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 a-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.

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.

Macrophage inflammatory protein-1 (MIP-1), MIP-1α (CCL3) and MIP-1β (CCL4) are chemokines crucial for immune responses towards infection and inflammation. Both MIP-1α and MIP-1β form high-molecular-weight aggregates. Our crystal structures reveal that MIP-1 aggregation is a polymerization process and human MIP-1α and MIP-1β form rod-shaped, double-helical polymers. Biophysical analyses and mathematical modelling show that MIP-1 reversibly forms a polydisperse distribution of rod-shaped polymers in solution. Polymerization buries receptor-binding sites of MIP-1α, thus depolymerization mutations enhance MIP-1α to arrest monocytes onto activated human endothelium. However, same depolymerization mutations render MIP-1α ineffective in mouse peritoneal cell recruitment. Mathematical modelling reveals that, for a long-range chemotaxis of MIP-1, polymerization could protect MIP-1 from proteases that selectively degrade monomeric MIP-1. Insulin-degrading enzyme (IDE) is identified as such a protease and decreased expression of IDE leads to elevated MIP-1 levels in microglial cells. Our structural and proteomic studies offer a molecular basis for selective degradation of MIP-1. The regulated MIP-1 polymerization and selective inactivation of MIP-1 monomers by IDE could aid in controlling the MIP-1 chemotactic gradient for immune surveillance.

Insulin is a hormone vital for glucose homeostasis, and insulindegrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our timedependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in its initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N-and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-Å resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity (ϳ100 nM) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages. IDE3 is an ϳ110-kDa zinc metalloprotease that is evolutionarily conserved from bacteria to humans (1, 2). It was first discovered based on its high affinity to bind insulin (ϳ100 nM) and degrade it into pieces (3, 4). Insulin is a 5.8-kDa hormone that plays a central role in glucose homeostasis and the development of diabetes in humans. Consistent with the in vitro activity of IDE for insulin degradation, loss-of-function mutations of IDE in rodents result in elevated insulin levels and glucose intolerance (5). In addition, a nucleotide polymorphism of the human IDE gene is linked to type 2 diabetes (6). Later studies showed that IDE can also degrade amyloid-␤ (A␤), a peptide vital to the progression of Alzheimer disease (7,8). Accumulating evidence from rodent models and human genetic analyses also indicate the physiological role of IDE in the clearance of A␤ (5, 9 -12).Despite nearly 60 years of studies on IDE, the molecular basis by which IDE binds, unfolds, and degrades insulin has only begun to be elucidated. Different from ATP-dependent proteases, IDE does not require the additional energy source such as ATP to unfold, bind, and cleave its substrates (4, 13). Insulin consists of the A and B chains that are held together by two inter-and one intra-chain disulfide bonds. Remarkably, IDE does not require disulfide bond isomerase activity to unfold and cleave insulin (4). Thus, IDE needs to overcome the stability created by the d...

Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-β (Aβ), peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulingrowth factor-II (IGF-II) and transforming growth factor-α (TGF-α) over IGF-I and epidermal growth factor (EGF), respectively. Here, we used high accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-α, amylin, reduced amylin, and Aβ by human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-α at 2.3 Å and IDE-amylin at 2.9 Å. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (aa 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and EGF families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their Nterminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.

The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system in studies of the role of protein dynamics in catalysis, but so far these studies were limited by the availability of structural information. Second, CbFDH and its mutants are of use in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in its commercial development. Here, we report the crystallization and structural determination of both holo-CbFDH and apo-CbFDH. The free energy barrier for the catalyzed reaction is computed, and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: The apo-enzyme crystal was obtained under co-crystallization conditions with a saturating concentration of both the cofactor (NAD+) and inhibitor (azide), which has a nM dissociation constant. It was found that the fraction of the apo-enzyme present in the solution is less than 1.7x10−7 (i.e. the solution is 99.9999% holo-enzyme). This is an extreme case where the crystal structure represents an insignificant fraction of enzyme in solution, and a mechanism rationalizing this phenomenon is presented.

Despite the availability of elaborate varieties of nanoparticles, their assembly into regular superstructures and photonic materials remains challenging. Here we show how flexible films of stacked polymer nanoparticles can be directly assembled in a roll-to-roll process using a bending-induced oscillatory shear technique. For sub-micron spherical nanoparticles, this gives elastomeric photonic crystals termed polymer opals showing extremely strong tunable structural colour. With oscillatory strain amplitudes of 300%, crystallization initiates at the wall and develops quickly across the bulk within only five oscillations. The resulting structure of random hexagonal close-packed layers is improved by shearing bidirectionally, alternating between two in-plane directions. Our theoretical framework indicates how the reduction in shear viscosity with increasing order of each layer accounts for these results, even when diffusion is totally absent. This general principle of shear ordering in viscoelastic media opens the way to manufacturable photonic materials, and forms a generic tool for ordering nanoparticles.

Bacillus anthracis, the etiologic agent for anthrax, secretes edema factor (EF) to disrupt intracellular signaling pathways. Upon translocation into host cells and association with a calcium sensor, calmodulin (CaM), EF becomes a highly active adenylyl cyclase (AC) that raises the intracellular concentration of cyclic AMP (cAMP). Growing evidence shows that EF plays a key role in anthrax pathogenesis by affecting cellular functions vital for host defense. This strategy is also used by Bordetella pertussis, a bacterium that causes whooping cough. Pertussis bacteria secrete the bifunctional toxin CyaA which raises the intracellular cAMP. Here, we discuss recent advances from structural analyses that reveal the molecular basis of the conserved mechanism of activation and catalysis of EF and CyaA by CaM even though these two toxins use the completely different sequences to bind CaM. Comparison of the biochemical and structural characteristics of these two AC toxins with host ACs reveal that they have diverse strategies of catalytic activation, yet use the same two-metal-ion catalytic mechanism. Keywordsanthrax edema factor; pertussis CyaA; class III adenylyl cyclase Edema factor (EF) is an 89-kDa protein secreted by Bacillus anthracis, the Gram-positive bacterium that cause anthrax. EF belongs to a family of bacterial toxins that can specifically elevate the intracellular cyclic AMP (cAMP) level, which is the prototypic second messenger that regulates diverse cellular responses. The biological effects of cAMP are mediated by the binding of cAMP to three families of signal transducers: cAMP-dependent protein kinases, cyclic nucleotide gated channels (Biel and Michalakis 2009), and EPAC, the guanine nucleotide exchange factor for Ras GTPase homologs Rap1 and Rap2 (Borland, Smith, and Yarwood 2009). There are two major mechanisms by which bacterial toxins can raise the intracellular cAMP level. The first mechanism is by the action of the bacterial adenylyl cyclase (AC) toxins, which possess AC activity, the enzymatic activity that converts ATP to cAMP. Their AC activities are activated only upon their entrance into host cells and association with the specific cellular proteins that serve as the activator. In addition to the aforementioned EF from Bacillus anthracis, two other examples of AC toxins include CyaA from Bordetella pertussis and ExoY from Pseudomonas aeruginosa (Leppla 1984;Wolff et al. 1980;Yahr et al. 1998). EF and CyaA share CaM as a common activator while the activator of ExoY is still unknown. The second mechanism by which intracellular cAMP is increased by bacteria is the ADP-ribosylation of heterotrimeric G proteins by bacterial toxins, resulting in increased catalytic activity of host membrane-bound AC (mAC). Cholera toxin from Vibrio cholera and labile toxin from Escherichia coli can ADP-ribosylate Gsα, rendering it constitutively active to stimulate host mAC. History of EF discovery and recent advancesEF was identified based on its ability to induce cutaneous edema and mortality of experi...