Wei-Jen Tang

Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin

Oedema factor, a calmodulin-activated adenylyl cyclase, is important in the pathogenesis of anthrax. Here we report the X-ray structures of oedema factor with and without bound calmodulin. Oedema factor shares no significant structural homology with mammalian adenylyl cyclases or other proteins. In the active site, 3′-deoxy-ATP and a single metal ion are well positioned for catalysis with histidine 351 as the catalytic base. This mechanism differs from the mechanism of two-metal-ion catalysis proposed for mammalian adenylyl cyclases. Four discrete regions of oedema factor form a surface that recognizes an extended conformation of calmodulin, which is very different from the collapsed conformation observed in other structures of calmodulin bound to effector peptides. On calmodulin binding, an oedema factor helical domain of relative molecular mass 15,000 undergoes a 15 Å translation and a 30° rotation away from the oedema factor catalytic core, which stabilizes a disordered loop and leads to enzyme activation. These allosteric changes provide the first molecular details of how calmodulin modulates one of its targets.

Gadd45|[beta]| mediates the NF-|[kappa]|B suppression of JNK signalling by targeting MKK7/JNKK2

NF-κB/Rel transcription factors control apoptosis, also known as programmed cell death. This control is crucial for oncogenesis, cancer chemo-resistance and for antagonizing tumour necrosis factor α (TNFα)-induced killing. With regard to TNFα, the anti-apoptotic activity of NF-κB involves suppression of the c-Jun N-terminal kinase (JNK) cascade. Using an unbiased screen, we have previously identified Gadd45β/Myd118, a member of the Gadd45 family of inducible factors, as a pivotal mediator of this suppressive activity of NF-κB. However, the mechanisms by which Gadd45β inhibits JNK signalling are not understood. Here, we identify MKK7/JNKK2 — a specific and essential activator of JNK — as a target of Gadd45β, and in fact, of NF-κB itself. Gadd45β binds to MKK7 directly and blocks its catalytic activity, thereby providing a molecular link between the NF-κB and JNK pathways. Importantly, Gadd45β is required to antagonize TNFα-induced cytotoxicity, and peptides disrupting the Gadd45β/MKK7 interaction hinder the ability of Gadd45β, as well as of NF-κB, to suppress this cytotoxicity. These findings establish a basis for the NF-κB control of JNK activation and identify MKK7 as a potential target for anti-inflammatory and anti-cancer therapy.

Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism.

Insulin-degrading enzyme (IDE), a Zn2+-metalloprotease, is involved in the clearance of insulin and amyloid-β (refs 1–3). Loss-of-function mutations of IDE in rodents cause glucose intolerance and cerebral accumulation of amyloid-β, whereas enhanced IDE activity effectively reduces brain amyloid-β (refs 4–7). Here we report structures of human IDE in complex with four substrates (insulin B chain, amyloid-β peptide (1–40), amylin and glucagon). The amino- and carboxy-terminal domains of IDE (IDE-N and IDE-C, respectively) form an enclosed cage just large enough to encapsulate insulin. Extensive contacts between IDE-N and IDE-C keep the degradation chamber of IDE inaccessible to substrates. Repositioning of the IDE domains enables substrate access to the catalytic cavity. IDE uses size and charge distribution of the substrate-binding cavity selectively to entrap structurally diverse polypeptides. The enclosed substrate undergoes conformational changes to form β-sheets with two discrete regions of IDE for its degradation. Consistent with this model, mutations disrupting the contacts between IDE-N and IDE-C increase IDE catalytic activity 40-fold. The molecular basis for substrate recognition and allosteric regulation of IDE could aid in designing IDE-based therapies to control cerebral amyloid-β and blood sugar concentrations.

Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice

Bacillus anthracis edema toxin (ET), an adenylyl cyclase, is an important virulence factor that contributes to anthrax disease. The role of ET in anthrax pathogenesis is, however, poorly understood. Previous studies using crude toxin preparations associated ET with subcutaneous edema, and ET-deficient strains of B. anthracis showed a reduction in virulence. We report the first comprehensive study of ET-induced pathology in an animal model. Highly purified ET caused death in BALB/cJ mice at lower doses and more rapidly than previously seen with the other major B. anthracis virulence factor, lethal toxin. Observations of gross pathology showed intestinal intralumenal fluid accumulation followed by focal hemorrhaging of the ileum and adrenal glands. Histopathological analyses of timed tissue harvests revealed lesions in several tissues including adrenal glands, lymphoid organs, bone, bone marrow, gastrointestinal mucosa, heart, and kidneys. Concomitant blood chemistry analyses supported the induction of tissue damage. Several cytokines increased after ET administration, including granulocyte colony-stimulating factor, eotaxin, keratinocyte-derived cytokine, MCP-1/JE, interleukin-6, interleukin-10, and interleukin-1beta. Physiological measurements also revealed a concurrent hypotension and bradycardia. These studies detail the extensive pathological lesions caused by ET and suggest that it causes death due to multiorgan failure.

Anthrax toxins suppress T lymphocyte activation by disrupting antigen receptor signaling

Anthrax is an infection caused by pathogenic strains of Bacillus anthracis, which secretes a three-component toxic complex consisting of protective antigen (PA), edema factor (EF), and lethal factor (LF). PA forms binary complexes with either LF or EF and mediates their entry into host cells. Although the initial phases of bacterial growth occur in the lymph node, the host fails to mount an effective immune response. Here, we show that LT and ET are potent suppressors of human T cell activation and proliferation triggered through the antigen receptor. Both LT and ET inhibit the mitogen-activated protein and stress kinase pathways, and both toxins inhibit activation of NFAT and AP-1, two transcription factors essential for cytokine gene expression. These data identify a novel strategy of immune evasion by B. anthracis, based on both effector subunits of the toxic complex, and targeted to a key cellular component of adaptive immunity.

Anthrax Edema Toxin Cooperates with Lethal Toxin to Impair Cytokine Secretion during Infection of Dendritic Cells

Bacillus anthracis secretes two critical virulence factors, lethal toxin (LT) and edema toxin (ET). In this study, we show that murine bone marrow-derived dendritic cells (DC) infected with B. anthracis strains secreting ET exhibit a very different cytokine secretion pattern than DC infected with B. anthracis strains secreting LT, both toxins, or a nontoxinogenic strain. ET produced during infection selectively inhibits the production of IL-12p70 and TNF-α, whereas LT targets IL-10 and TNF-α production. To confirm the direct role of the toxins, we show that purified ET and LT similarly disrupt cytokine secretion by DC infected with a nontoxinogenic strain. These effects can be reversed by specific inhibitors of each toxin. Furthermore, ET inhibits in vivo IL-12p70 and IFN-γ secretion induced by LPS. These results suggest that ET produced during infection impairs DC functions and cooperates with LT to suppress the innate immune response. This may represent a new strategy developed by B. anthracis to escape the host immune response.

Amyloid β-degrading cryptidases : insulin degrading enzyme, presequence peptidase, and neprilysin

Abstract.The accumulation of aggregates of amyloidogenic peptides is associated with numerous human diseases. One well studied example is the association between deposition of amyloid β (Aβ) and Alzheimer’s disease. Insulin degrading enzyme and neprilysin are involved in the clearance of Aβ, and presequence peptidase is suggested to play a role in the degradation of mitochondrial Aβ. Recent structural analyses reveal that these three peptidases contain a catalytic chamber (crypt) that selectively encapsulates and cleaves amyloidogenic peptides, hence the name cryptidase. The substrate selectivity of these cryptidases is determined by the size and charge distribution of their crypt as well as the conformational flexibility of substrates. The interaction of Aβ with the catalytic core of these cryptidases is controlled by conformational changes that make the catalytic chambers accessible for Aβ binding. These new structural and biochemical insights into cryptidases provide potential therapeutic strategies for the control of Aβ clearance.

Chemical screening by mass spectrometry to identify inhibitors of anthrax lethal factor

Mass spectrometry (MS) analysis is applicable to a broad range of biological analytes and has the important advantage that it does not require analytes to be labeled. A drawback of MS methods, however, is the need for chromatographic steps to prepare the analyte, precluding MS from being used in chemical screening and rapid analysis. Here, we report that surfaces that are chemically tailored for characterization by matrix-assisted laser-desorption ionization time-of-flight MS eliminate the need for sample processing and make this technique adaptable to parallel screening experiments. The tailored substrates are based on self-assembled monolayers that present ligands that interact with target proteins and enzymes. We apply this method to screen a chemical library against protease activity of anthrax lethal factor, and report a compound that inhibits lethal factor activity with a Ki of 1.1 μM and blocks the cleavage of MEK1 in 293 cells.

Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor.

Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen‐binding domain (PABD) and a calmodulin (CaM)‐activated adenylyl cyclase domain. Here, we report the crystal structures of CaM‐bound EF, revealing the architecture of EF PABD. CaM has N‐ and C‐terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF–CaM complex show how EF locks the N‐terminal domain of CaM into a closed conformation regardless of its calcium‐loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM and uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF–CaM complexed with nucleotides show that EF uses two‐metal–ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3′OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two‐metal–ion catalysis, suggesting the catalytic mechanism‐driven convergent evolution of two structurally diverse adenylyl cyclases.

Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin

CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C‐terminal domain of calmodulin. Four discrete regions of CyaA bind calcium‐loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an α‐helix of CyaA makes extensive contacts with the calcium‐induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin‐induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA–calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.