Dimer-tetramer Equilibrium Research Articles

Structural studies of an anti-inflammatory lectin from Canavalia boliviana seeds in complex with dimannosides.

Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.

Crystal structure of Escherichia coli SsuE: defining a general catalytic cycle for FMN reductases of the flavodoxin-like superfamily.

The Escherichia coli sulfur starvation utilization (ssu) operon includes a two-component monooxygenase system consisting of a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase, SsuE, and a monooxygenase, SsuD. SsuE is part of the flavodoxin-like superfamily, and we report here the crystal structures of its apo, FMN-bound, and FMNH2-bound forms at ∼2 Å resolution. In the crystals, SsuE forms a tetramer that is a dimer of dimers similar to those seen for homologous FMN reductases, quinone reductases, and the WrbA family of enzymes. A π-helix present at the tetramer building interface is unique to the reductases from two-component monooxygenase systems. Analytical ultracentrifugation studies of SsuE confirm a dimer-tetramer equilibrium exists in solution, with FMN binding favoring the dimer. As the active site includes residues from both subunits, at least a dimeric association is required for the function of SsuE. The structures show that one FMN binds tightly in a deeply held site, which makes available a second binding site, in which either a second FMN or the nicotinamide of NADPH can bind. The FMNH2-bound structure shows subtle changes consistent with its binding being weaker than that of FMN. Combining this information with published kinetic studies, we propose a general catalytic cycle for two-component reductases of the flavodoxin-like superfamily, by which the enzyme can potentially provide FMNH2 to its partner monooxygenase by different routes depending on the FMN concentration and the presence of a partner monooxygenase.

From knock-out phenotype to three-dimensional structure of a promising antibiotic target from Streptococcus pneumoniae.

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k cat = 22 s-1, K M PYR = 2.55 ± 0.05 mM and K M ASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T M app) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T M app = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K D 4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (K D 4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2) compared to its E. coli counterpart (500 Å2). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.

Evidence for dimer/tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer–tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in ΔCp, with NADP binding ΔCp closer to what is expected of crystallographic structures, while NADPH ΔCp is three times larger. The estimated dimer–tetramer association constant is 1.5·106M−1, and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.

Structure, Activity, and Substrate Selectivity of the Orf6 Thioesterase from Photobacterium profundum

Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

Characterization of the Unfolding Process of the Tetrameric and Dimeric Forms of Cratylia mollis Seed Lectin (CRAMOLL 1): Effects of Natural Fragmentation on Protein Stability

pCRAMOLL 1 is a major, non-glycosylated isolectin found in seeds of Cratylia mollis, which belongs to the Leguminosae family and the Diocleinae subtribe. The lectin (~25 kDa) consists of 236 amino acids, sharing 82% identity and virtually identical topological architecture with concanavalin A. Both lectins also share the same pH-dependent dimer-tetramer equilibrium and the ability to recognize Glc/Man moieties. Intricate post-translational events occurring in Diocleinae seed cotyledons result in a mixture of intact and fragmented monomers within the oligomeric assemblies of pCRAMOLL 1. In an earlier report, we demonstrated the production, purification, and characterization of the bacterially expressed form of CRAMOLL 1 (rCRAMOLL 1). The recombinant lectin retained sugar-binding activity and several other biophysical properties of pCRAMOLL 1, but its tetramers, which are composed of intact monomers only, show little enhancement in stability when probed with acidification, high temperatures, or hydrostatic pressure. Here we examined the urea-induced unfolding of the nonfragmented tetramers and dimers of rCRAMOLL 1 and compared this behavior with that of the mixed plant lectin counterparts. Using fluorescence, circular dichroism, size-exclusion chromatography, and chemical cross-linking experiments, we posited that the absence of fragmentation lent greater firmness to tetramers, but not to dimers. Dimeric and tetrameric pCRAMOLL 1 unfolded via a compact monomeric intermediate. In contrast, dimers of rCRAMOLL 1 behaved similarly to the plant dimer counterpart, but its tetrameric form remarkably showed no evidence of such partially unfolded monomers. By analyzing the crystal structure of pCRAMOLL 1, we were able to dissect the importance of the fragmentation to lectin stability.

Functional and Metabolic Effects of Adaptive Glycerol Kinase (GLPK) Mutants in Escherichia coli

Herein we measure the effect of four adaptive non-synonymous mutations to the glycerol kinase (glpK) gene on catalytic function and regulation, to identify changes that correlate to increased fitness in glycerol media. The mutations significantly reduce affinity for the allosteric inhibitor fructose-1,6-bisphosphate (FBP) and formation of the tetramer, which are structurally related, in a manner that correlates inversely with imparted fitness during growth on glycerol, which strongly suggests that these enzymatic parameters drive growth improvement. Counterintuitively, the glpK mutations also increase glycerol-induced auto-catabolite repression that reduces glpK transcription in a manner that correlates to fitness. This suggests that increased specific GlpK activity is attenuated by negative feedback on glpK expression via catabolite repression, possibly to prevent methylglyoxal toxicity. We additionally report that glpK mutations were fixed in 47 of 50 independent glycerol-adapted lineages. By far the most frequently mutated locus (nucleotide 218) was mutated in 20 lineages, strongly suggesting this position has an elevated mutation rate. This study demonstrates that fitness correlations can be used to interrogate adaptive processes at the protein level and to identify the regulatory constraints underlying selection and improved growth.

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli

The ternary complex comprising MutS, MutL, and DNA is a key intermediate in DNA mismatch repair. We used chemical cross-linking and fluorescence resonance energy transfer (FRET) to study the interaction between MutS and MutL and to shed light onto the structure of this complex. Via chemical cross-linking, we could stabilize this dynamic complex and identify the structural features of key events in DNA mismatch repair. We could show that in the complex between MutS and MutL the mismatch-binding and connector domains of MutS are in proximity to the N-terminal ATPase domain of MutL. The DNA- and nucleotide-dependent complex formation could be monitored by FRET using single cysteine variants labeled in the connector domain of MutS and the transducer domain of MutL, respectively. In addition, we could trap MutS after an ATP-induced conformational change by an intramolecular cross-link between Cys-93 of the mismatch-binding domain and Cys-239 of the connector domain.

Dimer–tetramer association equilibria of human adult hemoglobin and its mutants as observed by analytical ultracentrifugation

Dimer–tetramer equilibrium of human adult hemoglobin in CO form (COHb A) and its mutants were measured by sedimentation velocity and sedimentation equilibrium. In sedimentation velocity, the association constants were estimated by measuring the concentration dependence of the weight average sedimentation coefficients at pH 6 and 7 and fitting the data to the theoretical binding isotherms with association constants as a parameter. Association constants of wild type Hb A and three mutant Hbs, Hb Hirose(βW37S), recombinant (r)Hb(βW37H) and rHb(αY42S), in which an amino acid was replaced at the α 1β 2 interface, were measured in the presence and absence of inositol hexaphosphate (IHP). All the three mutations lowered the value of association constants, but the presence of IHP shifted the equilibrium toward tetramer. Although the association constant between dimer and tetramer of rHb(βW37H) and rHb(αY42S) were similar, sedimentation coefficient distribution function, c( s), analysis indicated that the association and dissociation rate constants of the former is higher than the latter.

A Fused α-β “Mini-spectrin” Mimics the Intact Erythrocyte Spectrin Head-to-head Tetramer

Head-to-head assembly of two spectrin heterodimers to form an actin-cross-linking tetramer is a physiologically dynamic interaction that contributes to red cell membrane integrity. Recombinant beta-spectrin C-terminal and alpha-spectrin N-terminal peptides can form tetramer-like univalent complexes, but they cannot evaluate effects of the open-closed dimer interactions or lateral associations of the two-spectrin strands on tetramer formation. In this study we produced and characterized a fused "mini-spectrin dimer" containing the beta-spectrin C-terminal region linked to the alpha-spectrin N-terminal region. This fused mini-spectrin mimics structural and functional properties of intact, full-length dimers and tetramers, including lateral association of the alpha and beta subunits in the dimer and formation of a closed dimer. High performance liquid chromatography gel filtration analyses of this mini-spectrin provide the first direct non-imaging experimental evidence for open and closed spectrin dimers and show that dimer-tetramer-oligomer interconversion is slow at low temperatures and accelerated at 30 degrees C, analogous to full-length spectrin. This protein exhibits wild type dimer-tetramer dissociation constants of approximately 1 mum at 30 degrees C, independent of initial oligomeric state. Conformational states of the mini-spectrin dimer were probed further using chemical cross-linking, which identified distinct groups of cross-links for "open" and "closed" dimers and confirmed the N-terminal region of alpha-spectrin remains highly flexible in the complex, exhibiting closely analogous structures to those observed for the isolated alpha-spectrin N-terminal using NMR (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) J. Biol. Chem. 278, 21837-21844). This fusion protein should serve as a useful template for structural and functional studies of the divalent tetramer site.

A single intersubunit salt bridge affects oligomerization and catalytic activity in a bacterial quinone reductase

YhdA, a thermostable NADPH:FMN oxidoreductase from Bacillus subtilis, reduces quinones via a ping-pong bi-bi mechanism with a pronounced preference for NADPH. The enzyme occurs as a stable tetramer in solution. The two extended dimer surfaces are packed against each other by a 90 rotation of one dimer with respect to the other. This assembly is stabilized by the formation of four salt bridges between K109 and D137 of the neighbouring protomers. To investigate the importance of the ion pair contacts, the K109L and D137L single replacement variants, as well as the K109L/D137L and K109D/D137K double replacement variants, were generated, expressed, purified, crystallized and biochemically characterized. The K109L and D137L variants form dimers instead of tetramers, whereas the K109L/D137L and K109D/D137K variants appear to exist in a dimer-tetramer equilibrium in solution. The crystal structures of the K109L and D137L variants confirm the dimeric state, with the K109L/D137L and K109D/D137K variants adopting a tetrameric assembly. Interestingly, all protein variants show a drastically reduced quinone reductase activity in steady-state kinetics. Detailed analysis of the two half reactions revealed that the oxidative half reaction is not affected, whereas reduction of the bound FMN cofactor by NADPH is virtually abolished. Inspection of the crystal structures indicates that the side chain of K109 plays a dual role by forming a salt bridge to D137, as well as stabilizing a glycine-rich loop in the vicinity of the FMN cofactor. In all protein variants, this glycine-rich loop exhibits a much higher mobility, compared to the wild-type. This appears to be incompatible with NADPH binding and thus leads to abrogation of flavin reduction.

Gas Phase and Solution Structures of 1-Methoxyallenyllithium

A combined computational and (13)C NMR study was used to determine the solution structures of 1-methoxyallenyllithium. The gas phase calculations indicated that this species is aggregated as a hexamer. The NMR spectra in THF solution, together with the calculated aggregation energies and chemical shifts, are consistent with a dimer-tetramer equilibrium.

Functional Roles of the Tetramer Organization of Malic Enzyme

Malic enzyme has a dimer of dimers quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In addition, the enzyme has distinct active sites within each subunit. The mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD(P)-ME) isoform behaves cooperatively and allosterically and exhibits a quaternary structure in dimer-tetramer equilibrium. The cytosolic NADP(+)-dependent malic enzyme (c-NADP-ME) isoform is noncooperative and nonallosteric and exists as a stable tetramer. In this study, we analyze the essential factors governing the quaternary structure stability for human c-NADP-ME and m-NAD(P)-ME. Site-directed mutagenesis at the dimer and tetramer interfaces was employed to generate a series of dimers of c-NADP-ME and m-NAD(P)-ME. Size distribution analysis demonstrated that human c-NADP-ME exists mainly as a tetramer, whereas human m-NAD(P)-ME exists as a mixture of dimers and tetramers. Kinetic data indicated that the enzyme activity of c-NADP-ME is not affected by disruption of the interface. There are no significant differences in the kinetic properties between AB and AD dimers, and the dimeric form of c-NADP-ME is as active as tetramers. In contrast, disrupting the interface of m-NAD(P)-ME causes the enzyme to be less active than wild type and to become less cooperative for malate binding; the k(cat) values of mutants decreased with increasing K(d,24) values, indicating that the dissociation of subunits at the dimer or tetramer interfaces significantly affects the enzyme activity. The above results suggest that the tetramer is required for a fully functional m-NAD(P)-ME. Taken together, the analytical ultracentrifugation data and the kinetic analysis of these interface mutants demonstrate the differential role of tetramer organization for the c-NADP-ME and m-NAD(P)-ME isoforms. The regulatory mechanism of m-NAD(P)-ME is closely related to the tetramer formation of this isoform.

The three-dimensional structure of diaminopimelate decarboxylase from Mycobacterium tuberculosis reveals a tetrameric enzyme organisation

The three-dimensional structure of the enzyme diaminopimelate decarboxylase from Mycobacterium tuberculosis has been determined in a new crystal form and refined to a resolution of 2.33 A. The monoclinic crystals contain one tetramer exhibiting D(2)-symmetry in the asymmetric unit. The tetramer exhibits a donut-like structure with a hollow interior. All four active sites are accessible only from the interior of the tetrameric assembly. Small-angle X-ray scattering indicates that in solution the predominant oligomeric species of the protein is a dimer, but also that higher oligomers exist at higher protein concentrations. The observed scattering data are best explained by assuming a dimer-tetramer equilibrium with about 7% tetramers present in solution. Consequently, at the elevated protein concentrations in the crowded environment inside the cell the observed tetramer may constitute the biologically relevant functional unit of the enzyme.

Functional role of fumarate site Glu59 involved in allosteric regulation and subunit–subunit interaction of human mitochondrial NAD(P)+‐dependent malic enzyme

Here we report on the role of Glu59 in the fumarate-mediated allosteric regulation of the human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME). In the present study, Glu59 was substituted by Asp, Gln or Leu. Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme. The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation constants, K(A,Fum) and alphaK(A,Fum). The E59D enzyme, although retaining the allosteric property, is quite different from the wild-type in enzyme activation. The K(A,Fum) and alphaK(A,Fum) of E59D are also much greater than those of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding. Analytical ultracentrifugation analysis shows that both the wild-type and E59Q enzymes exist as a dimer-tetramer equilibrium. In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface is changed by shortening one carbon side chain of Glu59 to Asp59. The E59L enzyme also shows a dimer-tetramer model similar to that of the wild-type, but it displays more dimers as well as monomers and polymers. Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecular geometry of the fumarate-binding site. Glu59 can precisely maintain the geometric specificity for the substrate cooperativity. According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation. Glu59 discriminates human m-NAD-ME from mitochondrial NADP+-dependent malic enzyme and cytosolic NADP+-dependent malic enzyme in fumarate activation and malate cooperativity.

3P-079 化学修飾ヘモグロビン酸素結合機能の蛋白濃度依存性(ヘム蛋白質,第47回日本生物物理学会年会)

3P-079 化学修飾ヘモグロビン酸素結合機能の蛋白濃度依存性(ヘム蛋白質,第47回日本生物物理学会年会)

Association Properties of βB1- and βA3-Crystallins: Ability To Form Heterotetramers

As major constituents of the mammalian lens, beta-crystallins associate into dimers, tetramers, and higher-order complexes to maintain lens transparency and refractivity. A previous study has shown that dimerization of betaB2- and betaA3-crystallins is energetically highly favored and entropically driven. While heterodimers further associate into higher-order complexes in vivo, a significant level of reversibly associated tetrameric crystallin has not been previously observed in vitro. To enhance our understanding of the interactions between beta-crystallins, we characterized the association of betaB1-crystallin, a major component of large beta-crystallin complexes (beta-high), with itself and with betaA3-crystallin. Mouse betaB1-crystallin and human betaA3-crystallin were expressed in Escherichia coli and purified chromatographically. Their association was then characterized using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and analytical sedimentation equilibrium centrifugation. When present alone, each beta-crystallin associates into homodimers; however, no tetramer formation is seen. Once mixing has taken place, formation of a heterocomplex between betaB1- and betaA3-crystallins is observed using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to results previously obtained after betaB2- and betaA3-crystallins had been mixed, mixed betaB1- and betaA3-crystallins show a dimer-tetramer equilibrium with a K d of 1.1 muM, indicating that these two beta-crystallins associate predominantly into heterotetramers in vitro. Thus, while each purified beta-crystallin associates only into homodimers and under the conditions studied mixed betaB2- and betaA3-crystallins form a mixture of homo- and heterodimers, mixed betaB1- and betaA3-crystallins associate predominantly into heterotetramers in equilibrium with heterodimers. These findings suggest a unique role for betaB1-crystallin in promoting higher-order crystallin association in the lens.

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.

Detection of Intermediates and Kinetic Control during Assembly of Bacteriophage P22 Procapsid

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter ≈ 50 nm). In this work, we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer–dimer–tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer–dimer equilibrium (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering, which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild-type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly.

Insights into the structural basis of the pH-dependent dimer–tetramer equilibrium through crystallographic analysis of recombinantDiocleinaelectins

The structural ground underlying the pH-dependency of the dimer-tetramer transition of Diocleinae lectins was investigated by equilibrium sedimentation and X-ray crystal structure determination of wild-type and site-directed mutants of recombinant lectins. Synthetic genes coding for the full-length alpha-chains of the seed lectins of Dioclea guianensis (termed r-alphaDguia) and Dioclea grandiflora (termed r-alphaDGL) were designed and expressed in Escherichia coli. This pioneering approach, which will be described in detail in the present paper, yielded recombinant lectins displaying carbohydrate-binding activity, dimer-tetramer equilibria and crystal structures indistinguishable from their natural homologues. Conversion of the pH-stable tetrameric r-alphaDGL into a structure exhibiting pH-dependent dimer-tetramer transition was accomplished through mutations that abolished the interdimeric interactions at the central cavity of the tetrameric lectins. Both the central and the peripheral interacting regions bear structural information for formation of the canonical legume lectin tetramer. We hypothesize that the strength of the ionic contacts at these sites may be modulated by the pH, leading to dissociation of those lectin structures that are not locked into a pH-stable tetramer through interdimeric contacts networking the central cavity loops.