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.

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.

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.

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.

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.

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.

Inhibition of tobacco etch virus protease activity by detergents.

Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.

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.

Crystallization and initial X-ray diffraction of BtuB, the integral membrane cobalamin transporter of Escherichia coli.

BtuB, the cobalamin transporter from Escherichia coli, has been overexpressed, purified and crystallized. The purified protein was solubilized in n-octyl tetraoxyethylene (C(8)E(4)) and was crystallized using sitting-drop vapor diffusion with PEG 3350 and magnesium acetate as precipitants (pH 6.5). Two crystal forms have been obtained. Crystal type I belongs to space group P3(1)21, with unit-cell parameters a = b = 81.6, c = 210.0 A, alpha = beta = 90, gamma = 120 degrees. Crystal type II belongs to space group P3(1)21, with unit-cell parameters a = b = 81.6, c = 226.0 A, alpha = beta = 90, gamma = 120 degrees. Each crystal form contains a monomer in the asymmetric unit. Diffraction for crystal type I extends to 2.0 A and diffraction for crystal type II extends to 2.7 A. Both crystal forms are suitable for structure determination.

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.

Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli.

Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella. The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.