On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Intrinsic pKas of ionizable residues in proteins: An explicit solvent calculation for lysozyme

Molecular dynamics simulations of triclinic hen egg white lysozyme in aqueous solution were performed to calculate the intrinsic pK_as of 14 ionizable residues. An all-atom model was used for both solvent and solute, and a single 180 ps simulation in conjunction with a Gaussian fluctuation analysis method was used. An advantage of the Gaussian fluctuation method is that it only requires a single simulation of the system in a reference state to calculate all the pK_as in the protein, in contrast to multiple simulations for the free energy perturbation method. pK_int shifts with respect to reference titratable residues were evaluated and compared to results obtained using a finite difference Poisson-Boltzmann (FDPB) method with a continuum solvent model; overall agreement with the direction of the shifts was generally observed, though the magnitude of the shifts was typically larger with the explicit solvent model. The contribution of the first solvation shell to the total charging free energies of the titratable groups was explicitly evaluated and found to be significant. Dielectric shielding between pairs of titratable groups was examined and found to be smaller than expected. The effect of the approximations used to treat the long-range interactions on the pK_int shifts is discussed. © 1994 Wiley-Liss, Inc.     

On the calculation of binding free energies using continuum methods: Application to MHC class I protein-peptide interactions

This paper describes a methodology to calculate the binding free energy (ΔG) of a protein-ligand complex using a continuum model of the solvent. A formal thermodynamic cycle is used to decompose the binding free energy into electrostatic and non-electrostatic contributions. In this cycle, the reactants are discharged in water, associated as purely nonpolar entities, and the final complex is then recharged. The total electrostatic free energies of the protein, the ligand, and the complex in water are calculated with the finite difference Poisson-Boltzmann (FDPB) method. The nonpolar (hydrophobic) binding free energy is calculated using a free energy-surface area relationship, with a single alkane/water surface tension coefficient (γ_aw). The loss in backbone and side-chain configurational entropy upon binding is estimated and added to the electrostatic and the nonpolar components of ΔG. The methodology is applied to the binding of the murine MHC class I protein H-2K^b with three distinct peptides, and to the human MHC class I protein HLA-A2 in complex with five different peptides. Despite significant differences in the amino acid sequences of the different peptides, the experimental binding free energy differences (ΔΔG_exp) are quite small (<0.3 and <2.7 kcal/mol for the H-2K^b and HLA-A2 complexes, respectively). For each protein, the calculations are successful in reproducing a fairly small range of values for ΔΔG_calc (<4.4 and <5.2 kcal/mol, respectively) although the relative peptide binding affinities of H-2K^b and HLA-A2 are not reproduced. For all protein-peptide complexes that were treated, it was found that electrostatic interactions oppose binding whereas nonpolar interactions drive complex formation. The two types of interactions appear to be correlated in that larger nonpolar contributions to binding are generally opposed by increased electrostatic contributions favoring dissociation. The factors that drive the binding of peptides to MHC proteins are discussed in light of our results.     

Concordance of residual dipolar couplings, backbone order parameters and crystallographic B-factors for a small alpha/beta protein: a unified picture of high probability, fast atomic motions in proteins

Using ensemble refinement of the third immunoglobulin binding domain (GB3) of streptococcal protein G (a small alpha/beta protein of 56 residues), we demonstrate that backbone (N-H, N-C', Calpha-Halpha, Calpha-C') residual dipolar coupling data in five independent alignment media, generalized order parameters from 15N relaxation data, and B-factors from a high-resolution (1.1A), room temperature crystal structure are entirely consistent with one another within experimental error. The optimal ensemble size representation is between four and eight, as assessed by complete cross-validation of the residual dipolar couplings. Thus, in the case of GB3, all three observables reflect the same low-amplitude anisotropic motions arising from fluctuations in backbone phi/psi torsion angles in the picosecond to nanosecond regime in both solution and crystalline environments, yielding a unified picture of fast, high-probability atomic motions in proteins. An understanding of these motions is crucial for understanding the impact of protein dynamics on protein function, since they provide part of the driving force for triggered conformational changes that occur, for example, upon ligand binding, signal transduction and enzyme catalysis.     

All-atom molecular dynamics simulations of an artificial sodium channel in a lipid bilayer: the effect of water solvation/desolvation of the sodium ion

All-atom molecular dynamics is used to investigate the transport of Na⁺ across a 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayer facilitated by a diazacrown hydraphile. Specifically, the free energy of Na⁺ passing through the bilayer is calculated using the adaptive biasing force method to study the free energy associated with the increase in Na⁺ transport in the presence of the hydraphile molecule. The results show that water interaction greatly influences Na⁺ transport through the lipid bilayer as water is pulled through the bilayer with Na⁺ forming a water channel. The hydraphile causes a reduction in the free energy barrier for the transport of Na⁺ through the head group part of the lipid bilayer since it complexes the Na⁺ reducing the necessity for water to be complexed and, therefore, dragged through with Na⁺, an energetically unfavorable process. The free energy associated with Na⁺ being desolvated within the bilayer is significantly decreased in the presence of the hydraphile molecule; the hydraphile increases the number of solvation states of Na⁺ that can be adopted, and this increase in the number of available configurations provides an entropic explanation for the success of the hydraphile.     

Energy functions for protein design I: efficient and accurate continuum electrostatics and solvation

Electrostatics and solvation energies are important for defining protein stability, structural specificity, and molecular recognition. Because these energies are difficult to compute quickly and accurately, they are often ignored or modeled very crudely in computational protein design. To address this problem, we have developed a simple, fast, and accurate approximation for calculating Born radii in the context of protein design calculations. When these approximate Born radii are used with the generalized Born continuum dielectric model, energies calculated by the 10(6)-fold slower finite difference Poisson-Boltzmann model are faithfully reproduced. A similar approach can be used for estimating solvent-accessible surface areas (SASAs). As an independent test, we show that these approximations can be used to accurately predict the experimentally determined pK(a)s of >200 ionizable groups from 15 proteins.     

Free energy determinants of tertiary structure and the evaluation of protein models

We develop a protocol for estimating the free energy difference between different conformations of the same polypeptide chain. The conformational free energy evaluation combines the CHARMM force field with a continuum treatment of the solvent. In almost all cases studied, experimentally determined structures are predicted to be more stable than misfolded "decoys." This is due in part to the fact that the Coulomb energy of the native protein is consistently lower than that of the decoys. The solvation free energy generally favors the decoys, although the total electrostatic free energy (sum of Coulomb and solvation terms) favors the native structure. The behavior of the solvation free energy is somewhat counterintuitive and, surprisingly, is not correlated with differences in the burial of polar area between native structures and decoys. Rather. the effect is due to a more favorable charge distribution in the native protein, which, as is discussed, will tend to decrease its interaction with the solvent. Our results thus suggest, in keeping with a number of recent studies, that electrostatic interactions may play an important role in determining the native topology of a folded protein. On this basis, a simplified scoring function is derived that combines a Coulomb term with a hydrophobic contact term. This function performs as well as the more complete free energy evaluation in distinguishing the native structure from misfolded decoys. Its computational efficiency suggests that it can be used in protein structure prediction applications, and that it provides a physically well-defined alternative to statistically derived scoring functions.     

Distance dependence and salt sensitivity of pairwise, coulombic interactions in a protein

Histidine pK(a) values were measured in charge-reversal (K78E, K97E, K127E, and K97E/K127E) and charge-neutralization (E10A, E101A, and R35A) mutants of staphylococcal nuclease (SNase) by (1)H-NMR spectroscopy. Energies of interaction between pairs of charges (DeltaG(ij)) were obtained from the shifts in pK(a) values relative to wild-type values. The data describe the distance dependence and salt sensitivity of pairwise coulombic interactions. Calculations with a continuum electrostatics method captured the experimental DeltaG(ij) when static structures were used and when the protein interior was treated empirically with a dielectric constant of 20. The DeltaG(ij) when r(ij) < or = 10 A were exaggerated slightly in the calculations. Coulomb's law with a dielectric constant near 80 and a Debye-Hückel term to account for screening by the ionic strength reproduced the salt sensitivity and distance dependence of DeltaG(ij) as well as the structure-based method. In their interactions with each other, surface charges behave as if immersed in water; the Debye length describes realistically the distance where interactions become negligible at a given ionic strength. On average, charges separated by distances (r(ij)) approximately 5 A interacted with DeltaG(ij) approximately 0.6 kcal/mole in 0.01 M KCl, but DeltaG(ij) decayed to < or =0.10 kcal/mole when r(ij) = 20 A. In 0.10 M KCl, DeltaG(ij) approximately 0.10 kcal/mole when r(ij) = 10 A. In 1.5 M KCl, only short-range interactions with r(ij) < or = 5 A persisted. Although at physiological ionic strengths the interactions between charges separated by more than 10 A are extremely weak, in situations where charge imbalance exists many weak interactions can cumulatively produce substantial effects.     

Genetic associations of body composition,flexibility and injury risk with ACE,ACTN3 and COL5A1 polymorphisms in Korean ballerinas

[Purpose]

The purpose of this study was to exam the association of body composition, flexibility, and injury risk to genetic polymorphisms including ACE ID, ACTN3 RX, and COL5A1 polymorphisms in ballet dancers in Korea.

[Methods]

For the purpose of this study, elite ballerinas (n = 97) and normal female adults (n = 203) aged 18 to 39 were recruited and these participants were tested for body weight, height, body fat, fat free mass, flexibility, injury risks on the joints and gene polymorphisms (ACE, ACTN3, COL5A1 polymorphism).

[Results]

As results, the ACE DD genotype in ballerinas was associated with higher body fat and percentage of body fat than the ACE II and ID genotypes (p < 0.05). In the study on the ACTN3 polymorphism and ballerinas, the XX genotype in ballerinas had lower body weight and lower fat-free mass than the RR and RX genotype (p < 0.005). Also, the means of sit and reach test for flexibility was lower in the ACTN3 XX genotype of ballerinas than the RR and RX genotype of ballerinas (p < 0.05). Among the sports injuries, the ankle injury of the XX-genotyped ballerinas was in significantly more prevalence than the RR and XX-genotyped ballerinas (p < 0.05). According to the odd ratio analysis, XX-genotyped ballerinas have the injury risk on the ankle about 4.7 (95% CI: 1.6~13.4, p < 0.05) times more than the RR and RX-genotyped ballerinas. Meanwhile, the COL5A1 polymorphism in ballerinas has no association with any factors including flexibility and injury risks.

[Conclusion]

In conclusion, ACE polymorphism and ACTN3 polymorphism were associated with ballerinas'' performance capacity; COL5A1 was not associated with any factors of performance of Ballerinas. The results suggested that the ACE DD genotype is associated with high body fat, the ACTN3 XX genotype is associated with low fat-free mass, low flexibility, and higher risk of ankle-joint injury.     

Crystal structure of the stationary phase survival protein SurE with metal ion and AMP

The stationary phase survival protein SurE is a metal ion-dependent phosphatase distributed among eubacteria, archaea, and eukaryotes. In Escherichia coli, SurE has activities as nucleotidase and exopolyphosphatase, and is thought to be involved in stress response. However, its physiological role and reaction mechanism are unclear. We report here the crystal structures of the tetramer of SurE from Thermus thermophilus HB8 (TtSurE) both alone and crystallized with Mn(2+) and substrate AMP. In the presence of Mn(2+) and AMP, differences between the protomers were observed in the active site and in the loop located near the active site; AMP-bound active sites with the loops in a novel open conformation were found in the two protomers, and AMP-free active sites with the loops in a conventional closed conformation were found in the other two protomers. The two loops in the open conformation are entwined with each other, and this entwining is suggested to be required for enzymatic activity by site-directed mutagenesis. TtSurE exists as an equilibrium mixture of dimer and tetramer in solution. The loop-entwined structure indicates that SurE acts as a tetramer. The structural features and the absence of negative cooperativity imply the half-of-the-sites reactivity mechanism resulting from a pre-existing tendency toward structural asymmetry.     

A CFF 91-based Continuum Solvation Model: Solvation Free Energies of Small Organic Molecules and Conformations of the Alanine Dipeptide in Solution

Abstract

For molecular mechanics simulations of solvated molecules, it is important to use a consistent approach for calculating both the force field energy and the solvation free energy. A continuum solvation model based upon the atomic charges provided with the CFF91 force field is derived. The electrostatic component of the solvation free energy is described by the Poisson-Bolzmann equation while the nonpolar comonent of the solvation energy is assumed to be proportional to the solvent accessible surface area of the solute. Solute atomic radii used to describe the interface between the solute and solvent are fitted to reproduce the energies of small organic molecules. Data for 140 compounds are presented and compared to experiment and to the results from the well-characterized quantum mechanical solvation model AM1-SM2. In particular, accurate results are obtained for amino acid neutral analogues (mean unsigned error of 0.3 kcal/mol). The conformational energetics of the solvated alanine dipeptide is discussed.     

Penconazole induced changes in photosynthesis, ion acquisition and protein profile of Mentha pulegium L. under drought stress

Effect of penconazole (PEN) treatment on drought-stressed Mentha pulegium L. plants was investigated. Six weeks after sowing, seedlings were grown under soil moisture corresponding to 100, 75, 50 and 25 % field capacity (FC) with or without PEN (15 mg l⁻¹) for 4 weeks. Results showed that the seedlings at 75 % FC showed maximum growth and water supply lower than 75 % FC was the threshold of drought-initiated negative effects on seedling growth. Drought stress significantly induced proline and carbohydrate contents and the decreased chlorophyll, photosynthesis parameters, soluble proteins and ion accumulations. Exogenous PEN increased the growth parameters, pigments, photosynthesis and ion accumulations in drought stressed and unstressed plants, but the effects of PEN were more significant under water deficit conditions. PEN also reduced the negative effects of drought by osmotic balance and protein accumulations. Electrophoretic patterns indicated that PEN treatment increased the intensity of some protein bands with the molecular weights of 30 kDa in shoot and 31 kDa in roots, and several new protein bands with the molecular masses between 116 and 14 kDa appeared in leaves, shoots and roots. These results suggest that the PEN application can be a useful tool in alleviation of effects of drought stress in M. pulegium plants.     

Insilico analysis and molecular docking of resuscitation promoting factor B (RpfB) protein of Mycobacterium tuberculosis

Invulnerability of Mycobacterium tuberculosis to various drugs and its persistency has stood as a hurdle in the race against eradication of the pathogenecity of the bacteria. Identification of novel antituberculosis compounds is highly demanding as the available drugs are resistant. The ability of the bacteria to surpass the body''s defenses and adapt itself to survive for disease reactivation is contributed by secreted proteins called resuscitating promoting factors (Rpfs). These factors aid in virulence and resuscitation from dormancy of the bacteria. Sequence analysis of RpfB was performed and compounds were first screened for toxicity and high-throughput virtual screening eliminating the toxic compounds. To understand the mechanism of ligand binding and interaction, molecular docking was performed for the compounds passing through the filter resulting with better docking studies predicting the possible binding mode of the inhibitors to the protein. Of all the active residues the binding conformation shows that residues Arg194, Arg196, Glu242, and Asn244 of the RpfB protein play vital role in the enzyme activity and interacts with the ligands. Promising compounds have been identified in the current study, thus holding promise for design of antituberculosis drugs.     

Influence of calcium ion on host cell invasion and intracellular replication by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of Ca2+ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and Ca2+ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the Ca2+ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular Ca2+ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.     

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function.Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 Å lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.     

Spontaneous formation of IpaB ion channels in host cell membranes reveals how Shigella induces pyroptosis in macrophages

The Gram-negative bacterium Shigella flexneri invades the colonic epithelium and causes bacillary dysentery. S. flexneri requires the virulence factor invasion plasmid antigen B (IpaB) to invade host cells, escape from the phagosome and induce macrophage cell death. The mechanism by which IpaB functions remains unclear. Here, we show that purified IpaB spontaneously oligomerizes and inserts into the plasma membrane of target cells forming cation selective ion channels. After internalization, IpaB channels permit potassium influx within endolysosomal compartments inducing vacuolar destabilization. Endolysosomal leakage is followed by an ICE protease-activating factor-dependent activation of Caspase-1 in macrophages and cell death. Our results provide a mechanism for how the effector protein IpaB with its ion channel activity causes phagosomal destabilization and induces macrophage death. These data may explain how S. flexneri uses secreted IpaB to escape phagosome and kill the host cells during infection and, may be extended to homologs from other medically important enteropathogenic bacteria.     

The interplay of stress and mowing disturbance for the intensity and importance of plant interactions in dry calcareous grasslands

Background and Aims There is still debate regarding the direction and strength of plant interactions under intermediate to high levels of stress. Furthermore, little is known on how disturbance may interact with physical stress in unproductive environments, although recent theory and models have shown that this interplay may induce a collapse of plant interactions and diversity. The few studies assessing such questions have considered the intensity of biotic interactions but not their importance, although this latter concept has been shown to be very useful for understanding the role of interactions in plant communities. The objective of this study was to assess the interplay between stress and disturbance for plant interactions in dry calcareous grasslands. Methods A field experiment was set up in the Dordogne, southern France, where the importance and intensity of biotic interactions undergone by four species were measured along a water stress gradient, and with and without mowing disturbance. Key Results The importance and intensity of interactions varied in a very similar way along treatments. Under undisturbed conditions, plant interactions switched from competition to neutral with increasing water stress for three of the four species, whereas the fourth species was not subject to any significant biotic interaction along the gradient. Responses to disturbance were more species-specific; for two species, competition disappeared with mowing in the wettest conditions, whereas for the two other species, competition switched to facilitation with mowing. Finally, there were no significant interactions for any species in the disturbed and driest conditions. Conclusions At very high levels of stress, plant performances become too weak to allow either competition or facilitation and disturbance may accelerate the collapse of interactions in dry conditions. The results suggest that the importance and direction of interactions are more likely to be positively related in stressful environments.     

Energetics of protein folding

The energetics of protein folding determine the 3D structure of a folded protein. Knowledge of the energetics is needed to predict the 3D structure from the amino acid sequence or to modify the structure by protein engineering. Recent developments are discussed: major factors are reviewed and auxiliary factors are discussed briefly. Major factors include the hydrophobic factor (burial of non-polar surface area) and van der Waals interactions together with peptide hydrogen bonds and peptide solvation. The long-standing model for the hydrophobic factor (free energy change proportional to buried non-polar surface area) is contrasted with the packing-desolvation model and the approximate nature of the proportionality between free energy and apolar surface area is discussed. Recent energetic studies of forming peptide hydrogen bonds (gas phase) are reviewed together with studies of peptide solvation in solution. Closer agreement is achieved between the 1995 values for protein unfolding enthalpies in vacuum given by Lazaridis-Archontis-Karplus and Makhatadze-Privalov when the solvation enthalpy of the peptide group is taken from electrostatic calculations. Auxiliary factors in folding energetics include salt bridges and side-chain hydrogen bonds, disulfide bridges, and propensities to form alpha-helices and beta-structure. Backbone conformational entropy is a major energetic factor which is discussed only briefly for lack of knowledge.     

Large variation among photoreceptors as the basis of visual flexibility in the common backswimmer

The common backswimmer, Notonecta glauca, uses vision by day and night for functions such as underwater prey animal capture and flight in search of new habitats. Although previous studies have identified some of the physiological mechanisms facilitating such flexibility in the animal''s vision, neither the biophysics of Notonecta photoreceptors nor possible cellular adaptations are known. Here, we studied Notonecta photoreceptors using patch-clamp and intracellular recording methods. Photoreceptor size (approximated by capacitance) was positively correlated with absolute sensitivity and acceptance angles. Information rate measurements indicated that large and more sensitive photoreceptors performed better than small ones. Our results suggest that backswimmers are adapted for vision in both dim and well-illuminated environments by having open-rhabdom eyes with large intrinsic variation in absolute sensitivity among photoreceptors, exceeding those found in purely diurnal or nocturnal species. Both electrophysiology and microscopic analysis of retinal structure suggest two retinal subsystems: the largest peripheral photoreceptors provide vision in dim light and the smaller peripheral and central photoreceptors function primarily in sunlight, with light-dependent pigment screening further contributing to adaptation in this system by dynamically recruiting photoreceptors with varying sensitivity into the operational pool.     

Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus,Medicago, and Phaseolus

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.