Bioorthogonal Catalysis for Treatment of Solid Tumors Using Thermostable, Self-Assembling, Single Enzyme Nanoparticles and Natural Product Conversion with Indole-3-acetic Acid.

Bioorthogonal catalysis (BC) generates chemical reactions not present in normal physiology for the purpose of disease treatment. Because BC catalytically produces the desired therapy only at the site of disease, it holds the promise of site-specific treatment with little or no systemic exposure or side effects. Transition metals are typically used as catalytic centers in BC; however, solubility and substrate specificity typically necessitate a coordinating enzyme and/or stabilizing superstructure for in vivo application. We report the use of self-assembling, porous exoshells (tESs) to encapsulate and deliver an iron-containing reaction center for the treatment of breast cancer. The catalytic center is paired with indole-3-acetic acid (IAA), a natural product found in edible plants, which undergoes oxidative decarboxylation, via reduction of iron(III) to iron(II), to produce free radicals and bioactive metabolites. The tES encapsulation is critical for endocytic uptake of BC reaction centers and, when followed by administration of IAA, results in apoptosis of MDA-MB-231 triple negative cancer cells and complete regression of in vivo orthotopic xenograft tumors (p < 0.001, n = 8 per group). When Renilla luciferase (rLuc) is substituted for horseradish peroxidase (HRP), whole animal luminometry can be used to monitor in vivo activity.

A general approach to protein folding using thermostable exoshells.

In vitro protein folding is a complex process which often results in protein aggregation, low yields and low specific activity. Here we report the use of nanoscale exoshells (tES) to provide complementary nanoenvironments for the folding and release of 12 highly diverse protein substrates ranging from small protein toxins to human albumin, a dimeric protein (alkaline phosphatase), a trimeric ion channel (Omp2a) and the tetrameric tumor suppressor, p53. These proteins represent a unique diversity in size, volume, disulfide linkages, isoelectric point and multi versus monomeric nature of their functional units. Protein encapsulation within tES increased crude soluble yield (3-fold to >100-fold), functional yield (2-fold to >100-fold) and specific activity (3-fold to >100-fold) for all the proteins tested. The average soluble yield was 6.5 mg/100 mg of tES with charge complementation between the tES internal cavity and the protein substrate being the primary determinant of functional folding. Our results confirm the importance of nanoscale electrostatic effects and provide a solution for folding proteins in vitro.

Redefining IL11 as a regeneration-limiting hepatotoxin and therapeutic target in acetaminophen-induced liver injury.

Acetaminophen (N-acetyl-p-aminophenol; APAP) toxicity is a common cause of liver damage. In the mouse model of APAP-induced liver injury (AILI), interleukin 11 (IL11) is highly up-regulated and administration of recombinant human IL11 (rhIL11) has been shown to be protective. Here, we demonstrate that the beneficial effect of rhIL11 in the mouse model of AILI is due to its inhibition of endogenous mouse IL11 activity. Our results show that species-matched IL11 behaves like a hepatotoxin. IL11 secreted from APAP-damaged human and mouse hepatocytes triggered an autocrine loop of NADPH oxidase 4 (NOX4)-dependent cell death, which occurred downstream of APAP-initiated mitochondrial dysfunction. Hepatocyte-specific deletion of Il11 receptor subunit alpha chain 1 (Il11ra1) in adult mice protected against AILI despite normal APAP metabolism and glutathione (GSH) depletion. Mice with germline deletion of Il11 were also protected from AILI, and deletion of Il1ra1 or Il11 was associated with reduced c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation and quickly restored GSH concentrations. Administration of a neutralizing IL11RA antibody reduced AILI in mice across genetic backgrounds and promoted survival when administered up to 10 hours after APAP. Inhibition of IL11 signaling was associated with the up-regulation of markers of liver regenerations: cyclins and proliferating cell nuclear antigen (PCNA) as well as with phosphorylation of retinoblastoma protein (RB) 24 hours after AILI. Our data suggest that species-matched IL11 is a hepatotoxin and that IL11 signaling might be an effective therapeutic target for APAP-induced liver damage.

Robust Performance of Potentially Functional SNPs in Machine Learning Models for the Prediction of Atorvastatin-Induced Myalgia.

Statins can cause muscle symptoms resulting in poor adherence to therapy and increased cardiovascular risk. We hypothesize that combinations of potentially functional SNPs (pfSNPs), rather than individual SNPs, better predict myalgia in patients on atorvastatin. This study assesses the value of potentially functional single nucleotide polymorphisms (pfSNPs) and employs six machine learning algorithms to identify the combination of SNPs that best predict myalgia. Methods: Whole genome sequencing of 183 Chinese, Malay and Indian patients from Singapore was conducted to identify genetic variants associated with atorvastatin induced myalgia. To adjust for confounding factors, demographic and clinical characteristics were also examined for their association with myalgia. The top factor, sex, was then used as a covariate in the whole genome association analyses. Variants that were highly associated with myalgia from this and previous studies were extracted, assessed for potential functionality (pfSNPs) and incorporated into six machine learning models. Predictive performance of a combination of different models and inputs were compared using the average cross validation area under ROC curve (AUC). The minimum combination of SNPs to achieve maximum sensitivity and specificity as determined by AUC, that predict atorvastatin-induced myalgia in most, if not all the six machine learning models was determined. Results: Through whole genome association analyses using sex as a covariate, a larger proportion of pfSNPs compared to non-pf SNPs were found to be highly associated with myalgia. Although none of the individual SNPs achieved genome wide significance in univariate analyses, machine learning models identified a combination of 15 SNPs that predict myalgia with good predictive performance (AUC >0.9). SNPs within genes identified in this study significantly outperformed SNPs within genes previously reported to be associated with myalgia. pfSNPs were found to be more robust in predicting myalgia, outperforming non-pf SNPs in the majority of machine learning models tested. Conclusion: Combinations of pfSNPs that were consistently identified by different machine learning models to have high predictive performance have good potential to be clinically useful for predicting atorvastatin-induced myalgia once validated against an independent cohort of patients.

Prioritizing Candidates of Post-Myocardial Infarction Heart Failure Using Plasma Proteomics and Single-Cell Transcriptomics.

BACKGROUND: Heart failure (HF) is the most common long-term complication of acute myocardial infarction (MI). Understanding plasma proteins associated with post-MI HF and their gene expression may identify new candidates for biomarker and drug target discovery. METHODS: We used aptamer-based affinity-capture plasma proteomics to measure 1305 plasma proteins at 1 month post-MI in a New Zealand cohort (CDCS [Coronary Disease Cohort Study]) including 181 patients post-MI who were subsequently hospitalized for HF in comparison with 250 patients post-MI who remained event free over a median follow-up of 4.9 years. We then correlated plasma proteins with left ventricular ejection fraction measured at 4 months post-MI and identified proteins potentially coregulated in post-MI HF using weighted gene co-expression network analysis. A Singapore cohort (IMMACULATE [Improving Outcomes in Myocardial Infarction through Reversal of Cardiac Remodelling]) of 223 patients post-MI, of which 33 patients were hospitalized for HF (median follow-up, 2.0 years), was used for further candidate enrichment of plasma proteins by using Fisher meta-analysis, resampling-based statistical testing, and machine learning. We then cross-referenced differentially expressed proteins with their differentially expressed genes from single-cell transcriptomes of nonmyocyte cardiac cells isolated from a murine MI model, and single-cell and single-nucleus transcriptomes of cardiac myocytes from murine HF models and human patients with HF. RESULTS: In the CDCS cohort, 212 differentially expressed plasma proteins were significantly associated with subsequent HF events. Of these, 96 correlated with left ventricular ejection fraction measured at 4 months post-MI. Weighted gene co-expression network analysis prioritized 63 of the 212 proteins that demonstrated significantly higher correlations among patients who developed post-MI HF in comparison with event-free controls (data set 1). Cross-cohort meta-analysis of the IMMACULATE cohort identified 36 plasma proteins associated with post-MI HF (data set 2), whereas single-cell transcriptomes identified 15 gene-protein candidates (data set 3). The majority of prioritized proteins were of matricellular origin. The 6 most highly enriched proteins that were common to all 3 data sets included well-established biomarkers of post-MI HF: N-terminal B-type natriuretic peptide and troponin T, and newly emergent biomarkers, angiopoietin-2, thrombospondin-2, latent transforming growth factor-ß binding protein-4, and follistatin-related protein-3, as well. CONCLUSIONS: Large-scale human plasma proteomics, cross-referenced to unbiased cardiac transcriptomics at single-cell resolution, prioritized protein candidates associated with post-MI HF for further mechanistic and clinical validation.

Predicting the Shapes of Protein Complexes through Collision Cross Section Measurements and Database Searches.

In structural biology, collision cross sections (CCSs) from ion mobility mass spectrometry (IM-MS) measurements are routinely compared to computationally or experimentally derived protein structures. Here, we investigate whether CCS data can inform about the shape of a protein in the absence of specific reference structures. Analysis of the proteins in the CCS database shows that protein complexes with low apparent densities are structurally more diverse than those with a high apparent density. Although assigning protein shapes purely on CCS data is not possible, we find that we can distinguish oblate- and prolate-shaped protein complexes by using the CCS, molecular weight, and oligomeric states to mine the Protein Data Bank (PDB) for potentially similar protein structures. Furthermore, comparing the CCS of a ferritin cage to the solution structures in the PDB reveals significant deviations caused by structural collapse in the gas phase. We then apply the strategy to an integral membrane protein by comparing the shapes of a prokaryotic and a eukaryotic sodium/proton antiporter homologue. We conclude that mining the PDB with IM-MS data is a time-effective way to derive low-resolution structural models.

Magnetic fields modulate metabolism and gut microbiome in correlation with Pgc-1α expression: Follow-up to an in vitro magnetic mitohormetic study.

Exercise modulates metabolism and the gut microbiome. Brief exposure to low mT-range pulsing electromagnetic fields (PEMFs) was previously shown to accentuate in vitro myogenesis and mitochondriogenesis by activating a calcium-mitochondrial axis upstream of PGC-1α transcriptional upregulation, recapitulating a genetic response implicated in exercise-induced metabolic adaptations. We compared the effects of analogous PEMF exposure (1.5 mT, 10 min/week), with and without exercise, on systemic metabolism and gut microbiome in four groups of mice: (a) no intervention; (b) PEMF treatment; (c) exercise; (d) exercise and PEMF treatment. The combination of PEMFs and exercise for 6 weeks enhanced running performance and upregulated muscular and adipose Pgc-1α transcript levels, whereas exercise alone was incapable of elevating Pgc-1α levels. The gut microbiome Firmicutes/Bacteroidetes ratio decreased with exercise and PEMF exposure, alone or in combination, which has been associated in published studies with an increase in lean body mass. After 2 months, brief PEMF treatment alone increased Pgc-1α and mitohormetic gene expression and after >4 months PEMF treatment alone enhanced oxidative muscle expression, fatty acid oxidation, and reduced insulin levels. Hence, short-term PEMF treatment was sufficient to instigate PGC-1α-associated transcriptional cascades governing systemic mitohormetic adaptations, whereas longer-term PEMF treatment was capable of inducing related metabolic adaptations independently of exercise.

Patients with acute and chronic coronary syndromes have elevated long-term thrombin generation.

Coronary artery disease is a leading cause of morbidity and mortality worldwide. Despite significant advances in revascularization strategies and antiplatelet therapy with aspirin and/or P2Y12 receptor antagonist, patients with acute coronary syndrome (ACS) continue to be at long-term risk of further cardiovascular events. Besides platelet activation, the role of thrombin generation (TG) in atherothrombotic complications is widely recognized. In this study, we hypothesized that there is an elevation of coagulation activation persists beyond 12 months in patients with ACS and chronic coronary syndrome (CCS) when compared with healthy controls. We measured TG profiles of patients within 72 h after percutaneous coronary intervention, at 6-month, 12-month and 24-month. Our results demonstrated that TG of patients with ACS (n = 114) and CCS (n = 40) were persistently elevated when compared to healthy individuals (n = 50) in peak thrombin (ACS 273.1 nM vs CCS 287.3 nM vs healthy 234.3 nM) and velocity index (ACS 110.2 nM/min vs CCS 111.0 nM/min vs healthy 72.9 nM/min) at 24-month of follow-up. Our results suggest a rationale for addition of anticoagulation to antiplatelet therapy in preventing long-term ischemic events after ACS. Further research could clarify whether the use of TG parameters to enable risk stratification of patients at heightened long-term procoagulant risk who may benefit most from dual pathway inhibition.

Surface protein engineering increases the circulation time of a cell membrane-based nanotherapeutic.

Mammalian cell membranes are often incompatible with chemical modifications typically used to increase circulation half-life. Using cellular nanoghosts as a model, we show that proline-alanine-serine (PAS) peptide sequences expressed on the membrane surface can extend the circulation time of a cell membrane derived nanotherapeutic. Membrane expression of a PAS 40 repeat sequence decreased protein binding and resulted in a 90% decrease in macrophage uptake when compared with non-PASylated controls (P ≤ 0.05). PASylation also extended circulation half-life (t1/2 = 37 h) compared with non-PASylated controls (t1/2 = 10.5 h) (P ≤ 0.005), resulting in ~7-fold higher in vivo serum concentrations at 24 h and 48 h (P ≤ 0.005). Genetically engineered membrane expression of PAS repeats may offer an alternative to PEGylation and provide extended circulation times for cellular membrane-derived nanotherapeutics.

Sources of variability in quantifying circulating thymosin beta-4: literature review and recommendations.

INTRODUCTION: Thymosin beta-4 (TB4) is an endogenous peptide with protective and regenerative effects in models of cellular and organ injury. TB4 is increasingly measured as a potential plasma or serum biomarker in human cardiovascular, liver, infectious, and autoimmune disease. AREAS COVERED: The focus of this review is the quantification of TB4 in clinical cohort studies and whether reported TB4 concentrations differ with respect to method of sample preparation. We survey current literature for studies measuring TB4 in human serum or plasma and compare reported concentrations in healthy controls. EXPERT OPINION: We find substantial intra- and inter- study variability in healthy controls, and a lack of protocol standardization. We further highlight three factors that may confound TB4 clinical measurements and should be considered in future study design: 1) residual platelets remaining in suspension after centrifugation, 2) TB4 release following ex vivo platelet activation, and 3) specificity of assays towards posttranslational modifications. Accordingly, we put forth our recommendations to minimize residual and activated platelets during sample collection, and to cross-validate TB4 measurements using both antibody-based and mass spectrometry-based methods.

Thermostable exoshells fold and stabilize recombinant proteins.

The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.

Thymosin Beta-4 Is Elevated in Women With Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Thymosin beta-4 (TB4) is an X-linked gene product with cardioprotective properties. Little is known about plasma concentration of TB4 in heart failure (HF), and its relationship with other cardiovascular biomarkers. We sought to evaluate circulating TB4 in HF patients with preserved (HFpEF) or reduced (HFrEF) ejection fraction compared to non-HF controls. METHODS AND RESULTS: TB4 was measured using a liquid chromatography and mass spectrometry assay in age- and sex-matched HFpEF (n=219), HFrEF (n=219) patients, and controls (n=219) from a prospective nationwide study. Additionally, a 92-marker multiplex proximity extension assay was measured to identify biomarker covariates. Compared with controls, plasma TB4 was elevated in HFpEF (985 [421-1723] ng/mL versus 1401 [720-2379] ng/mL, P<0.001), but not in HFrEF (1106 [556-1955] ng/mL, P=0.642). Stratifying by sex, only women (1623 [1040-2625] ng/mL versus 942 [386-1891] ng/mL, P<0.001), but not men (1238.5 [586-1967] ng/mL versus 1004 [451-1538] ng/mL, P=1.0), had significantly elevated TB4 in the setting of HFpEF. Adjusted for New York Heart Association class, N-terminal pro B-type natriuretic peptide, age, and myocardial infarction, hazard ratio to all-cause mortality is significantly higher in women with elevated TB4 (1.668, P=0.036), but not in men (0.791, P=0.456) with HF. TB4 is strongly correlated with a cluster of 7 markers from the proximity extension assay panel, which are either X-linked, regulated by sex hormones, or involved with NF-κB signaling. CONCLUSIONS: We show that plasma TB4 is elevated in women with HFpEF and has prognostic information. Because TB4 can preserve EF in animal studies of cardiac injury, the relation of endogenous, circulating TB4 to X chromosome biology and differential outcomes in female heart disease warrants further study.

Treatment with the MAO-A inhibitor clorgyline elevates monoamine neurotransmitter levels and improves affective phenotypes in a mouse model of Huntington disease.

Abnormal monoamine oxidase A and B (MAO-A/B) activity and an imbalance in monoamine neurotransmitters have been suggested to underlie the pathobiology of depression, a major psychiatric symptom observed in patients with neurodegenerative diseases, such as Huntington disease (HD). Increased MAO-A/B activity has been observed in brain tissue from patients with HD and in human and rodent HD neural cells. Using the YAC128 mouse model of HD, we studied the effect of an irreversible MAO-A inhibitor, clorgyline, on the levels of select monoamine neurotransmitters associated with affective function. We observed a decrease in striatal levels of the MAO-A/B substrates, dopamine and norepinephrine, in YAC128 HD mice compared with wild-type mice, which was accompanied by increased anxiety- and depressive-like behaviour at five months of age. Treatment for 26 days with clorgyline restored dopamine, serotonin, and norepinephrine neurotransmitter levels in the striatum and reduced anxiety- and depressive-like behaviour in YAC128 HD mice. This study supports a potential therapeutic use for MAO-A inhibitors in the treatment of depression and anxiety in patients with HD.

Ergothioneine, an adaptive antioxidant for the protection of injured tissues? A hypothesis.

Ergothioneine (ET) is a diet-derived, thiolated derivative of histidine with antioxidant properties. Although ET is produced only by certain fungi and bacteria, it can be found at high concentrations in certain human and animal tissues and is absorbed through a specific, high affinity transporter (OCTN1). In liver, heart, joint and intestinal injury, elevated ET concentrations have been observed in injured tissues. The physiological role of ET remains unclear. We thus review current literature to generate a specific hypothesis: that the accumulation of ET in vivo is an adaptive mechanism, involving the regulated uptake and concentration of an exogenous natural compound to minimize oxidative damage.

Microengineering in cardiovascular research: new developments and translational applications.

Microfluidic, cellular co-cultures that approximate macro-scale biology are important tools for refining the in vitro study of organ-level function and disease. In recent years, advances in technical fabrication and biological integration have provided new insights into biological phenomena, improved diagnostic measurements, and made major steps towards de novo tissue creation. Here we review applications of these technologies specific to the cardiovascular field, emphasizing three general categories of use: reductionist vascular models, tissue-engineered vascular models, and point-of-care diagnostics. With continued progress in the ability to purposefully control microscale environments, the detailed study of both primary and cultured cells may find new relevance in the general cardiovascular research community.

Real-time measurement of thrombin generation using continuous droplet microfluidics.

Thrombin, which has the leading role in the blood coagulation cascade, is an important biomarker in hemostasis and cardiovascular disease (CVD) development. In this study, a measurement system capable of continuously monitoring individual thrombin generation using droplet microfluidic technology is manipulated. The thrombin generation assay based on fluogenic substrate is performed within the droplets and the thrombin generation curve of plasma sample activated by tissue factor is measured in real-time to reflect the sample conditions dynamically. The injection of the inhibitor of thrombin generation is developed to assay the inhibited curve which relates to thrombin self-inhibition in biological systems. This microfluidic system is integrated with the microdialysis probe, which is useful to connect to the living animals for future in vivo real time thrombin measurements for rapid CVD diagnosis.

Superstructure based on ß-CD self-assembly induced by a small guest molecule.

The size, shape and surface chemistry of nanoparticles play an important role in cellular interaction. Thus, the main objective of the present study was the determination of the ß-cyclodextrin (ß-CD) self-assembly thermodynamic parameters and its structure, aiming to use these assemblies as a possible controlled drug release system. Light scattering measurements led us to obtain the ß-CD's critical aggregation concentration (cac) values, and consequently the thermodynamic parameters of the ß-CD spontaneous self-assembly in aqueous solution: Δ(agg)G(o) = -16.31 kJ mol(-1), Δ(agg)H(o) = -26.48 kJ mol(-1) and TΔ(agg)S(o) = -10.53 kJ mol(-1) at 298.15 K. Size distribution of the self-assembled nanoparticles below and above cac was 1.5 nm and 60-120 nm, respectively. The number of ß-CD molecules per cluster and the second virial coefficient were identified through Debye's plot and molecular dynamic simulations proposed the three-fold assembly for this system below cac. Ampicillin (AMP) was used as a drug model in order to investigate the key role of the guest molecule in the self-assembly process and the ß-CD:AMP supramolecular system was studied in solution, aiming to determine the structure of the supramolecular aggregate. Results obtained in solution indicated that the ß-CD's cac was not affected by adding AMP. Moreover, different complex stoichiometries were identified by nuclear magnetic resonance and isothermal titration calorimetry experiments.

In vivo prevention of arterial restenosis with paclitaxel-encapsulated targeted lipid-polymeric nanoparticles.

Following recent successes with percutaneous coronary intervention (PCI) for treating coronary artery disease (CAD), many challenges remain. In particular, mechanical injury from the procedure results in extensive endothelial denudation, exposing the underlying collagen IV-rich basal lamina, which promotes both intravascular thrombosis and smooth muscle proliferation. Previously, we reported the engineering of collagen IV-targeting nanoparticles (NPs) and demonstrated their preferential localization to sites of arterial injury. Here, we develop a systemically administered, targeted NP system to deliver an antiproliferative agent to injured vasculature. Approximately 60-nm lipid-polymeric NPs were surface functionalized with collagen IV-targeting peptides and loaded with paclitaxel. In safety studies, the targeted NPs showed no signs of toxicity and a ≥3.5-fold improved maximum tolerated dose versus paclitaxel. In efficacy studies using a rat carotid injury model, paclitaxel (0.3 mg/kg or 1 mg/kg) was i.v. administered postprocedure on days 0 and 5. The targeted NP group resulted in lower neointima-to-media (N/M) scores at 2 wk versus control groups of saline, paclitaxel, or nontargeted NPs. Compared with sham-injury groups, an ∼50% reduction in arterial stenosis was observed with targeted NP treatment. The combination of improved tolerability, sustained release, and vascular targeting could potentially provide a safe and efficacious option in the management of CAD.

Photo-response behavior of electrospun nanofibers based on spiropyran-cyclodextrin modified polymer.

Tunable and durable photochromic materials are a rapidly expanding area of interest, with applications ranging from biomedical devices to industrial-fields. Here we examine electrospun poly (methacrylic acid) PMAA nanofibers covalently modified with the highly photochromic molecule, spiropyran (SP) or a derivate SP which is firstly coupled to a cyclodextrin molecule (ßCDSP). The photochromic properties of the starting materials and of the nanofibers were investigated. ßCDSP, PMAASP and PMAA-ßCDSP polymers exhibited a reverse photochromism. The kinetic results revealed a faster isomerization process for the ßCDSP molecule, than that for the PMAA-ßCDSP and for the PMAASP, the slowest one. The fastest isomerization is attributed to the presence of a large number of hydroxyl groups of the ßCD which stabilizes the merocyanine form via hydrogen bonding, and the slowest isomerization is related to the PMAA chain structure that stabilizes the spiropyran form. Thus, combining the PMAA and ßCD properties the photo-isomerization can be modulated. The photoreversibility of this material was verified by UV-visible measurements cycling visible and UV light. Infrared spectroscopy and water contact angle were used for the nanofiber surface characterization, demonstrating the presence of the spiropyran on the mats surface and also showing a minimal effect on nanofiber size and shape when compared to PMAA fiber.

Structure of anthrax edema factor-calmodulin-adenosine 5'-(alpha,beta-methylene)-triphosphate complex reveals an alternative mode of ATP binding to the catalytic site.

Anthrax edema factor (EF) is a key virulence factor secreted by Bacillus anthracis. Here, we report a structure, at 3.0 A resolution, of the catalytic domain of EF (EF3) in complex with calmodulin (CaM) and adenosine 5'-(alpha,beta-methylene)-triphosphate (AMPCPP). Although the binding of the triphosphate of AMPCPP to EF3 can be superimposed on that of previously determined 3'deoxy-ATP (3'dATP) and 2'deoxy 3' anthraniloyl-ATP (2'd3' ANT-ATP) in EF3-CaM, the ribose and the adenine rings of AMPCPP are rotated approximately 105 and 180 degrees, respectively, relative to those of 3'dATP and 2'd3'ANT-ATP. Based on this model, K382 and F586 should play key roles in the recognition of adenine. However, mutations of these residues to alanine either separately or together cause only modest changes in Michaelis-Menten constants and IC50 values of AMPCPP and cAMP. Therefore, this alternate binding mode of the adenosine of AMPCPP binds to EF likely playing only a minor role in ATP binding and in catalysis.