Yuequan Shen

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.

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.

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.

Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins

Edema factor (EF) and CyaA are calmodulin (CaM)‐activated adenylyl cyclase exotoxins involved in the pathogenesis of anthrax and whooping cough, respectively. Using spectroscopic, enzyme kinetic and surface plasmon resonance spectroscopy analyses, we show that low Ca2+ concentrations increase the affinity of CaM for EF and CyaA causing their activation, but higher Ca2+ concentrations directly inhibit catalysis. Both events occur in a physiologically relevant range of Ca2+ concentrations. Despite the similarity in Ca2+ sensitivity, EF and CyaA have substantial differences in CaM binding and activation. CyaA has 100‐fold higher affinity for CaM than EF. CaM has N‐ and C‐terminal globular domains, each binding two Ca2+ ions. CyaA can be fully activated by CaM mutants with one defective C‐terminal Ca2+‐binding site or by either terminal domain of CaM while EF cannot. EF consists of a catalytic core and a helical domain, and both are required for CaM activation of EF. Mutations that decrease the interaction of the helical domain with the catalytic core create an enzyme with higher sensitivity to Ca2+–CaM activation. However, CyaA is fully activated by CaM without the domain corresponding to the helical domain of EF.

Structure of Substrate-Free Human Insulin Degrading Enzyme (Ide) and Biophysical Analysis of ATP-Induced Conformational Switch of Ide

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that hydrolyzes amyloid-β (Aβ) and insulin, which are peptides associated with Alzheimer disease (AD) and diabetes, respectively. Our previous structural analysis of substrate-bound human 113-kDa IDE reveals that the N- and C-terminal domains of IDE, IDE-N and IDE-C, make substantial contact to form an enclosed catalytic chamber to entrap its substrates. Furthermore, IDE undergoes a switch between the closed and open conformations for catalysis. Here we report a substrate-free IDE structure in its closed conformation, revealing the molecular details of the active conformation of the catalytic site of IDE and new insights as to how the closed conformation of IDE may be kept in its resting, inactive conformation. We also show that Aβ is degraded more efficiently by IDE carrying destabilizing mutations at the interface of IDE-N and IDE-C (D426C and K899C), resulting in an increase in Vmax with only minimal changes to Km. Because ATP is known to activate the ability of IDE to degrade short peptides, we investigated the interaction between ATP and activating mutations. We found that these mutations rendered IDE less sensitive to ATP activation, suggesting that ATP might facilitate the transition from the closed state to the open conformation. Consistent with this notion, we found that ATP induced an increase in hydrodynamic radius, a shift in electrophoretic mobility, and changes in secondary structure. Together, our results highlight the importance of the closed conformation for regulating the activity of IDE and provide new molecular details that will facilitate the development of activators and inhibitors of IDE.

Crystallization and preliminary X-ray study of the edema factor exotoxin adenylyl cyclase domain from Bacillus anthracis in the presence of its activator, calmodulin

Edema factor from Bacillus anthracis is a 92 kDa secreted adenylyl cyclase exotoxin and is activated by the host-resident protein calmodulin. Calmodulin is a ubiquitous intracellular calcium sensor in eukaryotes and activates edema factor nearly 1000-fold upon binding. While calmodulin has many known effectors, including kinases, phosphodiesterases, motor proteins, channels and type 1 adenylyl cyclases, no structures of calmodulin in complex with a functional enzyme have been solved. The crystallization and initial experimental phasing of crystals containing a complex of edema factor adenylyl cyclase domain and calmodulin are reported here. The edema factor-calmodulin complex crystallizes in three different space groups. A native data set in the I222 space group has been collected to 2.7 A and the self-rotation function solution suggests three edema factor-calmodulin complexes in each asymmetric unit. Initial 4 A phases were obtained by selenomethionyl MAD in combination with two heavy-atom derivatives. These phases were successfully extended to 2.7 A using NCS averaging.