core rather than to individual cores. From the point of view of a single monomer involvement of its residues in the shared (dimeric) core introduces deformations in the individual (monomeric) core.The role of individual helical folds varies. Three helixes (four in the B chain) follow the expected monomeric distribution. When considering the dimer as a whole, four folds remain accordant with theoretical predictions. Of particular note is the helix at 60–76, which diverges from the idealized distribution in each monomer while exerting a strong stabilizing influence on the dimer . This phenomenon can be explained by pointing out that the helix forms part of the interface zone and therefore plays a crucial part in quaternary stabilization.From a functional point of view, the complex contains a zinc finger-associated SCAN domain, which is a homologue of the HIV-1 capsid protein. Hydrophobic core analysis may therefore provide a new model for protein-protein interactions underpinning viral particle formation .This highly simplified, one-dimensional presentation of the presented case  reveals The charts shown in Staphylococcus aureus in the Zn2-form (1R1V).This category is represented by the metal-sensing transcriptional repressor from The dimer lacks a shared core while each monomer retains a well-defined core. The status of β-sheets both in the dimer and in each monomer separately indicates good accordance with the theoretical model. We can therefore conclude that β-sheets contribute to structural stability of both the dimer and each monomer.Most secondary folds in 1R1V appear to stabilize their parent monomer rather than the protein-protein complex. This is particularly true of the helixes at 25–38 and 41–50 .Some other folds (such as the β-fragment at 68–74) play an equal role in the stabilization of monomeric units and the dimer. Elimination of interface residues improves the dimer’s accordance, suggesting that, at least in the case of 1R1V, protein complexation is mediated by forces other than hydrophobic interactions. Further analysis indicates that strong electrostatic forces are at play since the interface is composed almost entirely of polar residues.2+ ions), which is critical for the protein’s biological function. The labile nature of both helixes is related to the required conformational changes when interacting with DNA strains [The helixes at 52–66 and 84–100 do not align with the hydrophobic core; instead, they are involved in binding ligands (Zn strains .This category is represented by 2CD0: human lambda-6 light chain dimer (Bence-Jones protein), a variable domain from the lambda-6 type immunoglobulin light chain .The protein is a homodimer consisting of two V immunoglobulin domains. Despite sharing a β-sandwich structure, both domains differ with respect to their FOD status. The dimer itself diverges from the model as does one of its monomeric subunits. Nevertheless, individual secondary folds appear to play a similar role in the stabilization of each monomer; for example, the upper β-sheet  is a good match for theoretical predictions in the dimer as well as in both monomers, while the lower β-sheet (lower core) significantly diverges from the idealized distribution in each case. 2CD0 also contains a disulfide bond which appears to further stabilize its tertiary conformation. The loop bounded by SS bond-forming Cys residues conforms to the idealized distribution with good accuracy, suggesting a measure of coaction between both stabilizing factors (disulfides and hydrophobic interactions). It seems that the formation of the SS bond is enabled by hydrophobic forces which guide Cys residues towards the center of the protein body and into close proximity to each other. The resulting bonds do not, however, contribute to the formation of a shared (dimeric) hydrophobic core .Of note is the variable status of β-folds: fragments 8–13, 19–26, 34–38, 45–48 and 69–76. Each of these folds contributes to the stability of either its parent monomer or the dimer.Interface residues do not play an important role in structural stabilization of 2CD0 since eliminating them does not appreciably change the RD value.Under natural conditions, the V domain of the immunoglobulin light chain undergoes complexation with the corresponding domain of the heavy chain. Pathological conditions lead to formation of an excessive number of light chain dimers. The asymmetric nature of the homodimer  may be aThe V domain dimer also plays an important role in amyloidogenesis research, given that products of the major human V kappa and V lambda gene families have been identified in AL deposits. One particular subgroup (lambda-6) has been found to be preferentially associated with this disease. Comparative analysis of the status of individual β-folds in immunoglobulin-like domains indicates that—Despite a high degree of structural similarity, they differ with respect to their adherence to the fuzzy oil drop model .An entirely unexpected form of interface is observed in the 1DXX and 1QAG homodimers. Here, fragments contributed by each monomer assemble to form a quasi-domain which is highly accordant with the idealized hydrophobicity distribution model and contributes to the protein’s biological activity. Note that both 1DXX and 1QAG are components of a cross-membrane link between the actin-based cytoskeleton of the muscle cell and the extracellular matrix—They must therefore adapt to external stimuli  and revert to their original conformation when such stimuli are not present.The “Modified” tag indicates domain structures identified on the basis of the FOD-based definition of a domain. “Modified” means deprived of the fragment of chain identified as participating in interface-domain. The leftmost column also lists mutations which cause Duchenne’s muscular dystrophy. The most detrimental mutations are underlined. Question marks indicate that a given mutation does not belong to the analyzed fragment, but is found in its immediate neighborhood.In addition to secondary folds the table also lists residues involved in interaction with actin. The helix at 119–134 is referred to as “Helix X” in .The four-chain complex (ABCD), taken as a whole, does not appear to include a hydrophobic core in the sense of the fuzzy oil drop model. Similar conditions are encountered in both sub-complexes (AB and CD), as well as in individual monomers . This strong discordance is caused by the presence of a cavity in the central part of the complex as well as in individual chains.The CH1 domain is highly accordant, unlike CH2, which does not contain a prominent core. The status of individual β-folds can be assessed on the basis of their RD coefficients, listed in Two fragments of CH1, which have been experimentally confirmed to mediate actin binding, retain accordance with the fuzzy oil drop model. It therefore seems likely that the act of complexation requires structural rearrangement of the entire CH1 domain as—Contrary to FOD predictions—No hydrophobic residues are exposed on the surface in the protein’s native form.Visual inspection reveals fragments with high  and low  accordance between both distributions. In-depth comparative analysis is provided in Visual inspection of the structure of the AB complex  suggestsAs a conclusion, we propose a new type of domain consisting of fragments which belong to separate chains .In Much like dystrophin, the utrophin chain—When considered as a standalone unit—Diverges from the idealized hydrophobicity density distribution model. The chain itself resembles dystrophin in that it consists of two distinct domains; however unlike in dystrophin both domains conform to the FOD model.In domain 1 (31–130 aa), the helical fragments at 63–75 and 85–103, as well as the adjoining loops (48–62 and 76–84) diverge from the model, whereas in domain 2 only the helix at 206–223 remains divergent. The inter-domain interface of utrophin differs from the one in dystrophin; only helical fragments are present and their spatial conformation is different. Nevertheless, calculations based on the FOD model indicate that the interface exhibits a hydrophobicity density distribution which is remarkably similar to its dystrophin counterpart and also in good agreement with the model .In general, the constituent domains of both proteins share similar FOD characteristics. Much like dystrophin, utrophin also contains a quasi-domain comprised of fragments contributed by individual chains (two helixes per chain). The chart depicted in The fuzzy oil drop model, which describes the hydrophobic core status in monomeric units (domains) and larger complexes (dimers), may assist researchers in the study of mechanisms responsible for quaternary structural stabilization. A dimeric complex may form around a shared hydrophobic core with a coherent distribution of hydrophobicity throughout the protein body. Alternatively, each monomer may retain its own hydrophobic core while interacting with its partner. An interesting phenomenon is observed in the case of dystrophin and utrophin: these proteins generate an independent quasi-domain in the interface zone, consisting of parts of both monomers and exhibiting very high accordance with the theoretical model. This solution appears to be correlated with the biological properties of both proteins: both dystrophin and utrophin bind actin filaments  with membrane glycoproteins  and must withstand substantial external forces, which produce structural deformations. Under these conditions structural stabilization by way of nonbinding interactions may prove insufficient, and a shared quasi-domain is needed to provide additional stability. This domain is not listed in protein domain databases since it is not entirely contained in either monomeric chain. It is also not recognized by CATH, which specifically focuses on individual polypeptide chains. Similarly, the C-terminal β-fold is not subject to formal classification as it lacks a suitable β-“partner” in the opposite chain (cf. secondary conformation scheme in PDBSum). In the case of utrophin and dystrophin the presented structural solution appears to be a consequence of these proteins’ biological properties: specifically, their susceptibility to stretching forces.Comparative analysis of the dystrophin and utrophin conformations presented in  underscoA similar relation between structure and function can be observed in the immunoglobulin-like domain of titin (1TIT) ,55. ThisIn summary, it should be noted that high concentrations of hydrophobicity density at the center of the domain exert a stabilizing influence on the domain as a whole. This observation is in agreement with the well-known relationship between hydrophobic interactions and tertiary structural stabilization.Pre-compute a set of high-affinity amino acid residue interactions with the target surface;Redesign natural protein scaffolds to incorporate a selection of these amino acids;Design the remainder of the interface to enhance binding affinity.The authors of  report tWe attempt to approach the presented issue by performing a holistic analysis of protein conformations on the basis of the fuzzy oil drop model.Some other publications, which deal with changes triggered by point mutations in the structure of protein-protein interfaces, also highlight the need for improved energy functions .Examples of structures characterized by variable FOD status can be found in the immunoglobulin-like domain family . DespiteThis work attempts to underscore the influence of global  hydrophobicity density distribution upon the protein’s biological properties; particularly its ability to form complexes. It should be noted that earlier methods which focus entirely on pairwise atom-atom interactions (including interactions between protein atoms and the surrounding water environment) only acknowledge the effects of immediate “interaction partners”, while the structure of interfaces is critically dependent on the overall distribution of hydrophobicity density throughout the protein body, as well as on local deviations from the idealized model. The interface does not form in separation from other parts of the protein; rather, the entire molecule must participate in this process, folding in such a way as to ensure that the contact area remains capable of interacting with the intended ligand. This process relies on correct distribution of hydrophobicity density, including local discordances. Point mutations, not necessarily located close to the interface zone, may disrupt the protein’s hydrophobic core and lead to structural deformations which may render the protein less susceptible—Or indeed entirely incapable of—Complexation. In light of this phenomenon, existing in silico folding models, which acknowledge the presence of ligands or external proteins, should undergo further evolution .All structures were taken from PDB Data Base . The ideThe 3D presentation of protein structures was generated using PyMol . Calcula