Chromophore carbonyl twisting in fluorescent biosensors encodes direct readout of protein conformations with multicolor switching.

Fluorescent labeling of proteins is a powerful tool for probing structure-function relationships with many biosensing applications. Structure-based rules for systematically designing fluorescent biosensors require understanding ligand-mediated fluorescent response mechanisms which can be challenging to establish. We installed thiol-reactive derivatives of the naphthalene-based fluorophore Prodan into bacterial periplasmic glucose-binding proteins. Glucose binding elicited paired color exchanges in the excited and ground states of these conjugates. X-ray structures and mutagenesis studies established that glucose-mediated color switching arises from steric interactions that couple protein conformational changes to twisting of the Prodan carbonyl relative to its naphthalene plane. Mutations of residues contacting the carbonyl can optimize color switching by altering fluorophore conformational equilibria in the apo and glucose-bound proteins. A commonly accepted view is that Prodan derivatives report on protein conformations via solvatochromic effects due to changes in the dielectric of their local environment. Here we show that instead Prodan carbonyl twisting controls color switching. These insights enable structure-based biosensor design by coupling ligand-mediated protein conformational changes to internal chromophore twists through specific steric interactions between fluorophore and protein.

Portable Gas Spring with Rachet and Pinion System as a Third Arm for Holding Endoscope in Two Handed Endoscopic ENT Surgery.

The role of endoscope has been changing from that being an adjuvant during microear surgery to the exclusive endoscopic middle ear surgery. However the only disadvantage of endoscopic ear surgery is its single handed technique as the non-dominant hand is used to hold the endoscope. We propose the concept and design of our portable endoscope holder for two handed endoscopic ear surgery. It is based on the gas spring action and rack and pinion system which act as a third arm to hold the endoscope. The novel portable endoscope holder bears the potential to provide benefits for various two handed endoscopic ear nose and throat surgeries. Level of evidence: Level V. Supplementary Information: The online version contains supplementary material available at 10.1007/s12070-022-03246-3.

Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY.

SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that SPY is distinct from ER-localized POFUTs and contains N-terminal tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain resembling the O-linked-N-acetylglucosamine (GlcNAc) transferases (OGTs). However, the structural feature that determines the distinct enzymatic selectivity of SPY remains unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of SPY and its complex with GDP-fucose, revealing distinct active-site features enabling GDP-fucose instead of UDP-GlcNAc binding. SPY forms an antiparallel dimer instead of the X-shaped dimer in human OGT, and its catalytic domain interconverts among multiple conformations. Analysis of mass spectrometry, co-IP, fucosylation activity, and cryo-EM data further demonstrates that the N-terminal disordered peptide in SPY contains trans auto-fucosylation sites and inhibits the POFUT activity, whereas TPRs 1-5 dynamically regulate SPY activity by interfering with protein substrate binding.

Experimental Investigations into Using Motion Capture State Feedback for Real-Time Control of a Humanoid Robot.

Regardless of recent advances, humanoid robots still face significant difficulties in performing locomotion tasks. Among the key challenges that must be addressed to achieve robust bipedal locomotion are dynamically consistent motion planning, feedback control, and state estimation of such complex systems. In this paper, we investigate the use of an external motion capture system to provide state feedback to an online whole-body controller. We present experimental results with the humanoid robot RH5 performing two different whole-body motions: squatting with both feet in contact with the ground and balancing on one leg. We compare the execution of these motions using state feedback from (i) an external motion tracking system and (ii) an internal state estimator based on inertial measurement unit (IMU), forward kinematics, and contact sensing. It is shown that state-of-the-art motion capture systems can be successfully used in the high-frequency feedback control loop of humanoid robots, providing an alternative in cases where state estimation is not reliable.

Evaluation of Mother's Complementary Feeding Knowledge and Occlusion.

Aim: Mothers' understanding of feeding methods and how they affect deciduous dentition were the focus of this study. Materials and Methods: This study looked at people from different parts of the population. In total, 230 moms of children aged 6 months to 5 years who completed an oral health questionnaire were included in the study. Mothers' awareness of feeding patterns and their impact on deciduous dentition were examined in the study. Results: Illiterate moms' children showed a significant difference in the quality of their occlusion compared to similarly raised children of literate mothers. Children who began supplementary feeding before the age of 6 months were more likely to have occlusion changes. Conclusion: Malocclusion prevalence was shown to be unrelated to meal behaviors. However, further research is required as there are just a few studies currently available.

Structural basis of NPR1 in activating plant immunity.

NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid1-4. Despite the important role of NPR1 in plant immunity5-7, understanding of its regulatory mechanisms has been hindered by a lack of structural information. Here we report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Cryo-electron microscopy analysis reveals that NPR1 is a bird-shaped homodimer comprising a central Broad-complex, Tramtrack and Bric-à-brac (BTB) domain, a BTB and carboxyterminal Kelch helix bundle, four ankyrin repeats and a disordered salicylic-acid-binding domain. Crystal structure analysis reveals a unique zinc-finger motif in BTB for interacting with ankyrin repeats and mediating NPR1 oligomerization. We found that, after stimulation, salicylic-acid-induced folding and docking of the salicylic-acid-binding domain onto ankyrin repeats is required for the transcriptional cofactor activity of NPR1, providing a structural explanation for a direct role of salicylic acid in regulating NPR1-dependent gene expression. Moreover, our structure of the TGA32-NPR12-TGA32 complex, DNA-binding assay and genetic data show that dimeric NPR1 activates transcription by bridging two fatty-acid-bound TGA3 dimers to form an enhanceosome. The stepwise assembly of the NPR1-TGA complex suggests possible hetero-oligomeric complex formation with other transcription factors, revealing how NPR1 reprograms the defence transcriptome.

Evaluation of Clinical Profile and Various Treatment Modalities in Oral Sub Mucous Fibrosis in North India: Our Experience.

Oral submucous fibrosis is a chronic disease affecting oral cavity and sometimes the pharynx. Etiology seems to be local irritants such as capsaicin, tobacco, areca nut and spicy foods. The main concern in this is the management of trismus and burning sensation of the oral mucosa. The aim of this study was to compare various medical treatment protocol of OSMF. 210 patients were divided randomly in 3 groups. In Group A, patients were given biweekly intralesional Hyaluronidase/Dexamethasone for 6 weeks. Group B patients were given tablet Pentoxifylline 400 mg TDS.Group C patients were given Eprisone hydrochloride. All three groups were given Lycopene 10,000 mcg for period of 6 weeks. All patients were given topical Triamcelone for local application. The examinations for mouth opening were repeated at weekly intervals for a period of 6 weeks.The most common complaint was burning sensation in 75.98% cases, difficulty in mouth opening in77.45% and difficulty in swallowing food in 61.76% cases. Group A showed improvement in 41.17% cases presenting with burning sensation followed by decreased mouth opening 39.70%. Group B showed improvement in 45.58% burning sensation, 17.64% with decreased mouth opening. Group C showed improvement in 48.52% patients having pain with spicy food, 32.35% with decreased mouth opening and 17.64% with difficulty in swallowing. We conclude that patients which received intralesional dexamethasone and hyaluronidase along with oral Lycopene showed better clinical and symptomatic improvement, and at present appears to be best non-surgical treatment.

Presentations of Pediatric Emergencies in a Tertiary Teaching Hospital in North India: An Overview.

Pediatric otorhinolaryngological emergencies constitute a major portion in emergency room presentations. The etiology of ENT problems in children is different from those in adults. Most of these can be managed conservatively while some need prompt management at a well equipped centre. To analyze the etiology, clinical profile and line of management of pediatric otorhinolaryngological emergencies. Retrospectively, records of 452 children up to the age of 16 years presenting with ENT complaints were included in the study. Out of 452 patients, 148 presented with aural complaints, 129 had nasal problems and 175 patients with throat complaints. They were classified into Trauma 69 (15.26%), Foreign body 278 (61.50%), Infective 82 (08.14%) and allergic/miscellaneous 23 (05.10%) cases. In aural complaints, foreign body insertion seen in 57 (12.17%) cases while earache in 55 (12.15%) patients. In patients with nasal complaints, foreign body was present in 78 (17.26%) cases. Nasal bleeding and discharge or pain and swelling around nose were the other presentations. Respiratory distress was present in 66 (14.60%) patients while 74 (16.37%) patients came with ingestion of some foreign body. Neck swellings were seen in 20 (04.42%) patients and 15 (03.31%) patients came with history of rashes, feeling of choking or allergic reactions. Surgical intervention after admission was the top most intervention in 202 (44.69%) patients followed by conservative management in 110 (24.33%) patients who were treated and then sent home from emergency centre itself. Minor surgical intervention was sufficient to treat 78 (17.25%) patients without admission. Most common ENT emergency was foreign bodies and these cases need operative intervention. Specialist ENT personnel is needed to handle these cases. Parents must be educated to keep likely causes of these foreign bodies out of reach of growing children and also need to be educated about signs like severe pain, dyspnoea, bleeding or unilateral nasal discharge for timely management.

Comparative Evaluation of Hearing in Cartilage Umbrella, Boomerang and Torp in Chronic Otitis Media Cases with Absent Stapes Suprastructure: Our Experience.

Ossicular discontinuity is one of the most common causes of conductive hearing loss. Ossicular chain reconstruction improves conductive hearing loss. With no additional cost, cartilage ossiculoplasty is easy to perform, and also the cartilage is well tolerated being an autograft. In this study we compared the audiological outcome in ossiculoplasty done by cartilage umbrella, cartilage boomerang and alloplastic TORP. 75 patients of age group 10-50 years clinically diagnosed with chronic otitis media with conductive hearing loss and an air bone gap (ABG) of at least 20 dB posted for surgery were included. Ossiculoplasty was done in three groups with autologous cartilage boomerang, cartilage umbrella and alloplastic TORP. In mucosal disease hearing gain was better in umbrella technique (17.66 ± 1.1) dB than Boomerang (16.9 ± 0.8) dB and TORP (10.68 ± 0.9) dB. ABG closure was higher in Boomerang and TORP. Hearing improvement in patients with squamosal disease managed by canal wall up surgery was 25.01 ± 1.1 dB, 27.73 ± 3.1 dB and 20.12 ± 1.8 dB in Boomerang, Umbrella and TORP group respectively showing that umbrella method gave maximum improvement. ABG closure was better in TORP group. In canal wall down surgery patient's maximum improvement was seen in Boomerang (29.51 ± 0.9) dB followed by Umbrella (26.67 ± 1.2) dB and TORP (25.27 ± 0.8) dB group. ABG closure was higher in Boomerang group. Cartilage ossiculoplasty is a reliable and effective method of ossicular chain reconstruction for both mucosal and squamosal disease. Cartilage ossiculoplasty has the added advantage of reduced chances of prosthesis extrusion as compared to TORP.

Oncogenic KRAS is dependent upon an EFR3A-PI4KA signaling axis for potent tumorigenic activity.

The HRAS, NRAS, and KRAS genes are collectively mutated in a fifth of all human cancers. These mutations render RAS GTP-bound and active, constitutively binding effector proteins to promote signaling conducive to tumorigenic growth. To further elucidate how RAS oncoproteins signal, we mined RAS interactomes for potential vulnerabilities. Here we identify EFR3A, an adapter protein for the phosphatidylinositol kinase PI4KA, to preferentially bind oncogenic KRAS. Disrupting EFR3A or PI4KA reduces phosphatidylinositol-4-phosphate, phosphatidylserine, and KRAS levels at the plasma membrane, as well as oncogenic signaling and tumorigenesis, phenotypes rescued by tethering PI4KA to the plasma membrane. Finally, we show that a selective PI4KA inhibitor augments the antineoplastic activity of the KRASG12C inhibitor sotorasib, suggesting a clinical path to exploit this pathway. In sum, we have discovered a distinct KRAS signaling axis with actionable therapeutic potential for the treatment of KRAS-mutant cancers.

Structure and function of the juxtamembrane GAF domain of potassium biosensor KdpD.

KdpD/KdpE two-component signaling system regulates expression of a high affinity potassium transporter responsible for potassium homeostasis. The C-terminal module of KdpD consists of a GAF domain linked to a histidine kinase domain. Whereas certain GAF domains act as regulators by binding cyclic nucleotides, the role of the juxtamembrane GAF domain in KdpD is unknown. We report the high-resolution crystal structure of KdpD GAF domain (KdpDG ) consisting of five α-helices, four ß-sheets and two large loops. KdpDG forms a symmetry-related dimer, wherein parallelly arranged monomers contribute to a four-helix bundle at the dimer-interface, SAXS analysis of KdpD C-terminal module reveals an elongated structure that is a dimer in solution. Substitution of conserved residues with various residues that disrupt the dimer interface produce a range of effects on gene expression demonstrating the importance of the interface in inactive to active transitions during signaling. Comparison of ligand binding site of the classic cyclic nucleotide-binding GAF domains to KdpDG reveals structural differences arising from naturally occurring substitutions in primary sequence of KdpDG that modifies the canonical NKFDE sequence motif required for cyclic nucleotide binding. Together these results suggest a structural role for KdpDG in dimerization and transmission of signal to the kinase domain.

Epicardial adipose tissue volume as a marker of coronary artery disease severity in patients with diabetes independent of coronary artery calcium: findings from the CTRAD study.

AIMS: The association between epicardial adipose tissue (EAT) volume and coronary artery disease (CAD) severity was evaluated, independent of traditional risk factors and coronary artery calcium (CAC) scores, in patients with diabetes type 2 (DM-2) using cardiac computed tomography angiography (CTA). METHODS: A multivariate analysis was utilized to assess for an independent association after calculating EAT volume, CAD severity, and calcium scores in 92 patients with DM-II from the CTRAD study. We graded CAD severity as none (normal coronaries), mild-moderate (<70% stenosis), and severe (70% or greater stenosis). RESULTS: A total of 39 (42.3%) asymptomatic patients with diabetes did not have CAD; 30.4% had mild/moderate CAD; and 27.1% had severe CAD. Mean EAT volume was highest in patients with severe CAD (143.14 cm(3)) as compared to mild/moderate CAD (112.7 cm(3)), and no CAD (107.5 cm(3)) (p = 0.003). After adjustment of clinical risk factors, notably, CAC score, multivariate regression analysis showed EAT volume was an independent predictor of CAD severity in this sample (odds ratio 11.2, 95% confidence interval 1.7-73.8, p = 0.01). CONCLUSIONS: Increasing EAT volume in asymptomatic patients with DM-II is associated with presence of severe CAD, independent of BMI and CAC, as well as traditional risk factors.

Comparison of epicardial adipose tissue volume and coronary artery disease severity in asymptomatic adults with versus without diabetes mellitus.

Epicardial adipose tissue (EAT) has been shown to have important effects on the development of coronary artery disease (CAD) through local paracrine influences on the vascular bed. We compared a cohort of asymptomatic patients with type II diabetes mellitus (DM) without known CAD to an age- and gender-matched group of asymptomatic patients without DM from the CTRAD (Cardiac CT's Role in Asymptomatic Patients with DM-II) study in which patients underwent a cardiac computed tomography angiogram, for early detection of CAD. Mean EAT volumes of 118.6 ± 43.0 and 70.0 ± 44.0 cm(3) were found in the DM and non-DM groups, respectively. When stratified by the presence and severity of CAD, it was found that in the DM (p = 0.003) and non-DM groups (p <0.001), there was a statistically significant increase in EAT volume as the patients were found to have increasingly severe CAD. After adjusting for age, race, gender, DM, hypertension, insulin use, body mass index, and coronary artery calcium (CAC) score, the presence of >120 cm(3) of EAT was found to be highly correlated with the presence of significant CAD (adjusted odds ratio 4.47, 95% confidence interval 1.35 to 14.82). We found that not only is EAT volume an independent predictor of CAD but that an increasing volume of EAT predicted increasing severity of CAD even after adjustment for CAC score.

An asymmetric heterodomain interface stabilizes a response regulator-DNA complex.

Two-component signal transduction systems consist of pairs of histidine kinases and response regulators, which mediate adaptive responses to environmental cues. Most activated response regulators regulate transcription by binding tightly to promoter DNA via a phosphorylation-triggered inactive-to-active transition. The molecular basis for formation of stable response regulator-DNA complexes that precede the assembly of RNA polymerases is unclear. Here, we present structures of DNA complexed with the response regulator KdpE, a member of the OmpR/PhoB family. The distinctively asymmetric complex in an active-like conformation reveals a unique intramolecular interface between the receiver domain (RD) and the DNA-binding domain (DBD) of only one of the two response regulators in the complex. Structure-function studies show that this RD-DBD interface is necessary to form stable complexes that support gene expression. The conservation of sequence and structure suggests that these findings extend to a large group of response regulators that act as transcription factors.

Flexibility of EF-hand motifs: structural and thermodynamic studies of Calcium Binding Protein-1 from Entamoeba histolytica with Pb2+, Ba2+, and Sr2+.

BACKGROUND: EF-hand proteins can be activated by the binding of various heavy metals other than calcium, and such complexes can disturb the calcium-signaling pathway and cause toxicity and disease causing state. So far, no comprehensive study has been done to understand different heavy metals binding to calcium signaling proteins. RESULTS: In this work, the flexibility of the EF-hand motifs are examined by crystallographic and thermodynamic studies of binding of Pb2+, Ba2+ and Sr2+ to Calcium Binding Protein-1 from Entamoeba histolytica (EhCaBP1). The structures of the EhCaBP1- heavy metal complexes are found to be overall similar, nevertheless specific differences in metal coordination, and small differences in the coordination distances between the metal and the ligands in the metal binding loop. The largest such distances occur for the Ba2+- EhCaBP1 complex, where two bariums are bound with partial occupancy at the EF2 motif. Thermodynamic studies confirm that EhCaBP1 has five binding sites for Ba2+ compared to four binding sites for the other metals. These structures and thermodynamic studies reveal that the EF-hand motifs can accommodate several heavy atoms with similar binding affinities. The binding of Ca2+ to the 1st, 2nd and 4th sites and the binding of Ba2+ to the 1st, 2nd, 4th and 5th sites are both enthalpically and entropically driven, whereas the binding of Sr2+ to the 1st, 2nd and 4th sites are simply enthalpy driven, interestingly in agreement with ITC data, Sr2+ do not coordinate with water in this structure. For all the metals, binding to the 3rd site is only entropy driven. CONCLUSION: Energetically, Ca2+ is preferred in three sites, while in one site Ba2+ has better binding energy. The Sr2+-coordination in the EF hand motifs is similar to that of the native Ca2+ bound structure, except for the lack of water coordination. Sr2+ coordination seems to be a pre-formed in nature since all seven coordinating atoms are from the protein itself, which also correlates with entropy contributions in Sr2+ binding. These findings improve our understanding of metal association with calcium binding proteins and of metal induced conformational changes.

Crystal structure and trimer-monomer transition of N-terminal domain of EhCaBP1 from Entamoeba histolytica.

EhCaBP1 is a well-characterized calcium binding protein from Entamoeba histolytica with four canonical EF-hand motifs. The crystal structure of EhCaBP1 reveals the trimeric organization of N-terminal domain. The solution structure obtained at pH 6.0 indicated its monomeric nature, similar to that of calmodulin. Recent domain-wise studies showed clearly that the N-terminal domain of EhCaBP1 is capable of performing most of the functions of the full-length protein. Additionally, the mode of target binding in the trimer is similar to that found in calmodulin. To study the dynamic nature of this protein and further validate the trimerization of N-terminal domain at physiological conditions, the crystal structure of N-terminal domain was determined at 2.5 A resolution. The final structure consists of EF-1 and EF-2 motifs separated by a long straight helix as seen in the full-length protein. The spectroscopic and stability studies, like far and near-ultraviolet circular dichroism spectra, intrinsic and extrinsic fluorescence spectra, acrylamide quenching, thermal denaturation, and dynamic light scattering, provided clear evidence for a conversion from trimeric state to monomeric state. As the pH was lowered from the physiological pH, a dynamic trimer-monomer transition was observed. The trimeric state and monomeric state observed in spectroscopic studies may represent the x-ray and NMR structures of the EhCaBP1. At pH 6.0, the endogenous kinase activation function was almost lost, indicating that the monomeric state of the protein, where EF-hand motifs are far apart, is not a functional state.

N- and C-terminal domains of the calcium binding protein EhCaBP1 of the parasite Entamoeba histolytica display distinct functions.

Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, and calcium signaling is thought to be involved in amoebic pathogenesis. EhCaBP1, a Ca(2+) binding protein of E. histolytica, is essential for parasite growth. High resolution crystal structure of EhCaBP1 suggested an unusual arrangement of the EF-hand domains in the N-terminal part of the structure, while C-terminal part of the protein was not traced. The structure revealed a trimer with amino terminal domains of the three molecules interacting in a head-to-tail manner forming an assembled domain at the interface with EF1 and EF2 motifs of different molecules coming close to each other. In order to understand the specific roles of the two domains of EhCaBP1, the molecule was divided into two halves, and each half was separately expressed. The domains were characterized with respect to their structure, as well as specific functional features, such as ability to activate kinase and bind actin. The domains were also expressed in E. histolytica cells along with green fluorescent protein. The results suggest that the N-terminal domain retains some of the properties, such as localization in phagocytic cups and activation of kinase. Crystal structure of EhCaBP1 with Phenylalanine revealed that the assembled domains, which are similar to Calmodulin N-terminal domain, bind to Phenylalanine revealing the binding mode to the target proteins. The C-terminal domain did not show any of the activities tested. However, over-expression in amebic cells led to a dominant negative phenotype. The results suggest that the two domains of EhCaBP1 are functionally and structurally different from each other. Both the domains are required for structural stability and full range of functional diversity.

Crystal structure of native O-acetyl-serine sulfhydrylase from Entamoeba histolytica and its complex with cysteine: structural evidence for cysteine binding and lack of interactions with serine acetyl transferase.

Cysteine plays a major role in the antioxidative defense mechanisms of the human parasite Entameoba histolytica. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine biosynthetic pathway involving two key enzymes O-acetyl-L-serine sulfhydrylase (OASS) and serine acetyl transferase (SAT). The crystal structure of native OASS from Entameoba histolytica (EhOASS) has been determined at 1.86 A resolution and in complex with its product cysteine at 2.4 A resolution. In comparison with other known OASS structures, insertion in the N-terminal region and C-terminal helix reveal critical differences, which may influence the protein-protein interactions. In spite of lacking chloride binding site at the dimeric interface, the N-terminal extension compared with other known cysteine synthases, participates in dimeric interactions in an interesting domain swapping manner, enabling it to form a stronger dimer. Sulfate is bound in the active site of the native structure, which is replaced by cysteine in the cysteine bound form causing reorientation of the small N-terminal domain and thus closure of the active site. Ligand binding constants of OAS, Cys, and Met with EhOASS are comparable with other known OASS indicating similar active site arrangement and dynamics. The cysteine complexed structure represents the snapshot of the enzyme just before releasing the final product with a closed active site. The C-terminal helix positioning in the EhOASS may effect its interactions with EhSAT and thus influencing the formation of the cysteine synthase complex in this organism.

Crystal structure of calcium binding protein-1 from Entamoeba histolytica: a novel arrangement of EF hand motifs.

Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Several EF-hand containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though these proteins have very high sequence similarity, they bind to different target proteins in a Ca2+ dependent manner, leading to different functional pathways (Yadava et al., Mol Biochem Parasito 1997;84:69-82; Chakrabarty et al., J Biol Chem 2004;279:12898-12908) The crystal structure of the Entamoeba histolytica calcium binding protein-1 (EhCaBP1) has been determined at 2.4 A resolution. The crystals were grown using MPD as precipitant and they belong to P6(3) space group with unit cell parameters of a = 95.25 A, b = 95.25 A, c = 64.99 A. Only two out of the four expected EF hand motifs could be modeled into the electron density map and the final model refined to R factor of 25.6% and Free_R of 28%. Unlike CaM, the first two EF hand motifs in EhCaBP1 are connected by a long helix and form a dumbbell shaped structure. Owing to domain swapping oligomerization three EhCaBP1 molecules interact in a head to tail manner to form a triangular trimer. This arrangement allows the EF-hand motif of one molecule to interact with that of an adjacent molecule to form a two EF-hand domain similar to that seen in the N-terminal domain of the NMR structure of CaBP1, calmodulin and troponin C. The oligomeric state of EhCaBP1 results in reduced flexibility between domains and may be responsible for the more limited set of targets recognized by EhCaBP1.