Real-world data on discordance between estrogen, progesterone, and HER2 receptor expression on diagnostic tumor biopsy versus tumor resection material.

PURPOSE: The estrogen (ER), progesterone (PR), and HER2 status are essential in guiding treatment decisions in breast cancer patients. In daily life, the ER/PR/HER2 status is expected to be commonly tested twice, i.e., at diagnosis using material from tumor needle biopsies, and after tumor resection using full tumor tissue material. This study explored the discordance of ER/PR/HER2 between tumor needle biopsies and full tumor resection material using real-world patient-level data from Dutch breast cancer patients. METHODS: Pathology reports of 11,054 breast cancer patients were derived from PALGA (Dutch Pathology Registry). Discordance was calculated for multiple combinations of the ER/PR/HER2 receptor status. The influence of patient and tumor characteristics on the probability of having discordant test results was analyzed using multiple logistic regression models (separately for ER, PR and HER2). RESULTS: For 1279 patients (14.4%), at least one of the receptors (ER/PR/HER2) was determined on both biopsy and tumor tissue material. The majority had concordant test results for ER (n = 916; 94.8%), PR (n = 1170; 86.7%), and HER2 (n = 881; 98.1%). Patients having an ER- and HER2-positive but PR-negative biopsy classification, BR grade III, and < 10% tumor tissue remaining after neoadjuvant therapy (NAT) have the highest probability of ER discordant test results (OR 4.991; p = 83.31%). The probability of discordance in PR is based on different sets of patient and tumor characteristics. Potential cost savings from omitting multiple tests if concordance can be perfectly predicted can be up to €205,000 yearly. CONCLUSIONS: Double testing of ER/PR/HER2 is less common than expected. Discordance in ER/PR/HER2 test results between tumor needle biopsy taken at the time of diagnosis and tumor resection material is very low, especially in patients not receiving any form of neoadjuvant therapy. These results imply that a substantial number of tests can potentially be omitted in specific subgroups of breast cancer patients.

Cost-effectiveness of procalcitonin testing to guide antibiotic treatment duration in critically ill patients: results from a randomised controlled multicentre trial in the Netherlands.

BACKGROUND: Procalcitonin (PCT) testing can help in safely reducing antibiotic treatment duration in intensive care patients with sepsis. However, the cost-effectiveness of such PCT guidance is not yet known. METHODS: A trial-based analysis was performed to estimate the cost-effectiveness of PCT guidance compared with standard of care (without PCT guidance). Patient-level data were used from the SAPS trial in which 1546 patients were randomised. This trial was performed in the Netherlands, which is a country with, on average, low antibiotic use and a short duration of hospital stay. As quality of life among sepsis survivors was not measured during the SAPS, this was derived from a Dutch follow-up study. Outcome measures were (1) incremental direct hospital cost and (2) incremental cost per quality-adjusted life year (QALY) gained from a healthcare perspective over a one-year time horizon. Uncertainty in outcomes was assessed with bootstrapping. RESULTS: Mean in-hospital costs were €46,081/patient in the PCT group compared with €46,146/patient with standard of care (i.e. - €65 (95% CI - €6314 to €6107); - 0.1%). The duration of the first course of antibiotic treatment was lower in the PCT group with 6.9 vs. 8.2 days (i.e. - 1.2 days (95% CI - 1.9 to - 0.4), - 14.8%). This was accompanied by lower in-hospital mortality of 21.8% vs. 29.8% (absolute decrease 7.9% (95% CI - 13.9% to - 1.8%), relative decrease 26.6%), resulting in an increase in mean QALYs/patient from 0.47 to 0.52 (i.e. + 0.05 (95% CI 0.00 to 0.10); + 10.1%). However, owing to high costs among sepsis survivors, healthcare costs over a one-year time horizon were €73,665/patient in the PCT group compared with €70,961/patient with standard of care (i.e. + €2704 (95% CI - €4495 to €10,005), + 3.8%), resulting in an incremental cost-effectiveness ratio of €57,402/QALY gained. Within this time frame, the probability of PCT guidance being cost-effective was 64% at a willingness-to-pay threshold of €80,000/QALY. CONCLUSIONS: Although the impact of PCT guidance on total healthcare-related costs during the initial hospitalisation episode is likely negligible, the lower in-hospital mortality may lead to a non-significant increase in costs over a one-year time horizon. However, since uncertainty remains, it is recommended to investigate the long-term cost-effectiveness of PCT guidance, from a societal perspective, in different countries and settings.

How to Agree on a CTC: Evaluating the Consensus in Circulating Tumor Cell Scoring.

For using counts of circulating tumor cells (CTCs) in the clinic to aid a physician's decision, its reported values will need to be accurate and comparable between institutions. Many technologies have become available to enumerate and characterize CTCs, thereby showing a large range of reported values. Here we introduce an Open Source CTC scoring tool to enable comparison of different reviewers and facilitate the reach of a consensus on assigning objects as CTCs. One hundred images generated from two different platforms were used to assess concordance between 15 reviewers and an expert panel. Large differences were observed between reviewers in assigning objects as CTCs urging the need for computer recognition of CTCs. A demonstration of a deep learning approach on the 100 images showed the promise of this technique for future CTC enumeration. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Health economic impact of liquid biopsies in cancer management.

INTRODUCTION: Liquid biopsies (LBs) are referred to as the sampling and analysis of non-solid tissue, primarily blood, as a diagnostic and monitoring tool for cancer. Because LBs are largely non-invasive, they are a less-costly alternative for serial analysis of tumor progression and heterogeneity to facilitate clinical management. Although a variety of tumor markers are proposed (e.g., free-circulating DNA), the clinical evidence for Circulating Tumor Cells (CTCs) is currently the most developed. Areas covered: This paper presents a health economic perspective of LBs in cancer management. We first briefly introduce the requirements in biomarker development and validation, illustrated for CTCs. Second, we discuss the state-of-art on the clinical utility of LBs in breast cancer in more detail. We conclude with a future perspective on the clinical use and reimbursement of LBs Expert commentary: A significant increase in clinical research on LBs can be observed and the results suggest a rapid change of cancer management. In addition to studies evaluating clinical utility of LBs, a smooth translation into clinical practice requires systematic assessment of the health economic benefits. This paper argues that (early stage) health economic research is required to facilitate its clinical use and to prioritize further evidence development.

Detecting Blood-Based Biomarkers in Metastatic Breast Cancer: A Systematic Review of Their Current Status and Clinical Utility.

Reviews on circulating biomarkers in breast cancer usually focus on one single biomarker or a selective group of biomarkers. An overview summarizing the discovery and evaluation of all blood-based biomarkers in metastatic breast cancer is lacking. This systematic review aims to identify the available evidence of known blood-based biomarkers in metastatic breast cancer, regarding their clinical utility and state-of-the-art position in the validation process. The initial search yielded 1078 original studies, of which 420 were assessed for eligibility. A total of 320 studies were included in the final synthesis. A Development, Evaluation and Application Chart (DEAC) of all biomarkers was developed. Most studies focus on identifying new biomarkers and search for relations between these biomarkers and traditional molecular characteristics. Biomarkers are usually investigated in only one study (68.8%). Only 9.8% of all biomarkers was investigated in more than five studies. Circulating tumor cells, gene expression within tumor cells and the concentration of secreted proteins are the most frequently investigated biomarkers in liquid biopsies. However, there is a lack of studies focusing on identifying the clinical utility of these biomarkers, by which the additional value still seems to be limited according to the investigated evidence.

Structural studies of the final enzyme in the alpha-aminoadipate pathway-saccharopine dehydrogenase from Saccharomyces cerevisiae.

The 1.64 A structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogenase, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.

Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase.

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.

Crystallization and preliminary X-ray diffraction studies of glycerol 3-phosphate cytidylyltransferase from Staphylococcus aureus.

Glycerol 3-phosphate cytidylyltransferase from Staphylococcus aureus (TarD(Sa)) has been expressed in Escherichia coli, purified to homogeneity and crystallized. The strategy used for determining crystallization conditions employed hanging-drop sparse-matrix screens and required a combination of three different optimization approaches. Specifically, the presence or absence of cofactors needed to be surveyed, the effects of small-molecule additives required exploration and the rate of vapour-diffusion had to be varied in order to obtain crystals of TarD(Sa) suitable for diffraction studies. Crystals thus obtained belong to the space group P3(1)21, with unit-cell parameters a = b = 92.2, c = 156.1 A, and contain four TarD(Sa) molecules per asymmetric unit. The resolution limit observed for these crystals using synchrotron radiation is 3.0 A.

Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose.

The three-dimensional structure of the lytic transglycosylase from bacteriophage lambda, also known as bacteriophage lambda lysozyme, complexed to the hexasaccharide inhibitor, hexa-N-acetylchitohexaose, has been determined by X-ray crystallography at 2.6 A resolution. The unit cell contains two molecules of the lytic transglycosylase with two hexasaccharides bound. Each enzyme molecule is found to interact with four N-acetylglucosamine units from one hexasaccharide (subsites A-D) and two N-acetylglucosamine units from the second hexasaccharide (subsites E and F), resulting in all six subsites of the active site of this enzyme being filled. This crystallographic structure, therefore, represents the first example of a lysozyme in which all subsites are occupied, and detailed protein-oligosaccharide interactions are now available for this bacteriophage lytic transglycosylase. Examination of the active site furthermore reveals that of the two residues that have been implicated in the reaction mechanism of most other c-type lysozymes (Glu35 and Asp52 in hen egg white lysozyme), only a homologous Glu residue is present. The lambda lytic transglycosylase is therefore functionally closely related to the Escherichia coli Slt70 and Slt35 lytic transglycosylases and goose egg white lysozyme which also lack the catalytic aspartic acid.

Crystal structures of homoserine dehydrogenase suggest a novel catalytic mechanism for oxidoreductases.

The structure of the antifungal drug target homoserine dehydrogenase (HSD) was determined from Saccharomyces cerevisiae in apo and holo forms, and as a ternary complex with bound products, by X-ray diffraction. The three forms show that the enzyme is a dimer, with each monomer composed of three regions, the nucleotide-binding region, the dimerization region and the catalytic region. The dimerization and catalytic regions have novel folds, whereas the fold of the nucleotide-binding region is a variation on the Rossmann fold. The novel folds impose a novel composition and arrangement of active site residues when compared to all other currently known oxidoreductases. This observation, in conjunction with site-directed mutagenesis of active site residues and steady-state kinetic measurements, suggest that HSD exhibits a new variation on dehydrogenase chemistry.

The COOH terminus of aminoglycoside phosphotransferase (3')-IIIa is critical for antibiotic recognition and resistance.

The aminoglycoside phosphotransferases (APHs) are widely distributed among pathogenic bacteria and are employed to covalently modify, and thereby detoxify, the clinically relevant aminoglycoside antibiotics. The crystal structure for one of these aminoglycoside kinases, APH(3')-IIIa, has been determined in complex with ADP and analysis of the electrostatic surface potential indicates that there is a large anionic depression present adjacent to the terminal phosphate group of the nucleotide. This region also includes a conserved COOH-terminal alpha-helix that contains the COOH-terminal residue Phe(264). We report here mutagenesis and computer modeling studies aimed at examining the mode of aminoglycoside binding to APH(3')-IIIa. Specifically, seven site mutants were studied, five from the COOH-terminal helix (Asp(261), Glu(262), and Phe(264)), and two additional residues that line the wall of the anionic depression (Tyr(55) and Arg(211)). Using a molecular modeling approach, six ternary complexes of APH(3')-IIIa.ATP with the antibiotics, kanamycin, amikacin, butirosin, and ribostamycin were independently constructed and these agree well with the mutagenesis data. The results obtained show that the COOH-terminal carboxylate of Phe(264) is critical for proper function of the enzyme. Furthermore, these studies demonstrate that there exists multiple binding modes for the aminoglycosides, which provides a molecular basis for the broad substrate- and regiospecificity observed for this enzyme.

Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.

BACKGROUND: The predominant mechanism of antibiotic resistance employed by pathogenic bacteria against the clinically used aminoglycosides is chemical modification of the drug. The detoxification reactions are catalyzed by enzymes that promote either the phosphorylation, adenylation or acetylation of aminoglycosides. Structural studies of these aminoglycoside-modifying enzymes may assist in the development of therapeutic agents that could circumvent antibiotic resistance. In addition, such studies may shed light on the development of antibiotic resistance and the evolution of different enzyme classes. RESULTS: The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside 6'-N-acetyltransferase type li (AAC(6')-li) in complex with the cofactor acetyl coenzyme A has been determined at 2.7 A resolution. The structure establishes that this acetyltransferase belongs to the GCN5-related N-acetyltransferase superfamily, which includes such enzymes as the histone acetyltransferases GCN5 and Hat1. CONCLUSIONS: Comparison of the AAC(6')-li structure with the crystal structures of two other members of this superfamily, Serratia marcescens aminoglycoside 3-N-acetyltransferase and yeast histone acetyltransferase Hat1, reveals that of the 84 residues that are structurally similar, only three are conserved and none can be implicated as catalytic residues. Despite the negligible sequence identity, functional studies show that AAC(6')-li possesses protein acetylation activity. Thus, AAC(6')-li is both a structural and functional homolog of the GCN5-related histone acetyltransferases.

Crystallization and preliminary X-ray diffraction studies of homoserine dehydrogenase from Saccharomyces cerevisiae.

Recombinant homoserine dehydrogenase from Saccharomyces cerevisiae has been crystallized in three different forms. Crystals of the apo-enzyme belong to the tetragonal space group P4 and have unit-cell-dimensions a = b = 130 and c = 240 A. The resolution limit for these crystals is 3.9 A. Crystals of homoserine dehydrogenase grown in the presence of the co-factor NAD+ have the tetragonal space group P41212 or its enantiomorph P43212. The unit-cell dimensions for these crystals are a = b = 80.4 and c = 250.2 A, and the observed resolution limit is 2.2 A. Protein crystals grown in the presence of the product L-homoserine and the inert NAD+ analogue 3-aminopyridine adenine dinucleotide belong to the monoclinic space group P21 with unit-cell parameters a = 58.8, b = 104.2, c = 120.7 A, beta = 91.9 degrees. This last crystal form has a diffraction limit of 2.7 A resolution.

Structure of an enzyme required for aminoglycoside antibiotic resistance reveals homology to eukaryotic protein kinases.

Bacterial resistance to aminoglycoside antibiotics is almost exclusively accomplished through either phosphorylation, adenylylation, or acetylation of the antibacterial agent. The aminoglycoside kinase, APH(3')-IIIa, catalyzes the phosphorylation of a broad spectrum of aminoglycoside antibiotics. The crystal structure of this enzyme complexed with ADP was determined at 2.2 A. resolution. The three-dimensional fold of APH(3')-IIIa reveals a striking similarity to eukaryotic protein kinases despite a virtually complete lack of sequence homology. Nearly half of the APH(3')-IIIa sequence adopts a conformation identical to that seen in these kinases. Substantial differences are found in the location and conformation of residues presumably responsible for second-substrate specificity. These results indicate that APH(3') enzymes and eukaryotic-type protein kinases share a common ancestor.

The role of a conserved water molecule in the redox-dependent thermal stability of iso-1-cytochrome c.

Eukaryotic cytochromes c contain a buried water molecule (Wat166) next to the heme that is associated through a network of hydrogen bonds to three invariant residues: tyrosine 67, asparagine 52, and threonine 78. Single-site mutations to two of these residues (Y67F, N52I, N52A) and the double-site mutation (Y67F/N52I) were introduced into Saccharomyces cerevisiae iso-1-cytochrome c to disrupt the hydrogen bonding network associated with Wat166. The N52I and Y67F/N52I mutations lead to a loss of Wat166 while N52A and Y67F modifications lead to the addition of a new water molecule (Wat166) at an adjacent site (Berghuis, A. M., Guillemette, J. G., McLendon, G., Sherman, F., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 236, 786-799; Berghuis, A. M., Guillemette, J. G., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 235, 1326-1341; Rafferty, S. P., Guillemette, J. G., Berghuis, A. M., Smith, M., Brayer, G. D., and Mauk, A. G. (1996) Biochemistry, 35, 10784-10792). We used differential scanning calorimetry (DSC) to determine the change in heat capacity (DeltaCp) and the temperature dependent enthalpy (DeltaHvH) for the thermal denaturation of both the oxidized and reduced forms of the iso-1 cytochrome c variants. The relative stabilities were expressed as the difference in the free energy of denaturation (DeltaGD) between the wild type and mutant proteins in both redox states. The disruption of the hydrogen bonding network results in increased stability for all of the mutant proteins in both redox states with the exception of the reduced Y67F variant which has approximately the same stability as the reduced wild type protein. For the oxidized proteins, DeltaGD values of 1.3, 4.1, 1.5, and 5.8 kcal/mol were determined for N52A, N52I, Y67F, and Y67F/N52I, respectively. The oxidized proteins were 8.2-11.5 kcal/mol less stable than the reduced proteins due to a redox-dependent increase in the entropy of unfolding.

Structure of the GDP-Pi complex of Gly203-->Ala gialpha1: a mimic of the ternary product complex of galpha-catalyzed GTP hydrolysis.

BACKGROUND: G proteins play a vital role in transmembrane signalling events. In their inactive form G proteins exist as heterotrimers consisting of an alpha subunit, complexed with GDP and a dimer of betagamma subunits. Upon stimulation by receptors, G protein alpha subunits exchange GDP for GTP and dissociate from betagamma . Thus activated, alphasubunits stimulate or inhibit downstream effectors. The duration of the activated state corresponds to the single turnover rate of GTP hydrolysis, which is typically in the range of seconds. In Gialpha1, the Gly203-->Ala mutation reduces the affinity of the substrate for Mg2+, inhibits a key conformational step that occurs upon GTP binding and consequently inhibits the release of betagamma subunits from the GTP complex. The structure of the Gly203-->Ala mutant of Gialpha1 (G203AGialpha1) bound to the slowly hydrolyzing analog of GTP (GTPgammaS) has been determined in order to elucidate the structural changes that take place during hydrolysis. RESULTS: We have determined the three dimensional structure of a Gly203-->Ala mutant of Gialpha1 at 2.6 A resolution. Although crystals were grown in the presence of GTPgammaS and Mg2+, the catalytic site contains a molecule of GDP and a phosphate ion, but no Mg2+. The phosphate ion is bound to a site near that occupied by the gamma-phosphate of GTPgammaS in the activated wild-type alpha subunit. A region of the protein, termed the Switch II helix, twists and bends to adopt a conformation that is radically different from that observed in other Gialpha1 subunit complexes. CONCLUSIONS: Under the conditions of crystallization, the Gly203-->Ala mutation appears to stabilize a conformation that may be similar, although perhaps not identical, to the transient ternary product complex of Gialpha1-catalyzed GTP hydrolysis. The rearrangement of the Switch II helix avoids a potential steric conflict caused by the mutation. However, it appears that dissociation of the gamma-phosphate from the pentacoordinate intermediate also requires a conformational change in Switch II. Thus, a conformational rearrangement of the Switch II helix may be required in Galpha-catalyzed GTP hydrolysis.

Mechanistic and structural contributions of critical surface and internal residues to cytochrome c electron transfer reactivity.

The influence of mutations in two conserved regions of yeast iso-1-cytochrome c believed to be critical to the mechanism of cytochrome c electron transfer reactions has been investigated. The variants Asn52Ala, Tyr67Phe, Ile75Met, and Thr78Gly involve perturbation of critical hydrogen-bonding interactions with an internal water molecule (Wat166) and have been studied in terms of their electrochemical properties and the kinetics with which they are reduced by Fe(EDTA)2- and oxidized by Co(phen)3(3+). In parallel studies, the Co(phen)3(3+) oxidation kinetics of Tyr, Leu, Ile, Ala, Ser, and Gly variants of the phylogenetically conserved residue Phe82 have been studied and correlated with previous electrochemical and kinetic results. To assist mechanistic interpretation of these results, the three-dimensional structures of the Asn52Ala and Ile75Met ferrocytochrome c variants have been determined. The reduction potentials of the variants modified in the region of Wat166 were at least 33 mV (pH 6, 25 degrees C, and mu = 0.1 M) lower than that of the wild-type protein. Electron transfer reactivity of this family of variants in both the oxidation and reduction reactions was increased as much as 10-fold over that of the wild-type cytochrome. On the other hand, the reactivity of the position-82 variants in both oxidation and reduction depended on the structural characteristics of the oxidation-reduction reagent with which they reacted, and this reactivity was related to the nature of the residue at this position. These findings have been interpreted as demonstrating that the principal influence of modification at position-82 arises from changes in the nature of reactant-protein interaction at the surface of the protein and in maintaining the high reduction potential of the cytochrome while the principal influence of internal modifications near Wat166 results from alteration of the reorganization energy for the oxidation state-linked conformational change defined by crystallographic analysis of the wild-type protein.

Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis.

Crystallographic analysis of 2.2 angstrom resolution shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein alpha subunit, Gi alpha 1. The switch II and switch III segments become disordered, and linker II connecting the Ras and alpha helical domains moves, thus altering the structures of potential effector and beta gamma binding regions. Contacts between the alpha-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and beta gamma binding determinants, are disordered in the complex with GTP, but are organized into a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring alpha subunits in the lattice, suggesting that multimers of alpha subunits or heterotrimers may play a role in signal transduction.

Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold.

C2 domains are regulatory sequence motifs that occur widely in nature. Synaptotagmin I, a synaptic vesicle protein involved in the Ca2+ regulation of exocytosis, contains two C2 domains, the first of which acts as a Ca2+ sensor. We now describe the three-dimensional structure of this C2 domain at 1.9 A resolution in both the Ca(2+)-bound and Ca(2+)-free forms. The C2 polypeptide forms an eight-stranded beta sandwich constructed around a conserved four-stranded motif designated as a C2 key. Ca2+ binds in a cup-shaped depression between two polypeptide loops located at the N- and C-termini of the C2-key motif.