A Study on Speech Analysis in Acquired Maxillary Defect Patients Treated with Maxillary Obturator.

Aim: The aim of the present study was to assess the speech analysis in acquired maxillary defect patients treated with maxillary obturator. Materials and Methods: Total of 16 patients were considered in the study. The age group of these patients ranged from 40 to 75 years with a mean age of 59.5 years, irrespective of their gender. The surgical obturator was fabricated using self-cure acrylic. The surgical obturator was delivered immediately after surgery. After a healing period of about 2 weeks, the surgical obturator was replaced by an interim prosthesis. This was processed with the help of heat-cure polymethylmethacrylate. The total number of patients was divided into two groups, namely, (A) Definitive obturator group and (B) Interim obturator group. The speech intelligibility (SI), was analyzed. Results: The mean scores for SI before prosthesis in definitive and interim groups were 19.13 ± 3.22 and 19.87 ± 1.72, respectively. This was increased after prosthesis insertion to 24.38 ± 1.30 and 22.37 ± 1.18, which further increased after adaptation period of 2 months to 28.75 ± 1.28 and 24.62 ± 1.59 in two groups. Conclusion: The present study concluded that speech was severely affected by maxillary resection and that rehabilitation with maxillary obturator was successful in restoring these aspects of speech.

Effectiveness and safety of optical coherence tomography-guided PCI in Indian patients with complex lesions: A multicenter, prospective registry.

BACKGROUND: Optical coherence tomography (OCT) is reported to be a feasible and safe imaging modality for the guidance of percutaneous coronary intervention (PCI) of complex lesions. METHODS: This multicenter, prospective registry assessed the minimum stent area (MSA) achieved under OCT guidance. A performance goal of 24% improvement in MSA over and above the recommendation set by the European Association of Percutaneous Cardiovascular Interventions Consensus 2018 (4.5 mm2 MSA for non-left main and 3.5 mm2 for small vessels). The incidence of contrast-induced nephropathy was also assessed. Core lab analysis was conducted. RESULTS: Five hundred patients (average age: 59.4 ± 10.1 years; 83% males) with unstable angina (36.8%), NSTEMI (26.4%), and STEMI (22%) were enrolled. The primary endpoint was achieved in 93% of lesions with stent diameter ≥2.75 mm (average MSA: 6.44 mm2) and 87% of lesions with stent diameter ≤2.5 mm (average MSA: 4.56 mm2). The average MSA (with expansion ≥80% cutoff) was 6.63 mm2 and 4.74 mm2 with a stent diameter ≥2.75 mm and ≤2.5 mm, respectively. According to the core lab analysis, the average MSA achieved with a stent diameter ≥2.75 mm and ≤2.5 mm was 6.23 mm2 and 3.95 mm2, respectively (with expansion ≥80% cutoff). Clinically significant serum creatinine was noted in two patients (0.45%). Major adverse cardiac events at 1 year were noted in 1.2% (n = 6) of the patients; all were cardiac deaths. CONCLUSION: PCI under OCT guidance improves procedural and long-term clinical outcomes in patients with complex lesions not just in a controlled trial environment but also in routine clinical practice.

Molecular studies in familial dilated cardiomyopathy - A pilot study.

Aim: To study genetic variants in patients of familial dilated cardiomyopathy. Methodology: Patients with reduced ejection fraction of less than 45% and dilated left ventricle are considered to have dilated cardiomyopathy. Clinical history was taken and possible secondary causes of dilated cardiomyopathy were excluded. Family history of ≥2 affected relatives or sudden cardiac death in a relative with age less than 35 years were included. Such patients blood sample were sent for next generation sequencing and analysed for presence of genetic variants. Results: As part of pilot study 20 patients (44% were female and 66% were male) were included. There was presence of 16 different pathogenic variants in 14 patients. Two patients had more than one variants in them. Most common of which were sarcomeric mutations constituting 32%. Titin followed by Filamin, Lamin and Desmosomal where the most commonly repeated mutations. Discussion: In our patients of familial dilated cardiomyopathy, 70% were detected to have pathogenic variants in them. Most common variations were seen on Titin gene. Thus those with familial dilated cardiomyopathy should be considered for next generation sequencing. First degree relatives of those with pathogenic variants should be screened using cascade testing for earlier detection and disease monitoring in them.

Diabetes Status After Lateral Pancreaticojejunostomy and Frey's Procedure in Chronic Calcific Pancreatitis: An Observational Study.

Introduction Diabetes secondary to pancreatic diseases is commonly referred to as pancreatogenic diabetes or type 3c diabetes mellitus. This study was conducted to determine the status of diabetes mellitus after Frey's procedure and lateral pancreaticojejunostomy (LPJ) in diabetic and nondiabetic patients with chronic calcific pancreatitis (CCP) and to discuss the clinicopathological course as well as diabetes in CCP. Materials and methods This study was designed as a retrospective observational study consisting of 27 patients with CCP who were surgically treated either with the pancreatic head coring Frey's procedure or with LPJ. Surgeries were performed in a tertiary care hospital of Eastern India by a team of surgeons following the same surgical principle. The diagnosis of CCP was made by clinical and radiological evaluations. Visual Analog Scale (VAS) scoring was used perioperatively to assess pain. Postoperatively, all the patients were monitored clinically; pain scoring and relevant investigations were done depending upon subjective and objective indications. Special attention was paid to diabetic patients through frequent follow-ups and tight glycemic control. All 27 patients were followed up with at least two outpatient follow-ups. Results The trends in fasting blood sugar values in the LPJ group showed a small spike in the early postoperative period (two weeks) with a p-value of >0.05, and later on, it improved over 18 months of follow-up, reaching below the preoperative values (mean 109.38). On the contrary, the fasting blood glucose levels in Frey's procedure revealed a significant spike in the early postoperative period (two weeks) with a mean sugar value of 148 mg/dl and a p-value of 0.01. The levels stayed well above the preoperative values over 18 months of follow-up. The trends in HbA1c showed marginal improvement in the LPJ group in a six-month follow-up period (p-value 0.008) from the preoperative levels. In Frey's procedure group, postoperative HbA1c levels at three months revealed an increase, which can be attributed to the minor but significant loss of pancreatic tissue from the head, which continued to be on the higher side at the six-month follow-up. Trends in mean insulin dosage showed a significant spike in the early postoperative period (two weeks) both in the LPJ (p-value 0.01) and Frey's procedure group (0.01); however, in the LPJ group, the insulin dose showed a reduction over the 18-month follow-up, reaching below the mean preoperative insulin dose. While in the Frey's procedure group, the postoperative insulin dose remained higher throughout the 18-month follow-up period (p-value <0.05). Conclusions LPJ has got a little effect on the diabetic status of nondiabetic patients. Frey's procedure leads to marginal deterioration of the diabetic status and increases in insulin dosage in both diabetic and nondiabetic patients.

Influence of Occlusal Stress on Implant Abutment Junction and Implant Bone Interface: A Finite Element Analysis Study.

AIM: The aim of the present study was to assess the occlusal stress on the implant-abutment junction and implant-bone interface of a long-span implant-supported prosthesis made of two different prosthetic materials. MATERIALS AND METHODS: A computerized tomography of the mandible was used to get the finite element model of the bone. The comparative groups were made as follows: S1 and S2 - 3.7 × 11 mm (44 region) and 4.5 × 11 mm (47 region), S1A and S2A - screw-retained porcelain-fused-to-metal prosthesis, S1B and S2B - cement-retained porcelain-fused-to-metal prosthesis, S1C and S2C - screw-retained zirconia prosthesis, and S1D - cement-retained zirconia prosthesis. Maximum stress generated on the implant-abutment interface of all the prostheses under vertical and oblique load was assessed. RESULTS: For all the comparative groups, maximum level of stress was generated at the cervical level of the implant-bone interface in comparison to the apical and middle-third level under both vertical and oblique load. No statistically significant difference between zirconia and porcelain-fused-to-metal prosthesis was seen at the implant-abutment interface and the cervical third of the implant-bone interface. A significant difference was found between all screw-retained and cement-retained groups. CONCLUSION: The present study concluded that the short implants in combination with standard-length implants using either porcelain-fused-to-metal or zirconia as prosthetic material in the form of long-span implant-supported prosthesis can be a viable treatment option in the posterior mandible. CLINICAL SIGNIFICANCE: The accuracy of the diagnosis, examination, and knowledge of the site where the implant must be inserted, and the choice of superstructure is important for the stability and lifespan of the implant prosthesis.

Discovery and Optimization of Pyrazole Amides as Inhibitors of ELOVL1.

Accumulation of very long chain fatty acids (VLCFAs) due to defects in ATP binding cassette protein D1 (ABCD1) is thought to underlie the pathologies observed in adrenoleukodystrophy (ALD). Pursuing a substrate reduction approach based on the inhibition of elongation of very long chain fatty acid 1 enzyme (ELOVL1), we explored a series of thiazole amides that evolved into compound 27─a highly potent, central nervous system (CNS)-penetrant compound with favorable in vivo pharmacokinetics. Compound 27 selectively inhibits ELOVL1, reducing C26:0 VLCFA synthesis in ALD patient fibroblasts, lymphocytes, and microglia. In mouse models of ALD, compound 27 treatment reduced C26:0 VLCFA concentrations to near-wild-type levels in blood and up to 65% in the brain, a disease-relevant tissue. Preclinical safety findings in the skin, eye, and CNS precluded progression; the origin and relevance of these findings require further study. ELOVL1 inhibition is an effective approach for normalizing VLCFAs in models of ALD.

An infrequent etiology of Infective Endocarditis.

Abiotrophia. defectiva is an infrequent but an important cause of infectious endocarditis (IE). IE caused by A. defectiva may actually be more common than what is reported in the literature because of its failure to grown on routine media like blood agar and MacConkey agar, besides difficulty in its identification by conventional methods. IE by A. defectiva generally have more fulminant course and is associated with higher mortality and morbidity due to frequent embolic episodes and valvular tissue destruction. Ceftriaxone or vancomycin in combination with gentamicin and surgical replacement of valve is the recommended management.

Early and midterm results of extended septal myectomy: Indian experience.

BACKGROUND AND OBJECTIVES: Hypertrophic obstructive cardiomyopathy (HOCM) is a genetic disorder, which is a cause of significant morbidity and sudden cardiac death. Extended septal myectomy (ESM) is the therapeutic gold standard to treat left ventricular outflow tract obstruction (LVOTO) in HOCM resulting in long-term symptomatic relief. The aim of the study was to assess the impact of ESM on midterm symptom relief, LVOTO, and survival in patients suffering from HOCM in the Indian population. METHODS: We retrospectively analyzed clinical data of 36 consecutive symptomatic patients with HOCM having symptoms refractory to medical treatment and LVOTO with resting gradient ≥ 50 mmHg and who underwent ESM at our institution from 2010 to 2019. Preoperative and postoperative transthoracic echocardiography was performed to assess left ventricular outflow tract (LVOT) gradient, septal thickness, and assessment of valvar and cardiac function. RESULTS: ESM was performed successfully in all 36 patients. The mean preoperative LVOT gradient was 113.06 ± 36.70 mmHg and decreased to 15.17 ± 7.30 mmHg (p < .0001) in the initial postoperative period. There were two in-hospital deaths (5.6%). There was no further mortality in the subsequent follow-up. The mean septal thickness was 23.89 ± 5.77 mm preoperatively and 13.17 ± 3.48 mm (p < .0001) postoperatively. During a mean follow-up of 2 years, the NYHA functional class improved from 3.50 ± 0.70 (III-IV) to 1.50 ± 0.70 (I) (p < .0001). CONCLUSIONS: ESM results in immediate abolition of mechanical obstruction to LVOT with normalization of left ventricular pressure and eliminates symptoms associated with HOCM.

A Comprehensive Assessment of Bone Losses in the Postoperative Phase of Single Implant Placed in Mandibular First Molar Regions: A Cone-Beam Computed Tomography-Based Clinical Study.

BACKGROUND AND AIM: Bone loss surrounding dental implant is an unavoidable phenomenon that occasionally leads to implant failure. Implant-related bone loss exhibits different patterns and rate as per oral milieu and hygiene habits. This study was aimed to clinically assess the crestal bone losses in the postoperative phase of single implant placed in mandibular first molar regions. MATERIALS AND METHODS: The present in vivo study was planned and completed on the patients selected from the Department of Prosthodontics of the institute. A total of twenty patients were selected randomly in which right mandibular first molar was rehabilitated by dental implants. All radiographic analysis was attempted by cone-beam computed tomography (CBCT). All twenty implants were placed by standard clinical protocols. CBCT analysis was attempted to measure existing bone levels on all four surfaces of implant. These measurements were completed at three different postoperative phases. Informed consent was obtained from all participating patients. STATISTICAL ANALYSIS AND RESULTS: Statistical analysis was completed by statistical software Statistical Package for the Social Sciences. P <0.05 was taken as statistically significant. Among all 20 patients, males were 14 and females were 6 in the age range of 25-45 years. Mean bone losses were in the range of 0.226-0.737 for Group I. Intergroup comparison by two-sample t-test showed a significant difference (0.01) for mesial surfaces. All mean values were maximum for mesial surfaces and minimum for lingual surfaces. CONCLUSION: The mean crestal bone loss at four implant surfaces evaluated at different postoperative phases was nonsignificant. However, there were significant differences in mean crestal bone loss at four surfaces of all studied implants in a particular group.

Implementation of Anthropometric Measurements in Anterior Teeth Selection in Two Ethnic Groups: An In Vivo Study.

AIM: The aim of this study was to evaluate whether there is any consistent relationship between interalar distance and six maxillary anterior teeth in two ethnic groups. MATERIALS AND METHODS: Two hundred subjects (Assamese 100 and Nyishi 100) of different age and sex ranging from 18 to 33 years were identified for this study. The distance between two widest points marked on the alae of nose was measured by means of sliding digital caliper. Statistical analysis of variance was the testing method to determine whether the nasal width and intercanine distance were different in various racial groups. RESULTS: The mean nasal width dimension in Assamese group was 36.38 mm (range 28.55-50.00 mm). The mean nasal width dimension in Nyishi group was 35.73 mm (range 31.34-41.90 mm). For the nasal width, no significant difference was observed between two races. For the canine cusp tip to cusp tip distance, the mean value of Assamese subjects was 43.63 mm (range 39.02-50.02 mm), whereas the Nyishi presented 41.77 mm (range 36.69-47.05 mm). CONCLUSION: For the two racial groups studied, the measurements of the interalar width showed a weak correlation with the canine to canine distance, not sufficiently high to be used as a predictive factor. It suggests to select wider artificial teeth. Practitioners can use ethnic norms as guides, not absolute values.

To study the effect of high dose Atorvastatin 40mg versus 80mg in patients with dyslipidemia.

OBJECTIVE: Primary objective was to compare the effects of atorvastatin 40mg vs 80mg on LDL-C in Indian patients with atherosclerotic dyslipidemia. Secondary objectives were to compare the effects of atorvastatin 40mg vs 80mg on HDL-C and triglycerides and also comparing of side effects (myopathy, hepatotoxicity and new onset diabetes mellitus) of both doses. METHOD: This Study is A Prospective, randomized, open-label, comparative study. This study was conducted on 240 patients of dyslipidemia (as per ACC/AHA 2013 lipid guidelines) attending the OPD/wards/CCU of department of cardiology, Sir Ganga Ram Hospital. They were randomly divided into 2 groups of 120 each. Group A consisted patients who received Atorvastatin 40mg daily and Group B Atorvastatin 80mg daily. The follow up period was 6 months. RESULTS: At 3 and 6 month follow up, Atorvastatin 40mg leads to mean LDL cholesterol reduction of 47.18±20.81 & 50.03±18.06 respectively. While Atorvastatin 80mg results in LDL reduction as 50.11±15.85 & 52.30±13.72. The comparison between two doses revealed a non-significant difference (p=.118 & p=.149 respectively). At 6 months of follow up, few patients reported myalgia (2 in group A and 7 in Group B). The difference between groups was significant (p=.045). Although none of our patient had significant elevation of CPK. CONCLUSION: This study concluded that both doses of atorvastatin (40 & 80mg) are equally efficacious in improving dyslipidemia but higher dose leads to more incidence of myalgia.

Anemia profile in patients with congestive heart failure a hospital based observational study.

OBJECTIVES: Iron deficiency (ID) is an emerging problem in patients with congestive heart failure (CHF) and can be a potential therapeutic target. As ID is highly prevalent in the society, it is hypothesized that Indian patients with CHF have high prevalence of ID. METHODS: CHF patients (n = 275) were selected and underwent laboratory evaluation including hemoglobin concentration, serum iron, transferrin, ferritin, B12 and folate level. RESULTS: Two hundred and seventy-five patients with heart failure (mean age - 62.72, mean Hb- 10.54 g/dl, 188 males [68.36%] and 87 females [31.64%]) were enrolled in the study. 211 out of 275 (76.7%) were found to be anemic. Out of 275 patients. 148 (53.8%) were diagnosed with iron deficiency. 12.7% (n = 35) were B12 deficient and 5.1% (n = 14) were folate deficient. In the anemic group, ID was present in 130 patients (61.61%), B12 deficiency in 32 patients (15.16%) and folate deficiency in 12 patients (5.68%). In the group of patients without anemia, ID was present in 18 patients (28.12%) while B12 and folate deficiency was present in 3 (4.68%) and 2 (3.12%) patients, respectively. CONCLUSION: Iron deficiency is present in substantial number while B12 and folate account for a few number of cases. Substantial number of patients without anemia were found to be iron deficient.

Refractory pediatric cardiogenic shock: A case for mechanical support.

Acute left ventricular dysfunction in children justifies aggressive treatment because of the high potential for complete recovery. The options for providing mechanical support to the failing heart in a child include extracorporeal membrane oxygenation, left ventricular assist devices, and the use of the intra-aortic balloon pump (IABP). The IABP is a commonly used method of temporary circulatory support in adults. However, despite the availability of pediatric size balloons, the usage of IABP for temporary circulatory support in children has not been widespread. Current case report, first from India in pediatric age group, aims to aware the pediatric intensivist about the role of IABP in providing temporary mechanical cardiovascular support in managing patients with refractory low cardiac output state.

Insights into regioselective metabolism of mefenamic acid by cytochrome P450 BM3 mutants through crystallography, docking, molecular dynamics, and free energy calculations.

Cytochrome P450 BM3 (CYP102A1) mutant M11 is able to metabolize a wide range of drugs and drug-like compounds. Among these, M11 was recently found to be able to catalyze formation of human metabolites of mefenamic acid and other nonsteroidal anti-inflammatory drugs (NSAIDs). Interestingly, single active-site mutations such as V87I were reported to invert regioselectivity in NSAID hydroxylation. In this work, we combine crystallography and molecular simulation to study the effect of single mutations on binding and regioselective metabolism of mefenamic acid by M11 mutants. The heme domain of the protein mutant M11 was expressed, purified, and crystallized, and its X-ray structure was used as template for modeling. A multistep approach was used that combines molecular docking, molecular dynamics (MD) simulation, and binding free-energy calculations to address protein flexibility. In this way, preferred binding modes that are consistent with oxidation at the experimentally observed sites of metabolism (SOMs) were identified. Whereas docking could not be used to retrospectively predict experimental trends in regioselectivity, we were able to rank binding modes in line with the preferred SOMs of mefenamic acid by M11 and its mutants by including protein flexibility and dynamics in free-energy computation. In addition, we could obtain structural insights into the change in regioselectivity of mefenamic acid hydroxylation due to single active-site mutations. Our findings confirm that use of MD and binding free-energy calculation is useful for studying biocatalysis in those cases in which enzyme binding is a critical event in determining the selective metabolism of a substrate.

Anomalous single coronary artery on low dose MDCT.

Coronary artery anomalies may involve the origin, course, and structure of epicardial coronary arteries and occur in less than 1% of the general population. Clinical presentation ranges from being completely asymptomatic to sudden death. Multi-detector computed tomography has come a long way in the diagnosis of coronary artery anomalies since the introduction of 4 rows of detectors in scanners, considering its non invasive nature and the benefits of 3D reconstruction. Defining the coronary anatomy helps in clinical decision making and timely intervention. Since repeated angiographies may be required, low dose CT is an excellent investigation for diagnosis and post interventional follow up rather than repeated invasive catheter angiographies or high dose CT examination. We report two cases of clinically significant single coronary artery anomalies; a case of single Right coronary artery and another case of single Left coronary artery (Anomaly of origin & course).

Refolding and characterization of a soluble ectodomain complex of the calcitonin gene-related peptide receptor.

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of two membrane proteins: calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a class B G-protein-coupled receptor (GPCR), possessing a characteristic large amino-terminal extracellular domain (ECD) important for ligand recognition and binding. Dimerization of CLR with RAMP1 provides specificity for CGRP versus related agonists. Here we report the expression, purification, and refolding of a soluble form of the CGRP receptor comprising a heterodimer of the CLR and RAMP1 ECDs. The extracellular protein domains corresponding to residues 23-133 of CLR and residues 26-117 of RAMP1 were shown to be sufficient for formation of a stable, monodisperse complex. The binding affinity of the purified ECD complex for the CGRP peptide was significantly lower than that of the native receptor (IC(50) of 12 microM for the purified ECD complex vs 233 pM for membrane-bound CGRP receptor), indicating that other regions of CLR and/or RAMP1 are important for peptide agonist binding. However, high-affinity binding to known potent and specific nonpeptide antagonists of the CGRP receptor, including olcegepant and telcagepant (K(D) < 0.02 muM), as well as N-terminally truncated peptides and peptide analogues (140 nM to 1.62 microM) was observed.

Membrane protein expression and production: effects of polyhistidine tag length and position.

Polyhistidine tags enable the facile purification of proteins by immobilized metal affinity chromatography (IMAC). Both the type and position of purification tags can affect significantly properties of a protein such as its expression level, behavior in solution, and its ability to form suitable samples (esp. suitable crystals for X-ray crystallography). We investigated systematically the effects of polyhistidine tag length and position on many properties related to expression and purification of recombinant integral membrane proteins. Specifically, modified Escherichia coli pET expression vectors were built that placed 6- or 10-histidine tags at the N- or C-termini of the subcloned gene. The E. coli water channel AqpZ was subcloned into this suite of vectors and its expression, purification, solution properties, and yield were characterized. These studies show that: (1) all vectors yield similar expression levels, (2) tag length has a greater effect than tag position upon yield, (3) neither tag length nor position affects significantly detergent solubilization of the protein, (4) the length of the tag affects the oligomerization state of the purified protein, and (5) the tag length and position change chromatographic behavior of the detergent-solubilized protein. In addition, substitution of the lysine codon AAA at the second position, previously shown to have some effect upon soluble protein expression levels, did not have a large effect on AqpZ production. We are currently producing approximately 12 mg of purified AqpZ per liter of shake-flask culture, and preliminary crystals that diffract to approximately 5A resolution have been obtained.

Enzymatic E-colicins bind to their target receptor BtuB by presentation of a small binding epitope on a coiled-coil scaffold.

Toxins and viruses often initiate their attacks by binding to specific proteins on the surfaces of target cells. Bacterial toxins (e.g. bacteriocins) and viruses (bacteriophages) targeting Gram-negative bacteria typically bind to outer membrane proteins. Bacterial E-colicins target Escherichia coli by binding to the outer membrane cobalamin transporter BtuB. Colicins are tripartite molecules possessing receptor-binding, translocation, and toxin domains connected by long coiled-coil alpha-helices. Surprisingly, the crystal structure of colicin E3 does not possess a recognizable globular fold in its receptor-binding domain. We hypothesized that the binding epitope of enzymatic E-colicins is a short loop connecting the two alpha-helices that comprise the coiled-coil region and that this flanking coiled-coil region serves to present the loop in a binding-capable conformation. To test this hypothesis, we designed and synthesized a 34-residue peptide (E-peptide-1) corresponding to residues Ala366-Arg399 of the helix-loop-helix region of colicin E3. Cysteines placed near the ends of the peptide (I372C and A393C) enabled crosslinking for reduction of conformational entropy and formation of a peptide structure that would present the loop epitope. A fluorescent analog was also made for characterization of binding by measurement of fluorescence polarization. Our analysis shows the following. (i). E-peptide-1 is predominantly random coil in aqueous solution, but disulfide bond formation increases its alpha-helical content in both aqueous buffer and solvents that promote helix formation. (ii). Fluorescein-labeled E-peptide-1 binds to purified BtuB in a calcium-dependent manner with a Kd of 43.6 +/- 4.9 nm or 2370 +/- 670 nm in the presence or absence of calcium, respectively. (iii). In the presence of calcium, cyanocobalamin (CN-Cbl) displaces E-peptide-1 with a nanomolar inhibition constant (Ki = 78.9 +/- 5.6 nm). We conclude that the BtuB binding sites for cobalamins and enzymatic E-colicins are overlapping but inequivalent and that the distal loop and (possibly) the short alpha-helical flanking regions are sufficient for high affinity binding.

Substrate-induced transmembrane signaling in the cobalamin transporter BtuB.

The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients. In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed. We have solved crystal structures of BtuB, the outer membrane cobalamin transporter from Escherichia coli, in the absence and presence of cyanocobalamin (vitamin B(12)). In these structures, the Ton box is ordered and undergoes a conformational change in the presence of bound substrate. Calcium has been implicated as a necessary factor for the high-affinity binding (K(d) approximately 0.3 nM) of cyanocobalamin to BtuB. We observe two bound calcium ions that order three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium.