Extending the understanding of mutagenicity: structural insights into primer-extension past a benzo[a]pyrene diol epoxide-DNA adduct

DNA polymerase enzymes employ a number of innate fidelity mechanisms to ensure the faithful replication of the genome. However, when confronted with DNA damage, their fidelity mechanisms can be evaded, resulting in a mutation that may contribute to the carcinogenic process. The environmental carcinogen benzo[a]pyrene is metabolically activated to reactive intermediates, including the tumorigenic (+)-anti-benzo[a]pyrene diol epoxide, which can attack DNA at the exocyclic amino group of guanine to form the major (+)-trans-anti-[BP]-N(2)-dG adduct. Bulky adducts such as (+)-trans-anti-[BP]-N(2)-dG primarily block DNA replication, but are occasionally bypassed and cause mutations if paired with an incorrect base. In vitro standing-start primer-extension assays show that the preferential insertion of A opposite (+)-trans-anti-[BP]-N(2)-dG is independent of the sequence context, but the primer is extended preferentially when dT is positioned opposite the damaged base in a 5'-CG*T-3' sequence context. Regardless of the base positioned opposite (+)-trans-anti-[BP]-N(2)-dG, extension of the primer past the lesion site poses the greatest block to polymerase progression. In order to gain insight into primer-extension of each base opposite (+)-trans-anti-[BP]-N(2)-dG, we carried out molecular modeling and 1.25 ns unrestrained molecular dynamics simulations of the adduct in the +1 position of the template within the replicative pol I family T7 DNA polymerase. Each of the four bases was modeled at the 3' terminus of the primer, incorporated opposite the adduct, and the next-to-be replicated base was in the active site with its Watson-Crick partner as the incoming nucleotide. As in our studies of nucleotide incorporation, (+)-trans-anti-[BP]-N(2)-dG was modeled in the syn conformation in the +1 position, with the BP moiety on the open major groove side of the primer-template duplex region, leaving critical protein-DNA interactions intact. The present work revealed that the efficiency of primer-extension past this bulky adduct opposite each of the four bases in the 5'-CG*T-3' sequence can be rationalized by the stability of interactions between the polymerase protein, primer-template DNA and incoming nucleotide. However, the relative stabilization of each nucleotide opposite (+)-trans-anti-[BP]-N(2)-dG in the +1 position (T > G > A > or = C) differed from that when the adduct and partner were the nascent base-pair (A > T > or = G > C). In addition, extension past (+)-trans-anti-[BP]-N(2)-dG may pose a greater block to a high fidelity DNA polymerase than does nucleotide incorporation opposite the adduct because the presence of the modified base-pair in the +1 position is more disruptive to the polymerase-DNA interactions than it is within the active site itself. The dN:(+)-trans-anti-[BP]-N(2)-dG base-pair is strained to shield the bulky aromatic BP moiety from contact with the solvent in the +1 position, causing disruption of protein-DNA interactions that would likely result in decreased extension of the base-pair. These studies reveal in molecular detail the kinds of specific structural interactions that determine the function of a processive DNA polymerase when challenged by a bulky DNA adduct.     

Inhibition ofin-vitro amino acid incorporation by the carcinogen N-methyl N’-nitro N-nitrosoguanidine

The addition of the carcinogen, N-methyl N’-nitro N-nitrosoguanidine, to a cell-free system consisting of purified polysome and ‘pH 5 enzyme’ fraction resulted in a marked inhibition of incorporation of (¹⁴C)-leucine into polypeptides. The extent of inhibition was remarkably high if the cell-free system contained limiting amount of ‘pH 5 enzyme’ fraction. Under this condition, the rate of inhibition was dependent on the concentration of carcinogen. Some component present in the ‘pH 5 enzyme’ fraction was inferred to be the susceptible factor, since the inhibition at low concentration of carcinogen could be reversed by increasing the amount of this fraction in the polysomal system. It was ascertained that tRNA was the primary target of carcinogenic action. Evidence suggested that functions attributed to tRNA such as aminoacylation and ribosomal transfer were both affected in a characteristic way by the action of the carcinogenic N-nitroso compound.     

Analysis of single nucleotide incorporation reactions by capillary electrophoresis

Single nucleotide incorporation assays have been used to probe the kinetic parameters of many DNA and RNA polymerases. Traditionally, oligonucleotide primers are 5'-(32)P labeled using T4 kinase and annealed to a complementary template with a 5' overhang. To quantify the reaction kinetics, the products of the primer extension reactions are usually separated using denaturing polyacrylamide gel electrophoresis and quantified using a phosphorimager or other method to measure radioactivity. We have developed a method using a 5' fluorescently labeled oligonucleotide to examine the kinetics of single nucleotide incorporation catalyzed by recombinant human mitochondrial polymerase gamma (Pol gamma) holoenzyme. Using laser-induced fluorescence detection in the P/ACE MDQ instrument, primers 5' labeled with fluorescent probes such as 6-carboxyfluorescein can be rapidly separated and quantified. However, we also show that only select probes can be used, presumably due to unfavorable interactions between Pol gamma and certain 5' labels.     

Structural insights into the first incision reaction during nucleotide excision repair

Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3' incision reaction. Here we report the crystal structure of the N-terminal catalytic domain of UvrC at 1.5 A resolution, which catalyzes the 3' incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY-YIG endonuclease domains. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond.     

Study of the incorporation of selenium into peroxidase isozyme of wheat seedling

The effects of selenium on the activity of peroxidase (POD) of wheat seedling and its isozyme pattern were studied using a greenhouse hydroponic experiment. The results show that the activity of POD is increased in response to higher Se concentration (approx 5.0 mg/L) in culture medium. The electrophoretic pattern of the POD isozyme was altered by growth of the wheat in a selenium medium. Se could incorporate into some POD isozymes during either seed germination or the seedling growth period. There is a dose-responsive incorporation of selenium in isozyme of POD and selenium content in the isozyme increase along with the increase of selenium concentration in the medium.     

Effects of rGM-CSF on leukemia cell proliferation and on the incorporation of cytosine arabinoside into DNA.

In vitro studies of the effects of recombinant granulocyte macrophage-colony stimulating factor (rGM-CSF) on freshly obtained human leukemia cells were conducted to determine if there is a relationship between the effects of this growth factor on the proliferative characteristics of leukemia cells and on their incorporation of cytosine arabinoside (araC) into DNA. While rGM-CSF was found to be able to stimulate both leukemia cell proliferation and araC incorporation, for individual leukemia specimens there was no consistent relationship among these effects. In some specimens proliferation was stimulated without an increase in araC incorporation. The reverse was also observed. These studies demonstrate the difficulty in identifying assays capable of predicting the clinical effects of growth factors on leukemia cells in patients since the effect in vitro vary with the assay.     

Nickel incorporation into hydrogenase 3 from Escherichia coli requires the precursor form of the large subunit

A mutant derivative of hycE, the gene for the large subunit of hydrogenase 3 from Escherichia coli, was constructed that lacks the 3′-terminal part encoding the C-terminal portion of the HycE polypeptide, which is proteolytically removed during maturation of the hydrogenase. The truncated gene was transferred to the in situ position on the chromosome. Although the mutant possessed HycE in its "mature" form, it was devoid of hydrogenase 3 activity. The activity was not restored by high nickel concentrations in the medium. The mutated HycE was not associated with detectable radioactivity when the strain was grown in the presence of ⁶³Ni²⁺. These results indicate that the C-terminal extension in the precursor form of the large subunit keeps the protein in a conformation required for the coordination of the metal. Received: 31 July 1995 / Accepted: 3 November 1995     

Induction of lipid peroxidation in hamster organs by the carcinogen cadmium: melioration by melatonin

Cadmium is a well-known human carcinogen. Lipid peroxidation is involved in cadmium-related toxicity and carcinogenesis. Melatonin is an effective antioxidant and free radical scavenger. The potential protective effects of melatonin against cadmium-induced lipid peroxidation in hamster brain, heart, kidney, testes, lung, and liver were examined. Lipid peroxidation was induced by intraperitoneal injection of cadmium chloride [single dose of 1 mg/kg body weight (bw)]. To test whether melatonin would protect against the toxicity of the carcinogen, the melatonin was injected peritoneally at a dose of either 15 mg/kg bw or 5 mg/kg bw, 0.5 h before cadmium treatment and thereafter at 8 h intervals during the day in the 48 h interval following the cadmium injection. One group of hamsters received only a single melatonin injection (a dose of 15 mg/kg bw, 30 min prior to cadmium). Forty-eight hours after cadmium injection, lipid peroxidation increased in brain, heart, kidney, testes, and lung. Either multiple injections of melatonin at both the 5 and 15 mg/kg bw doses, or a single injection of 15 mg/kg bw, prevented the cadmium-related increases in lipid peroxidation in brain, heart and lung. Cadmium-induced lipid peroxidation in kidney was prevented by melatonin when it was given as a single dose of 15 mg/kg bw. Melatonin slightly, but not significantly, reduced cadmium-induced lipid peroxidation in testes. It is concluded that cadmium toxicity, at least with regard to the resulting lipid peroxidation, is reduced by administering melatonin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structural studies on MtRecA-nucleotide complexes: insights into DNA and nucleotide binding and the structural signature of NTP recognition

RecA protein plays a crucial role in homologous recombination and repair of DNA. Central to all activities of RecA is its binding to Mg(+2)-ATP. The active form of the protein is a helical nucleoprotein filament containing the nucleotide cofactor and single-stranded DNA. The stability and structure of the helical nucleoprotein filament formed by RecA are modulated by nucleotide cofactors. Here we report crystal structures of a MtRecA-ADP complex, complexes with ATPgammaS in the presence and absence of magnesium as well as a complex with dATP and Mg+2. Comparison with the recently solved crystal structures of the apo form as well as a complex with ADP-AlF4 confirms an expansion of the P-loop region in MtRecA, compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP. The ligand bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve in maintaining the network of interactions crucial for nucleotide binding. The nucleotide binding site itself, however, remains relatively unchanged. The analysis also reveals that ATPgammaS rather than ADP-AlF4 is structurally a better mimic of ATP. From among the complexed structures, a definition for the two DNA-binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA. The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide binding pocket, at the molecular level. Modeling studies on the mutants again point to the relative rigidity of the nucleotide binding site. Comparison with other NTP binding proteins reveals many commonalties in modes of binding by diverse members in the structural family, contributing to our understanding of the structural signature of NTP recognition.     

Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes

The copper content of recombinant CotA laccase from Bacillus subtilis produced by Escherichia coli cells is shown to be strongly dependent on the presence of copper and oxygen in the culture media. In copper-supplemented media, a switch from aerobic to microaerobic conditions leads to the synthesis of a recombinant holoenzyme, while the maintenance of aerobic conditions results in the synthesis of a copper-depleted population of proteins. Strikingly, cells grown under microaerobic conditions accumulate up to 80-fold more copper than aerobically grown cells. In vitro copper incorporation into apoenzymes was monitored by optical and electron paramagnetic resonance (EPR) spectroscopy. This analysis reveals that copper incorporation into CotA laccase is a sequential process, with the type 1 copper center being the first to be reconstituted, followed by the type 2 and the type 3 copper centers. The copper reconstitution of holoCotA derivatives depleted in vitro with EDTA results in the complete recovery of the native conformation as monitored by spectroscopic, kinetic and thermal stability analysis. However, the reconstitution of copper to apo forms produced in cultures under aerobic and copper-deficient conditions resulted in incomplete recovery of biochemical properties of the holoenzyme. EPR and resonance Raman data indicate that, presumably, folding in the presence of copper is indispensable for the correct structure of the trinuclear copper-containing site.     

Functional impacts of non-synonymous single nucleotide polymorphisms: selective constraint and structural environments

In this work, we studied the correlations between selective constraint, structural environments and functional impacts of non-synonymous single nucleotide polymorphisms (nsSNPs). We found that the relation between solvent accessibility and functional impacts of nsSNPs is not as simple as generally thought. Finer structural classifications need to be taken into account to reveal the complex relations between the characteristics of a structure environment and its influence on the functional impacts of nsSNPs. We introduced two parameters for each structural environment, consensus residue percentage and residue distribution distance, to characterize the selective constraint imposed by the environment. Both parameters significantly correlate with the functional bias of nsSNPs across the structural environments. This result shows that selective constraint underlies the bias of a structural environment towards a certain type of nsSNPs (disease-associated or benign).     

Toward an individual-level understanding of vigilance: the role of social information

A gregarious lifestyle affords the benefit of collective detectionof predators through the many-eyes effect. Studies of vigilanceare generally concerned with exploring the relationship betweenvigilance rates and group size. However, a mechanistic understandingof the rules individual animals use to achieve this group-levelbehavior is lacking. Building on a previous modeling approach,we suggest that individuals reconcile their own private informationagainst the social information they receive from their groupmates in order to decide whether to feed or be vigilant at anyone time. We present a novel modeling approach utilizing a Markovchain Monte Carlo process to describe the transition betweenvigilant and nonvigilant states. Many of our assumptions arebased qualitatively on recently published experimental observations.We vary the amount of social information and the fidelity withwhich individuals process this information and show that thishas a profound effect on the individual vigilance rate, theindividual vigilant bout length, and the proportion of vigilantindividuals at any one time. A wide range of group-level vigilancepatterns can be obtained by varying simple behavioral characteristicsof individual animals. We find that generally, increasing theamount of, and sensitivity to, social information generatesa more cooperative vigilance behavior. This model potentiallyprovides a theoretical and conceptual framework for examiningspecific real-life systems. We propose analyzing individual-baseddata from real animals by considering their group to be a connectednetwork of individuals, with information transfer between them.     

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO₂ at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.     

Mesophile versus thermophile: insights into the structural mechanisms of kinetic stability

Obtaining detailed knowledge of folding intermediate and transition state (TS) structures is critical for understanding protein folding mechanisms. Comparisons between proteins adapted to survive extreme temperatures with their mesophilic homologs are likely to provide valuable information on the interactions relevant to the unfolding transition. For kinetically stable proteins such as alpha-lytic protease (alphaLP) and its family members, their large free energy barrier to unfolding is central to their biological function. To gain new insights into the mechanisms that underlie kinetic stability, we have determined the structure and high temperature unfolding kinetics of a thermophilic homolog, Thermobifida fusca protease A (TFPA). These studies led to the identification of a specific structural element bridging the N and C-terminal domains of the protease (the "domain bridge") proposed to be associated with the enhanced high temperature kinetic stability in TFPA. Mutagenesis experiments exchanging the TFPA domain bridge into alphaLP validate this hypothesis and illustrate key structural details that contribute to TFPA's increased kinetic thermostability. These results lead to an updated model for the unfolding transition state structure for this important class of proteases in which domain bridge undocking and unfolding occurs at or before the TS. The domain bridge appears to be a structural element that can modulate the degree of kinetic stability of the different members of this class of proteases.     

Prototropic tautomerism and basic molecular principles of hypoxanthine mutagenicity: an exhaustive quantum-chemical analysis

The molecular structures, relative stability order, and dipole moments of a complete family of 21 planar hypoxanthine (Hyp) prototropic molecular–zwitterionic tautomers including ylidic forms were computationally investigated at the MP2/6–311++G(2df,pd)//B3LYP/6–311++G(d,p) level of theory in vacuum and in three different surrounding environments: continuum with a low dielectric constant (??=?4) corresponding to a hydrophobic interface of protein–nucleic acid interactions, dimethylsulfoxide (DMSO), and water. The keto-N1HN7H tautomer was established to be the global minimum in vacuum and in continuum with ??=?4, while Hyp molecule exists as a mixture of the keto-N1HN9H and keto-N1HN7H tautomers in approximately equal amounts in DMSO and in water at T?=?298.15?K. We found out that neither intramolecular tautomerization by single proton transfer in the Hyp base, nor intermolecular tautomerization by double proton transfer in the most energetically favorable Hyp·Hyp homodimer (symmetry C _2h ), stabilized by two equivalent N1H…O6 H-bonds, induces the formation of the enol tautomer (marked with an asterisk) of Hyp with cis-oriented O6H hydroxyl group relative to neighboring N1C6 bond. We first discovered a new scenario of the keto–enol tautomerization of Hyp?·?Hyp homodimer (C _2h ) via zwitterionic near-orthogonal transition state (TS), stabilized by N1⁺H…N1^? and O6⁺H…N1^? H-bonds, to heterodimer Hyp^??·?Hyp (C _s ), stabilized by O6H…O6 and N1H…N1 H-bonds. We first showed that Hyp^??·?Thy mispair (C _s ), stabilized by O6H…O4, N3H…N1, and C2H…O2 H-bonds, mimicking Watson–Crick base pairing, converts to the wobble Hyp?·?Thy base pair (C _s ), stabilized by N3H…O6 and N1H…O2 H-bonds, via high- and low-energy TSs and intermediate Hyp?·?Thy^?, stabilized by O4H…O6, N1H…N3, and C2H…O2 H-bonds. The most energetically favorable TS is the zwitterionic pair Hyp⁺?·?Thy^? (C _s ), stabilized by O6⁺H…O4^?, O6⁺H…N3^?, N1⁺H…N3^?, and N1⁺H…O2^? H-bonds. The authors expressed and substantiated the hypothesis, that the keto tautomer of Hyp is a mutagenic compound, while enol tautomer Hyp^? does not possess mutagenic properties. The lifetime of the nonmutagenic tautomer Hyp^? exceeds by many orders the time needed to complete a round of DNA replication in the cell. For the first time purine–purine planar H-bonded mispairs containing Hyp in the anti-orientation with respect to the sugar moiety – Hyp?·?Ade _syn , Hyp?·?Gua^? _syn , and Hyp?·?Gua _syn , that closely resembles the geometry of the Watson–Crick base pairs, have been suggested as the source of transversions. An influence of the surrounding environment (??=?4) on the stability of studied complexes and corresponding TSs was estimated by means of the conductor-like polarizable continuum model. Electron-topological, structural, vibrational, and energetic characterictics of all conventional and nonconventional H-bonds in the investigated structures are presented. Presented data are key to understanding elementary molecular mechanisms of mutagenic action of Hyp as a product of the adenine deamination in DNA.     

Lactoperoxidase: structural insights into the function,ligand binding and inhibition

Lactoperoxidase (LPO) is a member of a large group of mammalian heme peroxidases that include myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The LPO is found in exocrine secretions including milk. It is responsible for the inactivation of a wide range of micro-organisms and hence, is an important component of defense mechanism in the body. With the help of hydrogen peroxide, it catalyzes the oxidation of halides, pseudohalides and organic aromatic molecules. Historically, LPO was isolated in 1943, nearly seventy years ago but its three-dimensional crystal structure has been elucidated only recently. This review provides various details of this protein from its discovery to understanding its structure, function and applications. In order to highlight species dependent variations in the structure and function of LPO, a detailed comparison of sequence, structure and function of LPO from various species have been made. The structural basis of ligand binding and distinctions in the modes of binding of substrates and inhibitors have been analyzed extensively.     

长期秸秆黑炭施加对石灰性潮土肥力、固碳及氨挥发的影响

通过5年10季的小麦/谷子轮作盆栽试验,持续观测了每季0、2.25和22.5 t·hm^-2黑炭施用下作物生长和土壤性质变化及氨挥发数量.结果表明:相同NPK肥施用下,与不施黑炭处理相比,黑炭处理能促进作物生长,提高土壤养分供应.22.5 t·hm^-2黑炭处理下5年作物累积籽粒和秸秆产量分别增加24.1%和74.1%;相应地上部N、P和K累计吸收量分别增加93.5%、71.2%和46.3%;轮作结束后土壤有效P、K含量及阳离子交换量分别提高262%、274%和58.3%,有机碳提高843%,C/N增至25,容重降低46.6%,土壤pH值则无明显变化.2.25和22.5 t·hm^-2黑炭处理的黑炭表观分解率为每年3.5%~5.7%.高量黑炭施用可导致氨挥发数量增加,22.5 t·hm^-2黑炭处理下10季氨挥发总量增加102%.     

Asymmetry in structural defenses: insights into selective predation in the wild

Assessment of geographical patterns in fluctuating asymmetry (small, random differences between sides of bilateral characters) among populations shows promise as a tool to resolve the relative biomechanical importance of traits, in addition to being a possible indicator of habitat quality. We used 115 endemic freshwater populations of threespine stickleback (Gasterosteus aculeatus) from Haida Gwaii (Queen Charlotte Islands), British Columbia, Canada, to explore the degree of concordance between geographical variation of asymmetry in a predator defense structure (bony lateral plates) and geographical variation in several indirect measures of predation regime as well as several abiotic habitat variables. We found a geographical cline in the population frequency of lateral plate asymmetries, with reduced asymmetry in the southern clear-water regions of the archipelago characterized by long reaction distances and greater chance of capture by predators, and elevated asymmetry in the northern stained-water regions with poor visibility and low chances of capture. Lateral plate asymmetry was strongly correlated with expression of several defensive armor traits, including total plate numbers among populations, mean cross-sectional diameter of stickleback with the dorsal and pelvic spines erect, and mean degree of overlap between the plates and spine supports. There were no significant correlations between frequency of asymmetric fish and any of our abiotic habitat variables. Stickleback with structural plate asymmetries had fewer trout-induced scars than symmetric fish in the significant majority of populations, and there was a decrease in structural plate asymmetry with age in stained-water habitats, suggesting that trout predators may be selectively removing asymmetric fish in some lakes. This study provides evidence that geographical variation in developmental stability of threespine stickleback, as seen in the frequencies of asymmetry, reflects differences among populations in the importance of structural defenses to fitness rather than differences in habitat quality, and that asymmetry may be a target of selection by predators in wild populations.     

Crystal structure of Dioclea rostrata lectin: insights into understanding the pH-dependent dimer-tetramer equilibrium and the structural basis for carbohydrate recognition in Diocleinae lectins

The legume lectins from the subtribe Diocleinae, often referred to as concanavalin A-like lectins, are a typical example of highly similar proteins that show distinct biological activities. The pH-dependent oligomerization that some of these lectins undergo and the relative position of amino acids within the carbohydrate-binding site are factors that have been reported to contribute to these differences in the activities of Diocleinae lectins. In the present work, we determined the amino acid sequence and the crystal structure of the lectin of Dioclea rostrata seeds (DRL), with the aim of investigating the structural bases of the different behavior displayed by this lectin in comparison to other Diocleinae lectins and determining the reason for the distinct pH-dependent dimer-tetramer equilibrium. In addition, we discovered a novel multimeric arrangement for this lectin.