Allosteric regulation of nitrate transporter NRT via the signaling protein PII.

Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.

Fine structure and assembly pattern of a minimal myophage Pam3.

The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 Å in diameter, connected via a three-section neck to an 840-Å-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. In vitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology.

Inhibition of phosphorylated calcium/calmodulin-dependent protein kinase IIα relieves streptozotocin-induced diabetic neuropathic pain through regulation of P2X3 receptor in dorsal root ganglia.

Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1st to 3rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2nd to 3rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2nd and 3rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management.

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7.

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate), a nonspecific P2X1-7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4-L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.

Analgesic effect of electroacupuncture on bone cancer pain in rat model: the role of peripheral P2X3 receptor.

Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 104 cells/3 µL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4-6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,ß-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca2+ imaging analysis revealed that the EA stimulation decreased the percentage of α,ß-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,ß-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA's analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.

Electroacupuncture may alleviate diabetic neuropathic pain by inhibiting the microglia P2X4R and neuroinflammation.

Diabetic neuropathic pain (DNP) is a common and destructive complication of diabetes mellitus. The discovery of effective therapeutic methods for DNP is vitally imperative because of the lack of effective treatments. Although 2 Hz electroacupuncture (EA) was a successful approach for relieving DNP, the mechanism underlying the effect of EA on DNP is still poorly understood. Here, we established a rat model of DNP that was induced by streptozotocin (STZ) injection. P2X4R was upregulated in the spinal cord after STZ-injection. The upregulation of P2X4R was mainly expressed on activated microglia. Intrathecal injection of a P2X4R antagonist or microglia inhibitor attenuated STZ-induced nociceptive thermal hyperalgesia and reduced the overexpression of brain-derived neurotrophic factor (BDNF), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the spinal cord. We also assessed the effects of EA treatment on the pain hypersensitivities of DNP rats, and further investigated the possible mechanism underlying the analgesic effect of EA. EA relieved the hyperalgesia of DNP. In terms of mechanism, EA reduced the upregulation of P2X4R on activated microglia and decreased BDNF, IL-1ß and TNF-α in the spinal cord. Mechanistic research of EA's analgesic impact would be beneficial in ensuring its prospective therapeutic effect on DNP as well as in extending EA's applicability.

Rubisco accumulation factor 1 (Raf1) plays essential roles in mediating Rubisco assembly and carboxysome biogenesis.

Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis.

Capsid Structure of Anabaena Cyanophage A-1(L).

A-1(L) is a freshwater cyanophage with a contractile tail that specifically infects Anabaena sp. PCC 7120, one of the model strains for molecular studies of cyanobacteria. Although isolated for half a century, its structure remains unknown, which limits our understanding on the interplay between A-1(L) and its host. Here we report the 3.35 Å cryo-EM structure of A-1(L) capsid, representing the first near-atomic resolution structure of a phage capsid with a T number of 9. The major capsid gp4 proteins assemble into 91 capsomers, including 80 hexons: 20 at the center of the facet and 60 at the facet edge, in addition to 11 identical pentons. These capsomers further assemble into the icosahedral capsid, via gradually increasing curvatures. Different from the previously reported capsids of known-structure, A-1(L) adopts a noncovalent chainmail structure of capsid stabilized by two kinds of mortise-and-tenon inter-capsomer interactions: a three-layered interface at the pseudo 3-fold axis combined with the complementarity in shape and electrostatic potential around the 2-fold axis. This unique capsomer construction enables A-1(L) to possess a rigid capsid, which is solely composed of the major capsid proteins with an HK97 fold. IMPORTANCE Cyanobacteria are the most abundant photosynthetic bacteria, contributing significantly to the biomass production, O2 generation, and CO2 consumption on our planet. Their community structure and homeostasis in natural aquatic ecosystems are largely regulated by the corresponding cyanophages. In this study, we solved the structure of cyanophage A-1(L) capsid at near-atomic resolution and revealed a unique capsid construction. This capsid structure provides the molecular details for better understanding the assembly of A-1(L), and a structural platform for future investigation and application of A-1(L) in combination with its host Anabaena sp. PCC 7120. As the first isolated freshwater cyanophage that infects the genetically tractable model cyanobacterium, A-1(L) should become an ideal template for the genetic engineering and synthetic biology studies.

Coordinating carbon and nitrogen metabolic signaling through the cyanobacterial global repressor NdhR.

The coordination of carbon and nitrogen metabolism is essential for bacteria to adapt to nutritional variations in the environment, but the underlying mechanism remains poorly understood. In autotrophic cyanobacteria, high CO2 levels favor the carboxylase activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) to produce 3-phosphoglycerate, whereas low CO2 levels promote the oxygenase activity of RuBisCO, leading to 2-phosphoglycolate (2-PG) production. Thus, the 2-PG level is reversely correlated with that of 2-oxoglutarate (2-OG), which accumulates under a high carbon/nitrogen ratio and acts as a nitrogen-starvation signal. The LysR-type transcriptional repressor NAD(P)H dehydrogenase regulator (NdhR) controls the expression of genes related to carbon metabolism. Based on genetic and biochemical studies, we report here that 2-PG is an inducer of NdhR, while 2-OG is a corepressor, as found previously. Furthermore, structural analyses indicate that binding of 2-OG at the interface between the two regulatory domains (RD) allows the NdhR tetramer to adopt a repressor conformation, whereas 2-PG binding to an intradomain cleft of each RD triggers drastic conformational changes leading to the dissociation of NdhR from its target DNA. We further confirmed the effect of 2-PG or 2-OG levels on the transcription of the NdhR regulon. Together with previous findings, we propose that NdhR can sense 2-OG from the Krebs cycle and 2-PG from photorespiration, two key metabolites that function together as indicators of intracellular carbon/nitrogen status, thus representing a fine sensor for the coordination of carbon and nitrogen metabolism in cyanobacteria.

Crystal structure of a novel fold protein Gp72 from the freshwater cyanophage Mic1.

Cyanophages, widespread in aquatic systems, are a class of viruses that specifically infect cyanobacteria. Though they play important roles in modulating the homeostasis of cyanobacterial populations, little is known about the freshwater cyanophages, especially those hypothetical proteins of unknown function. Mic1 is a freshwater siphocyanophage isolated from the Lake Chaohu. It encodes three hypothetical proteins Gp65, Gp66, and Gp72, which share an identity of 61.6% to 83%. However, we find these three homologous proteins differ from each other in oligomeric state. Moreover, we solve the crystal structure of Gp72 at 2.3 Å, which represents a novel fold in the α + ß class. Structural analyses combined with redox assays enable us to propose a model of disulfide bond mediated oligomerization for Gp72. Altogether, these findings provide structural and biochemical basis for further investigations on the freshwater cyanophage Mic1.

Structural and functional insights into the Asp1/2/3 complex mediated secretion of pneumococcal serine-rich repeat protein PsrP.

The accessory sec system consisting of seven conserved components is commonly distributed among pathogenic Gram-positive bacteria for the secretion of serine-rich-repeat proteins (SRRPs). Asp1/2/3 protein complex in the system is responsible for both the O-acetylation of GlcNAc and delivering SRRPs to SecA2. However, the molecular mechanism of how Asp1/2/3 transport SRRPs remains unknown. Here, we report the complex structure of Asp1/2/3 from Streptococcus pneumoniae at 2.9 Å. Further functional assays indicated that Asp1/2/3 can stimulate the ATPase activity of SecA2. In addition, the deletion of asp1/2/3 gene resulted in the accumulation of a secreted version of PsrP with an altered glycoform in protoplast fraction of the mutant cell, which suggested the modification/transport coupling of the substrate. Altogether, these findings not only provide structural basis for further investigations on the transport process of SRRPs, but also uncover the indispensable role of Asp1/2/3 in the accessory sec system.

The model cyanobacteria Anabaena sp. PCC 7120 possess an intact but partially degenerated gene cluster encoding gas vesicles.

BACKGROUND: Bacterial gas vesicles, composed of two major gas vesicle proteins and filled with gas, are a unique class of intracellular bubble-like nanostructures. They provide buoyancy for cells, and thus play an essential role in the growth and survival of aquatic and soil microbes. Moreover, the gas vesicle could be applied to multimodal and noninvasive biological imaging as a potential nanoscale contrast agent. To date, cylinder-shaped gas vesicles have been found in several strains of cyanobacteria. However, whether the functional gas vesicles could be produced in the model filamentous cyanobacteria Anabaena sp. PCC 7120 remains controversial. RESULTS: In this study, we found that an intact gvp gene cluster indeed exists in the model filamentous cyanobacteria Anabaena sp. PCC 7120. Real-time PCR assays showed that the gvpA gene is constitutively transcribed in vivo, and its expression level is upregulated at low light intensity and/or high growth temperature. Functional expression of this intact gvp gene cluster enables the recombinant Escherichia coli to gain the capability of floatation in the liquid medium, thanks to the assembly of irregular gas vesicles. Furthermore, crystal structure of GvpF in combination with enzymatic activity assays of GvpN suggested that these two auxiliary proteins of gas vesicle are structurally and enzymatically conserved, respectively. CONCLUSIONS: Our findings show that the laboratory strain of model filamentous cyanobacteria Anabaena sp. PCC 7120 possesses an intact but partially degenerated gas vesicle gene cluster, indicating that the natural isolate might be able to produce gas vesicles under some given environmental stimuli for better floatation.

Multi-functional regulator MapZ controls both positioning and timing of FtsZ polymerization.

The tubulin-like GTPase protein FtsZ, which forms a discontinuous cytokinetic ring at mid-cell, is a central player to recruit the division machinery to orchestrate cell division. To guarantee the production of two identical daughter cells, the assembly of FtsZ, namely Z-ring, and its precise positioning should be finely regulated. In Streptococcus pneumoniae, the positioning of Z-ring at the division site is mediated by a bitopic membrane protein MapZ (mid-cell-anchored protein Z) through direct interactions between the intracellular domain (termed MapZ-N (the intracellular domain of MapZ)) and FtsZ. Using nuclear magnetic resonance titration experiments, we clearly assigned the key residues involved in the interactions. In the presence of MapZ-N, FtsZ gains a shortened activation delay, a lower critical concentration for polymerization and a higher cooperativity towards GTP hydrolysis. On the other hand, MapZ-N antagonizes the lateral interactions of single-stranded filaments of FtsZ, thus slows down the formation of highly bundled FtsZ polymers and eventually maintains FtsZ at a dynamic state. Altogether, we conclude that MapZ is not only an accelerator to trigger the polymerization of FtsZ, but also a brake to tune the velocity to form the end-product, FtsZ bundles. These findings suggest that MapZ is a multi-functional regulator towards FtsZ that controls both the precise positioning and proper timing of FtsZ polymerization.

Structural characterization of the redefined DNA-binding domain of human XPA.

XPA (xeroderma pigmentosum complementation group A), a key scaffold protein in nucleotide excision repair (NER) pathway, is important in DNA damage verification and repair proteins recruitment. Earlier studies had mapped the minimal DNA-binding domain (MBD) of XPA to a region corresponding to residues 98-219. However, recent studies indicated that the region involving residues 98-239 is the redefined DNA-binding domain (DBD), which binds to DNA substrates with a much higher binding affinity than MBD and possesses a nearly identical binding affinity to the full-length XPA protein. However, the structure of the redefined DBD domain of XPA (XPA-DBD) remains to be investigated. Here, we present the crystal structure of XPA-DBD at 2.06 Šresolution. Structure of the C-terminal region of XPA has been extended by 21 residues (Arg211-Arg231) as compared with previously reported MBD structures. The structure reveals that the C-terminal extension (Arg211-Arg231) is folded as an α-helix with multiple basic residues. The positively charged surface formed in the last C-terminal helix suggests its critical role in DNA binding. Further structural analysis demonstrates that the last C-terminal region (Asp217-Thr239) of XPA-DBD might undergo a conformational change to directly bind to the DNA substrates. This study provides a structural basis for understanding the possible mechanism of enhanced DNA-binding affinity of XPA-DBD.

Crystal structure of the choline-binding protein CbpJ from Streptococcus pneumoniae.

The choline-binding proteins play essential roles in pneumococcal colonization and virulence. It has been suggested that the choline-binding protein J (termed CbpJ; encoded by the gene sp_0378) from Streptococcus pneumoniae TIGR4 involves in the colonization in host and contributes to evasion of neutrophil killing. Here we report the 2.0 Šcrystal structure of CbpJ in complex with choline. CbpJ consists of an N-terminal putative functional domain (N-domain) followed by a C-terminal choline-binding domain (CBD). The N-domain harbors four degenerated choline-binding repeats (CBRs) that lose the capacity of binding to choline, whereas the CBD is composed of seven typical CBRs. Further functional assays showed that the CBD contributes to the pneumococcal adhesion to human lung epithelial cell A549. These findings provide insights into the pneumococcal pathogenesis and broaden our understanding on the functions of choline-binding proteins.

Crystal structure of pentameric shell protein CsoS4B of Halothiobacillus neapolitanus α-carboxysome.

Carboxysome, encapsulating an enzymatic core within an icosahedral-shaped semipermeable protein shell, could enhance CO2 fixation under low CO2 conditions in the environment. The shell of Halothiobacillus neapolitanus α-carboxysome possesses two 38% sequence-identical pentameric proteins, namely CsoS4A and CsoS4B. However, the functions of two paralogous pentameric proteins in α-carboxysome assembly remain unknown. Here we report the crystal structure of CsoS4B at 2.15 Šresolution. It displays as a stable pentamer, each subunit of which consists of a ß-barrel core domain, in addition to an insertion of helix α1 that forms the central pore. Structural comparisons and multiple-sequence alignment strongly indicate that CsoS4A and CsoS4B differ from each other in interacting with various components of α-carboxysome, despite they share a similar overall structure. These findings provide the structural basis for further investigations on the self-assembly process of carboxysome.

Structural insights into the catalysis and substrate specificity of cyanobacterial aspartate racemase McyF.

L-amino acids represent the most common amino acid form, most notably as protein residues, whereas D-amino acids, despite their rare occurrence, play significant roles in many biological processes. Amino acid racemases are enzymes that catalyze the interconversion of L- and/or D-amino acids. McyF is a pyridoxal 5'-phosphate (PLP) independent amino acid racemase that produces the substrate D-aspartate for the biosynthesis of microcystin in the cyanobacterium Microcystis aeruginosa PCC7806. Here we report the crystal structures of McyF in complex with citrate, L-Asp and D-Asp at 2.35, 2.63 and 2.80 Å, respectively. Structural analyses indicate that McyF and homologs possess highly conserved residues involved in substrate binding and catalysis. In addition, residues Cys87 and Cys195 were clearly assigned to the key catalytic residues of "two bases" that deprotonate D-Asp and L-Asp in a reaction independent of PLP. Further site-directed mutagenesis combined with enzymatic assays revealed that Glu197 also participates in the catalytic reaction. In addition, activity assays proved that McyF could also catalyze the interconversion of L-MeAsp between D-MeAsp, the precursor of another microcystin isoform. These findings provide structural insights into the catalytic mechanism of aspartate racemase and microcystin biosynthesis.

The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian cGMP-regulated phosphodiesterases, Anabaenaadenylyl cyclases and Escherichia coli transcription activator FhlA) domain containing bifunctional enzyme DcpA (diguanylate cyclase and phosphodiesterase A) that catalyzes the synthesis and hydrolysis of c-di-GMP. Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments.

Defining the enzymatic pathway for polymorphic O-glycosylation of the pneumococcal serine-rich repeat protein PsrP.

Protein O-glycosylation is an important post-translational modification in all organisms, but deciphering the specific functions of these glycans is difficult due to their structural complexity. Understanding the glycosylation of mucin-like proteins presents a particular challenge as they are modified numerous times with both the enzymes involved and the glycosylation patterns being poorly understood. Here we systematically explored the O-glycosylation pathway of a mucin-like serine-rich repeat protein PsrP from the human pathogen Streptococcus pneumoniae TIGR4. Previous works have assigned the function of 3 of the 10 glycosyltransferases thought to modify PsrP, GtfA/B, and Gtf3 as catalyzing the first two reactions to form a unified disaccharide core structure. We now use in vivo and in vitro glycosylation assays combined with hydrolytic activity assays to identify the glycosyltransferases capable of decorating this core structure in the third and fourth steps of glycosylation. Specifically, the full-length GlyE and GlyG proteins and the GlyD DUF1792 domain participate in both steps, whereas full-length GlyA and the GlyD GT8 domain catalyze only the fourth step. Incorporation of different sugars to the disaccharide core structure at multiple sites along the serine-rich repeats results in a highly polymorphic product. Furthermore, crystal structures of apo- and UDP-complexed GlyE combined with structural analyses reveal a novel Rossmann-fold "add-on" domain that we speculate to function as a universal module shared by GlyD, GlyE, and GlyA to forward the peptide acceptor from one enzyme to another. These findings define the complete glycosylation pathway of a bacterial glycoprotein and offer a testable hypothesis of how glycosyltransferase coordination facilitates glycan assembly.

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.

Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a ß-prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore-forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish-specific defense molecule.