Three-dimensional solution structure of the cytoplasmic B domain of the mannitol transporter IImannitol of the Escherichia coli phosphotransferase system

The solution structure of the cytoplasmic B domain of the mannitol (Mtl) transporter (II(Mtl)) from the mannitol branch of the Escherichia coli phosphotransferase system has been solved by multidimensional NMR spectroscopy with extensive use of residual dipolar couplings. The ordered IIB(Mtl) domain (residues 375-471 of II(Mtl)) consists of a four-stranded parallel beta-sheet flanked by two helices (alpha(1) and alpha(3)) on one face and helix alpha(2) on the opposite face with a characteristic Rossmann fold comprising two right-handed beta(1)alpha(1)beta(2) and beta(3)alpha(2)beta(4) motifs. The active site loop is structurally very similar to that of the eukaryotic protein tyrosine phosphatases, with the active site cysteine (Cys-384) primed in the thiolate state (pK(a) < 5.6) for nucleophilic attack at the phosphorylated histidine (His-554) of the IIA(Mtl) domain through stabilization by hydrogen bonding interactions with neighboring backbone amide groups at positions i + 2/3/4 from Cys-384 and with the hydroxyl group of Ser-391 at position i + 7. Modeling of the phosphorylated state of IIB(Mtl) suggests that the phosphoryl group can be readily stabilized by hydrogen bonding interactions with backbone amides in the i + 2/4/5/6/7 positions as well as with the hydroxyl group of Ser390 at position i + 6. Despite the absence of any significant sequence identity, the structure of IIB(Mtl) is remarkably similar to the structures of bovine protein tyrosine phosphatase (which contains two long insertions relative to IIB(Mtl)) and the cytoplasmic B component of enzyme II(Chb), which fulfills an analogous role to IIB(Mtl) in the N,N'-diacetylchitobiose branch of the phosphotransferase system. All three proteins utilize a cysteine residue in the nucleophilic attack of a phosphoryl group covalently bound to another protein.     

Two regions of the tail are necessary for the isoform-specific functions of nonmuscle myosin IIB

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB(-/-) fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament.     

The transport/phosphorylation of N,N'-diacetylchitobiose in Escherichia coli. Characterization of phospho-IIB(Chb) and of a potential transition state analogue in the phosphotransfer reaction between the proteins IIA(Chb) AND IIB(Chb)

Enzyme II permeases of the phosphoenolpyruvate:glycose phosphotransferase system comprise one to five separately encoded polypeptides, but most contain similar domains (IIA, IIB, and IIC). The phosphoryl group is transferred from one domain to another, with histidine as the phosphoryl acceptor in IIA and cysteine as the acceptor in certain IIB domains. IIB(Chb) is a phosphocarrier in the uptake/phosphorylation of the chitin disaccharide, (GlcNAc)(2) by Escherichia coli and is unusual because it is separately encoded and soluble. Both the crystal and solution structures of a IIB(Chb) mutant (C10S) have been reported. In the present studies, homogeneous phospho-IIB(Chb) was isolated, and the phosphoryl-Cys linkage was established by (31)P NMR spectroscopy. Rate constants for the hydrolysis of phospho-IIB(Chb) plotted versus pH gave the same shape peak reported for the model compound, butyl thiophosphate, but was shifted about 4 pH units. Evidence is presented for a stable complex between homogeneous Cys10SerIIB(Chb) (which cannot be phosphorylated) and phospho-IIA(Chb), but not with IIA(Chb). The complex (a tetramer (3)) contains equimolar quantities of the two proteins and has been chemically cross-linked. It appears to be an analogue of the transition state complex in the reaction: phospho-IIA(Chb) + IIB(Chb) <--> IIA(Chb) + phospho-IIB(Chb). This is apparently the first report of the isolation of a transition state analogue in a protein-protein phosphotransfer reaction.     

Crystal structure of the IIB(Sor) domain of the sorbose permease from Klebsiella pneumoniae solved to 1.75A resolution

The phosphoenolpyruvate transferase system (PTS) is the major pathway by which bacteria import hexose sugars across the plasma membrane. The PTS transfers a phosphoryl group sequentially via several components from the glycolytic intermediate phosphoenolpyruvate (PEP) to the translocated sugar. It is comprised of the two general proteins enzyme I and HPr, and a sugar-specific enzyme II complex. Sugar translocation is through the membrane domain of the enzyme II complex. The enzyme II complex can belong to one of six families based upon sequence similarity, with the sorbose transporter from Klebsiella pneumoniae a member of the mannose family.The structure of the IIB(Sor) domain was solved to 1.75A resolution by molecular replacement. It has a central core of seven parallel beta-strands surrounded by a total of six alpha-helices. Three helices cover the front face, one the back face with the remaining two capping the central beta-sheet at the top and bottom. The catalytic His15 residue is situated on the surface-exposed loop between strand 1 and helix 1. In addition to the features previously observed in the homologous IIB(Lev) domain from Bacillus subtilis we see new features in the IIB(Sor) structure. First, the catalytic His15 side-chain is fixed in a specific conformation by forming a short hydrogen bond with Asp10, which in turn makes a salt-bridge with Arg8. Second, as observed in other phosphoproteins, an arginine residue (Arg12) is well poised to stabilize a phosphoryl group on His15. Third, we see an Asp/His pair reminiscent of that observed in the IIA(Man) domain from Escherichia coli. Finally, docking of IIA(Man) to IIB(Sor) shows that Arg12 in its current conformation is well positioned to assist the subsequent transfer of the phosphoryl group onto the sugar in line with previous mutagenesis studies.     

Spatial structure of zervamicin IIB bound to DPC micelles: implications for voltage-gating.

Zervamicin IIB is a 16-amino acid peptaibol that forms voltage-dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. The spatial structure of zervamicin IIB bound to dodecylphosphocholine micelles was studied by nuclear magnetic resonance spectroscopy. The set of 20 structures obtained has a bent helical conformation with a mean backbone root mean square deviation value of approximately 0.2 A and resembles the structure in isotropic solvents (Balashova et al., 2000. NMR structure of the channel-former zervamicin IIB in isotropic solvents. FEBS Lett 466:333-336). The N-terminus represents an alpha-helix, whereas the C-terminal part has a mixed 3(10)/alpha(R) hydrogen-bond pattern. In the anisotropic micelle environment, the bending angle on Hyp10 (23 degrees) is smaller than that (47 degrees) in isotropic solvents. In the NOESY (Nuclear Overhauser Effect Spectroscopy) spectra, the characteristic attenuation of the peptide signals by 5- and 16-doxylstearate relaxation probes indicates a peripheral mode of the peptaibol binding to the micelle with the N-terminus immersed slightly deeper into micelle interior. Analysis of the surface hydrophobicity reveals that the zervamicin IIB helix is amphiphilic and well suited to formation of a tetrameric transmembrane bundle, according to the barrel-stave mechanism. The results are discussed in a context of voltage-driven peptaibol insertion into membrane.     

Solution NMR structures of productive and non-productive complexes between the A and B domains of the cytoplasmic subunit of the mannose transporter of the Escherichia coli phosphotransferase system

Solution structures of complexes between the isolated A (IIA(Man)) and B (IIB(Man)) domains of the cytoplasmic component of the mannose transporter of Escherichia coli have been solved by NMR. The complex of wild-type IIA(Man) and IIB(Man) is a mixture of two species comprising a productive, phosphoryl transfer competent complex and a non-productive complex with the two active site histidines, His-10 of IIA(Man) and His-175 of IIB(Man), separated by approximately 25A. Mutation of the active site histidine, His-10, of IIA(Man) to a glutamate, to mimic phosphorylation, results in the formation of a single productive complex. The apparent equilibrium dissociation constants for the binding of both wild-type and H10E IIA(Man) to IIB(Man) are approximately the same (K(D) approximately 0.5 mM). The productive complex can readily accommodate a transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the Nepsilon2 atom of His-10 of IIA(Man) and the Ndelta1 atom of His-175 of IIB(Man) with negligible (<0.2A) local backbone conformational changes in the immediate vicinity of the active site. The non-productive complex is related to the productive one by a approximately 90 degrees rotation and approximately 37A translation of IIB(Man) relative to IIA(Man), leaving the active site His-175 of IIB(Man) fully exposed to solvent in the non-productive complex. The interaction surface on IIA(Man) for the non-productive complex comprises a subset of residues used in the productive complex and in both cases involves both subunits of IIA(Man). The selection of the productive complex by IIA(Man)(H10E) can be attributed to neutralization of the positively charged Arg-172 of IIB(Man) at the center of the interface. The non-productive IIA(Man)-IIB(Man) complex may possibly be relevant to subsequent phosphoryl transfer from His-175 of IIB(Man) to the incoming sugar located on the transmembrane IIC(Man)-IID(Man) complex.     

Genetic organization of ascB-dapE internalin cluster serves as a potential marker for Listeria monocytogenes sublineages IIA, IIB, and IIC

Listeria monocytogenes is an important foodborne pathogen that comprises four genetic lineages: I, II, III, and IV. Of these, lineage II is frequently recovered from foods and environments and responsible for the increasing incidence of human listeriosis. In this study, the phylogenetic structure of lineage II was determined through sequencing analysis of the ascB-dapE internalin cluster. Fifteen sequence types proposed by multilocus sequence typing based on nine housekeeping genes were grouped into three distinct sublineages, IIA, IIB, and IIC. Organization of the ascBdapE internalin cluster could serve as a molecular marker for these sublineages, with inlGHE, inlGC2DE, and inlC2DE for IIA, IIB, and IIC, respectively. These sublineages displayed specific genetic and phenotypic characteristics. IIA and IIC showed a higher frequency of recombination (rho/theta). However, recombination events had greater effect (r/m) on IIB, leading to its high nucleotide diversity. Moreover, IIA and IIB harbored a wider range of internalin and stress-response genes, and possessed higher nisin tolerance, whereas IIC contained the largest portion of low-virulent strains owing to premature stop codons in inlA. The results of this study indicate that IIA, IIB, and IIC might occupy different ecological niches, and IIB might have a better adaptation to a broad range of environmental niches.     

Reaching Out to Send a Message: Proteins Associated with Neurite Outgrowth and Neurotransmission are Altered with Age in the Long-Lived Naked Mole-Rat

Aging is the greatest risk factor for developing neurodegenerative diseases, which are associated with diminished neurotransmission as well as neuronal structure and function. However, several traits seemingly evolved to avert or delay age-related deterioration in the brain of the longest-lived rodent, the naked mole-rat (NMR). The NMR remarkably also exhibits negligible senescence, maintaining an extended healthspan for ~75 % of its life span. Using a proteomic approach, statistically significant changes with age in expression and/or phosphorylation levels of proteins associated with neurite outgrowth and neurotransmission were identified in the brain of the NMR and include: cofilin-1; collapsin response mediator protein 2; actin depolymerizing factor; spectrin alpha chain; septin-7; syntaxin-binding protein 1; synapsin-2 isoform IIB; and dynamin 1. We hypothesize that such changes may contribute to the extended lifespan and healthspan of the NMR.     

Non‐muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC)

Non‐muscle myosin II (NM II) helps mediate survival and apoptosis in response to TNF‐alpha (TNF), however, NM II's mechanism of action in these processes is not fully understood. NM II isoforms are involved in a variety of cellular processes and differences in their enzyme kinetics, localization, and activation allow NM II isoforms to have distinct functions within the same cell. The present study focused on isoform specific functions of NM IIA and IIB in mediating TNF induced apoptosis. Results show that siRNA knockdown of NM IIB, but not NM IIA, impaired caspase cleavage and nuclear condensation in response to TNF. NM II's function in promoting cell death signaling appears to be independent of actomyosin contractility (AMC) since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF induced caspase cleavage. Immunoprecipitation studies revealed associations of NM IIB with clathrin, FADD, and caspase 8 in response to TNF suggesting a role for NM IIB in TNFR1 endocytosis and the formation of the death inducing signaling complex (DISC). These findings suggest that NM IIB promotes TNF cell death signaling in a manner independent of its force generating property. J. Cell. Biochem. 9999: 1365–1375, 2010. © 2010 Wiley‐Liss, Inc.     

Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structure-activity relationship

Bacitracin is a widely used metal-dependent peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(II) for its biological activity, and has been reported to bind several other transition metal ions, including Co(II), Ni(II), and Cu(II). Despite the wide use of bacitracin, a structure-activity relationship for this drug has not been established, and the structure of its metal complexes has not been fully determined. We report here one- and two-dimensional nuclear magnetic resonance (NMR) studies of the structure of the metal complexes of several bacitracin analogues by the use of paramagnetic Co(II) as a probe. The Co(II) complex of this antibiotic exhibits many well-resolved isotropically shifted (1)H NMR signals in a large spectral window ( approximately 200 ppm) due to protons near the metal, resulting from both contact and dipolar shift mechanisms. The assignment of the isotropically shifted (1)H NMR features concludes that bacitracin A(1), the most potent component of the bacitracin mixture, binds to Co(II) via the His-10 imidazole ring N(epsilon), the thiazoline nitrogen, and the monodentate Glu-4 carboxylate to form a labile complex in aqueous solutions. The free amine of Ile-1 does not bind Co(II). Several different analogues of bacitracin have also been isolated or prepared, and the studies of their Co(II) binding properties further indicate that the antimicrobial activity of these derivatives correlates directly to their metal binding mode. For example, the isotropically shifted (1)H NMR spectral features of the high-potent bacitracin analogues, including bacitracins A(1), B(1), and B(2), are virtually identical. However, Glu-4 and/or the thiazoline ring does not bind Co(II) in the bacitracin analogues with low antibiotic activities, including bacitracins A(2) and F.     

Structure of the topoisomerase VI-B subunit: implications for type II topoisomerase mechanism and evolution

Type IIA and type IIB topoisomerases each possess the ability to pass one DNA duplex through another in an ATP-dependent manner. The role of ATP in the strand passage reaction is poorly understood, particularly for the type IIB (topoisomerase VI) family. We have solved the structure of the ATP-binding subunit of topoisomerase VI (topoVI-B) in two states: an unliganded monomer and a nucleotide-bound dimer. We find that topoVI-B is highly structurally homologous to the entire 40-43 kDa ATPase region of type IIA topoisomerases and MutL proteins. Nucleotide binding to topoVI-B leads to dimerization of the protein and causes dramatic conformational changes within each protomer. Our data demonstrate that type IIA and type IIB topoisomerases have descended from a common ancestor and reveal how ATP turnover generates structural signals in the reactions of both type II topoisomerase families. When combined with the structure of the A subunit to create a picture of the intact topoisomerase VI holoenzyme, the ATP-driven motions of topoVI-B reveal a simple mechanism for strand passage by the type IIB topoisomerases.     

NMR structure of cysteinyl-phosphorylated enzyme IIB of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli

The determination by NMR of the solution structure of the phosphorylated enzyme IIB (P-IIB(Chb)) of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is presented. Most of the backbone and side-chain resonances were assigned using a variety of mostly heteronuclear NMR experiments. The remaining resonances were assigned with the help of the structure calculations.NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addition, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous restraints into new ambiguous restraints, an error function was constructed that was less sensitive to information loss caused by assignment uncertainties. The final set of structures had a pairwise rmsd of 0.59 A and 1.16 A for the heavy atoms of the backbone and side-chains, respectively.Comparing the P-IIB(Chb) solution structure with the previously determined NMR and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are limited to the active-site region. The phosphoryl group at the active-site cysteine residue is surrounded by a loop formed by residues 10 through 16. NOE and chemical shift data suggest that the phosphoryl group makes hydrogen bonds with the backbone amide protons of residues 12 and 15. The binding mode of the phosphoryl group is very similar to that of the protein tyrosine phosphatases. The differences observed are in accordance with the presumption that IIB(Chb) has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cysteine SH proton and the solvent via the hydroxyl group of Thr16.     

Studying metal ion binding properties of a three-way junction RNA by heteronuclear NMR

Self-splicing group II introns are highly structured RNA molecules, containing a characteristic secondary and catalytically active tertiary structure, which is formed only in the presence of Mg(II). Mg(II) initiates the first folding step governed by the κζ element within domain 1 (D1κζ). We recently solved the NMR structure of D1κζ derived from the mitochondrial group II intron ribozyme Sc.ai5γ and demonstrated that Mg(II) is essential for its stabilization. Here, we performed a detailed multinuclear NMR study of metal ion interactions with D1κζ, using Cd(II) and cobalt(III)hexammine to probe inner- and outer-sphere coordination of Mg(II) and thus to better characterize its binding sites. Accordingly, we mapped ¹H, ¹⁵N, ¹³C, and ³¹P spectral changes upon addition of different amounts of the metal ions. Our NMR data reveal a Cd(II)-assisted macrochelate formation at the 5′-end triphosphate, a preferential Cd(II) binding to guanines in a helical context, an electrostatic interaction in the ζ tetraloop receptor and various metal ion interactions in the GAAA tetraloop and κ element. These results together with our recently published data on Mg(II) interaction provide a much better understanding of Mg(II) binding to D1κζ, and reveal how intricate and complex metal ion interactions can be.     

Visualization of the phosphorylated active site loop of the cytoplasmic B domain of the mannitol transporter II(Mannitol) of the Escherichia coli phosphotransferase system by NMR spectroscopy and residual dipolar couplings

The solution structure of a stably phosphorylated form of the cytoplasmic B domain of the mannitol-specific transporter (IIB(Mtl)) of the Escherichia coli phosphotransferase system, containing a mutation of the active site Cys384 to Ser, has been solved by NMR. The strategy employed relies principally on backbone residual dipolar couplings recorded in three different alignment media, supplemented by nuclear Overhauser enhancement data and torsion angle restraints related specifically to the active site loop (residues 383-393). As judged from the dipolar coupling data, the remainder of the structure is unchanged upon phosphorylation within the errors of the coordinates of the previously determined solution structure of unphosphorylated wild-type IIB(Mtl). Thus, only the active site loop was refined. Phosphorylation results in a backbone atomic rms shift of approximately 0.7 angstroms in the active site loop. The resulting conformation is less than 0.5 angstroms away from the equivalent P-loop in both the low and high molecular mass eukaryotic tyrosine phosphatases. 3J(NP) coupling constant measurements using quantitative J-correlation spectroscopy provide a direct demonstration of a hydrogen bond between the phosphoryl group and the backbone amide of Ser391 at position i + 7 from phospho-Ser384, with an approximately linear P-O-H(N) bond angle. The structure also reveals additional hydrogen bonding interactions involving the backbone amides of residues at positions i + 4 and i + 5, and the hydroxyl groups of two serine residues at positions i + 6 and i + 7 that stabilize the phosphoryl group.     

Cooperative folding of tau peptide by coordination of group IIB metal cations during heparin-induced aggregation

The group IIB elements, especially Cd(II) and Hg(II), are increasingly considered as potential environmental neurotoxins. This study demonstrates that the Alzheimer’s tau fragment R2, corresponding to the second repeat of the microtubule-binding domain, can bind to Zn(II), Cd(II) and Hg(II). Isothermal titration calorimetry experiments suggest that the most likely coordination site is the thiol group of Cys291, and this is further confirmed by a control experiment using a C291A mutant peptide. Circular dichroism spectrum reveals that the coordination of group IIB cations, especially Hg(II), can induce pronounced conformational conversions in natively unfolded R2, from random coil to other ordered structures. ThS fluorescence assays and electron microscopy indicate that the group IIB cations promote heparin-induced aggregation of R2, giving relatively small R2 filaments. The efficiency in promoting aggregation, as well as inducing conformational conversion, varies strongly with the cation’s polarizability. Based on these results, a model is proposed in which the cooperative folding of R2 through cross-bridging of group IIB cations is suggested to be a key factor in promoting aggregation, in addition to the effective neutralization of coulombic charge–charge repulsion by heparin, the poly-anion inducer. Our results provide clues to understanding the potential pathogenic role of group IIB metals in the development of neurofibrillary tangles, a typical hallmark of Alzheimer’s disease.     

Effects of maturation and co-culture treatments on the developmental capacity of early bovine embryos.

A total of 901 cumulus-oocyte complexes (COCs) were collected from bovine ovaries obtained at a local abattoir. COCs randomly assigned to Treatment I (n = 451), were cultured in TCM-199 + 10% fetal bovine serum (FBS) and hormones, while oocytes in Treatment II (n = 450) were cultured in TCM-199 + 20% estrous cow serum (ECS). Assessment of maturation revealed that 91.3% (42/46) of oocytes in Treatment I had reached metaphase II of meiosis, which was greater (P less than 0.05) than the 73.3% (33/45) in Treatment II. Following in vitro fertilization, 203 oocytes from Treatment I were co-cultured on bovine granulosa cells (Treatment IA) while the remaining 202 oocytes were co-cultured on bovine oviductal cells (Treatment IB). Similarly, 203 oocytes from Treatment II were co-cultured on granulosa cells (Treatment IIA) or oviductal cells (Treatment IIB, n = 202). Co-culture was maintained for 8 days. The proportion of cleaved zygotes was higher (P less than 0.05) in Treatment IB (86.6%) compared to Treatments IA (78.8%), IIA (58.1%), and IIB (64.8%). The proportion of cleaved zygotes that progressed beyond the 16-cell stage was also greater (P less than 0.001) in Treatment IB (71.4%) compared to Treatments IA (50.0%), IIA (35.4%) and IIB (55.8%). Treatment IB also produced the highest proportion of blastocysts (P less than 0.0001) (41.1%) versus 24.6% (IA), 11.3% (IIA) and 18.3% (IIB). The proportion of day 6 morulae that progressed to form day 8 blastocysts was similar for both co-culture treatments (IA, 70.1%; IB 70.2%; IIA, 51.5%; IIB 50.8%) and varied only between in vitro maturation groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Major histocompatibility complex class IIA and IIB genes of Nile tilapia Oreochromis niloticus: Genomic structure,molecular polymorphism and expression patterns

Major histocompatibility complex (MHC) is a large genomic region characterized by extremely high polymorphism, and it plays an important role in the immune response of vertebrates. In the present study, we isolated MHC class II genes from Nile tilapia in order to investigate the immune mechanism in tilapia and develop better strategies for disease prevention. Moreover, we cloned the full-length cDNA sequences of MHC IIA and IIB from Nile tilapia by the RACE approach. In addition, the genomic structure, molecular polymorphism and expression patterns of MHC II genes in Nile tilapia were also examined. Compared with that of other teleosts, Nile tilapia MHC class IIA contained four exons and three introns. The deduced amino acid sequence of the MHC IIA molecule shared 25.4–64.5% similarity with those of other teleosts and mammals. Six exons and five introns were identified from Nile tilapia MHC IIB, and the deduced amino acid sequence shared 26.9–74.7% similarity with those of other teleosts and mammals. All the characteristic features of MHC class II chain structure could be identified in the deduced sequences of MHC IIA and IIB molecules, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. A total of 12 MHC IIA alleles were identified from six individuals. Four alleles originating from a single individual suggested that at least four MHC IIA loci existed. Moreover, 10 MHC IIB alleles were identified, among which four were detected in a single individual, suggesting that at least four MHC IIB loci existed. The expression of MHC IIA and IIB at the mRNA level in 10 types of normal tissues was determined using quantitative real-time PCR analysis. The highest expression level was detected in stomach and gill, whereas the lowest expression was detected in muscle and brain. Furthermore, MHC IIA and IIB were probably two candidate immune molecules involved in the resistance against streptococcosis, because their expression was significantly up-regulated in gill, kidney, intestine and spleen after the intraperitoneal injection of Streptococcus agalactiae.