Loop Truncation Research Articles

First crystal structures of 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) from Mycobacterium tuberculosis indicate a distinct mechanism of intermediate stabilization

The development of drug resistance by Mycobacterium tuberculosis and other pathogenic bacteria emphasizes the need for new antibiotics. Unlike animals, most bacteria synthesize isoprenoid precursors through the MEP pathway. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the first reaction of the MEP pathway and is an attractive target for the development of new antibiotics. We report here the successful use of a loop truncation to crystallize and solve the first DXPS structures of a pathogen, namely M. tuberculosis (MtDXPS). The main difference found to other DXPS structures is in the active site where a highly coordinated water was found, showing a new mechanism for the enamine-intermediate stabilization. Unlike other DXPS structures, a “fork-like” motif could be identified in the enamine structure, using a different residue for the interaction with the cofactor, potentially leading to a decrease in the stability of the intermediate. In addition, electron density suggesting a phosphate group could be found close to the active site, provides new evidence for the D-GAP binding site. These results provide the opportunity to improve or develop new inhibitors specific for MtDXPS through structure-based drug design.

Catalytic and structural effects of flexible loop deletion in organophosphorus hydrolase enzyme: A thermostability improvement mechanism

Thermostability improvement of enzymes used industrially or commercially would develop their capacity and commercial potential due to increased enzymatic competence and cost-effectiveness. Several stabilizing factors have been suggested to be the base of thermal stability, like proline replacements, disulfide bonds, surface loop truncation and ionic pair networks creation. This research evaluated the mechanism of increasing the rigidity of organophosphorus hydrolase enzyme by flexible loop truncation. Bioinformatics analysis revealed that the mutated protein retains its stability after loop truncation (five amino acids deleted). The thermostability of the wild-type (OPH-wt) and mutated (OPH-D5) enzymes were investigated by half-life, Delta Gi, and fluorescence and far-UV CD analysis. Results demonstrated an increase half-life and Delta Gi in OPH-D5 compared to OPH-wt. These results were confirmed by extrinsic fluorescence and circular dichroism (CD) spectrometry experiments, therefore, as rigidity increased in OPHD5 after loop truncation, half-life and Delta Gi also increased. Based on these findings, a strong case is presented for thermostability improvement of OPH enzyme by flexible loop truncation after bioinformatics analysis.

An NMR Confirmation for Increased Folded State Entropy Following Loop Truncation.

Previous studies conducted on flexible loop regions in proteins revealed that the energetic consequences of changing loop length predominantly arise from the entropic cost of ordering a loop during folding. However, in an earlier study of human acylphosphatase (hmAcP) using experimental and computational approaches, we showed that thermodynamic stabilization upon loop truncation can be attributed mainly to the increased entropy of the folded state. Here, using 15N NMR spectroscopy, we studied the effect of loop truncation on hmAcP backbone dynamics on the picosecond-nanosecond timescale with the aim of confirming the effect of folded state entropy on protein stability. NMR-relaxation-derived N-H squared generalized order parameters reveal that loop truncation results in a significant increase in protein conformational flexibility. Comparison of these results with previously acquired all-atom molecular dynamics simulation, analyzed here in terms of squared generalized NMR order parameters, demonstrates general agreement between the two methods. The NMR study not only provides direct evidence for the enhanced conformational entropy of the folded state of hmAcP upon loop truncation but also gives a quantitative measure of the observed effects.

Probing the Impact of Local Structural Dynamics of Conformational Epitopes on Antibody Recognition.

Antibody-antigen interactions are governed by recognition of specific residues and structural complementarity between the antigen epitope and antibody paratope. While X-ray crystallography has provided detailed insights into static conformations of antibody-antigen complexes, factors such as conformational flexibility and dynamics, which are not readily apparent in the structures, can also have an impact on the binding event. Here we investigate the contribution of dynamics in the HIV-1 gp120 glycoprotein to antibody recognition of conserved conformational epitopes, including the CD4- and coreceptor-binding sites, and an inner domain site that is targeted by ADCC-active antibodies. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) was used to measure local structural dynamics across a panel of variable loop truncation mutants of HIV-1 gp120, including full-length gp120, ΔV3, ΔV1/V2, and extended core, which includes ΔV1/V2 and V3 loop truncations. CD4-bound full-length gp120 was also examined as a reference state. HDX-MS revealed a clear trend toward an increased level of order of the conserved subunit core resulting from loop truncation. Combined with biolayer interferometry and enzyme-linked immunosorbent assay measurements of antibody-antigen binding, we demonstrate that an increased level of ordering of the subunit core was associated with better recognition by an array of antibodies targeting complex conformational epitopes. These results provide detailed insight into the influence of structural dynamics on antibody-antigen interactions and suggest the importance of characterizing the structural stability of vaccine candidates to improve antibody recognition of complex epitopes.

The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals

The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.

Vocal Cord Dysfunction Related to Water-Damaged Buildings

Vocal cord dysfunction (VCD) is the intermittent paradoxical adduction of the vocal cords during respiration, resulting in variable upper airway obstruction. Exposure to damp indoor environments is associated with adverse respiratory health outcomes, including asthma, but its role in the development of VCD is not well described. We describe the spectrum of respiratory illness in occupants of 2 water-damaged office buildings. The National Institute for Occupational Safety and Health conducted a health hazard evaluation that included interviews with managers, a maintenance officer, a remediation specialist who had evaluated the 2 buildings, employees, and consulting physicians. In addition, medical records and reports of building evaluations were reviewed. Diagnostic evaluations for VCD had been conducted at the Asthma and Allergy Center of the Medical College of Wisconsin. Two cases of VCD were temporally related to occupancy of water-damaged buildings. The patients experienced cough, chest tightness, dyspnea, wheezing, and hoarseness when in the buildings. Spirometry was normal. Methacholine challenge did not show bronchial hyperreactivity but did elicit symptoms of VCD and inspiratory flow-volume loop truncation. Direct laryngoscopy revealed vocal cord adduction during inspiration. Coworkers developed upper and lower respiratory symptoms; their diagnoses included sinusitis and asthma, consistent with recognized effects of exposure to indoor dampness. Building evaluations provided evidence of water damage and mold growth. VCD can occur with exposure to water-damaged buildings and should be considered in exposed patients with asthma-like symptoms.

The Natively Disordered Loop of Bcl-2 Undergoes Phosphorylation-Dependent Conformational Change and Interacts with Pin1

Bcl-2 plays a central role in the regulation of apoptosis. Structural studies of Bcl-2 revealed the presence of a flexible and natively disordered loop that bridges the Bcl-2 homology motifs, BH3 and BH4. This loop is phosphorylated on multiple sites in response to a variety of external stimuli, including the microtubule-targeting drugs, paclitaxel and colchicine. Currently, the underlying molecular mechanism of Bcl-2 phosphorylation and its biological significance remain elusive. In this study, we investigated the molecular characteristics of this anti-apoptotic protein. To this end, we generated synthetic peptides derived from the Bcl-2 loop, and multiple Bcl-2 loop truncation mutants that include the phosphorylation sites. Our results demonstrate that S87 in the flexible loop of Bcl-2 is the primary phosphorylation site for JNK and ERK2, suggesting some sequence or structural specificity for the phosphorylation by these kinases. Our NMR studies and molecular dynamics simulation studies support indicate that phosphorylation of S87 induces a conformational change in the peptide. Finally, we show that the phosphorylated peptides of the Bcl-2 loop can bind Pin1, further substantiating the phosphorylation-mediated conformation change of Bcl-2.

V3 Loop Truncations in HIV-1 Envelope Impart Resistance to Coreceptor Inhibitors and Enhanced Sensitivity to Neutralizing Antibodies

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (ΔV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.

Structure/Function Analysis of α2A-Adrenergic Receptor Interaction with G Protein-coupledReceptor Kinase 2

G protein-coupled receptors (GPCRs) mediate the ability of a diverse array of extracellular stimuli to control intracellular signaling. Many GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs), a process that mediates agonist-specific desensitization in many cells. Although GRK binding to activated GPCRs results in kinase activation and receptor phosphorylation, relatively little is known about the mechanism of GRK/GPCR interaction or how this interaction results in kinase activation. Here, we used the alpha2A-adrenergic receptor (alpha(2A)AR) as a model to study GRK/receptor interaction because GRK2 phosphorylation of four adjacent serines within the large third intracellular loop of this receptor is known to mediate desensitization. Various domains of the alpha(2A)AR were expressed as glutathione S-transferase fusion proteins and tested for the ability to bind purified GRK2. The second and third intracellular loops of the alpha(2A)AR directly interacted with GRK2, whereas the first intracellular loop and C-terminal domain did not. Truncation mutagenesis identified three discrete regions within the third loop that contributed to GRK2 binding, the membrane proximal N- and C-terminal regions as well as a central region adjacent to the phosphorylation sites. Site-directed mutagenesis revealed a critical role for specific basic residues within these regions in mediating GRK2 interaction with the alpha(2A)AR. Mutation of these residues within the holo-alpha(2A)AR diminished GRK2-promoted phosphorylation of the receptor as well as the ability of the kinase to be activated by receptor binding. These studies provide new insight into the mechanism of interaction and activation of GRK2 by GPCRs and suggest that GRK2 binding is critical not only for receptor phosphorylation but also for full activity of the kinase.

Efficient Insertion of Odd-numbered Transmembrane Segments of the Tetracycline Resistance Protein Requires Even-numbered Segments

Functional membrane insertion elements in the pBR322 tetracycline resistance protein were identified by comparing the ability of odd-numbered transmembrane segments and their attached periplasmic loops to insert into the membrane individually or when combined with the next even-numbered segment in the tetracycline resistance protein sequence. The efficiency with which individual odd-numbered segments and periplasmic loops inserted was probed by treating proteins truncated at the distal ends of periplasmic loops P2-P6 with carboxypeptidases and endoproteases in inside-out membrane vesicles. Insertion of odd-numbered segments and attached loops is inefficient when they occupy a C-terminal position in the protein. The C-terminal odd-numbered segment and loop sequences of 34-54% of the molecules of periplasmic loop truncation mutants could be removed by carboxypeptidase Y. In contrast, odd-numbered segments and loops insert efficiently if the next even-numbered segment in the sequence is present. In such cytoplasmic loop truncation mutants, only the cytoplasmic tail sequences of the proteins could be removed by carboxypeptidases. Remarkably, insertion of individual odd-numbered segments and loops is inefficient even though free energies for insertion of these sequences are highly favorable. The results indicate that pairs of adjacent segments, possibly "helical hairpins," are necessary for efficient membrane insertion of the tetracycline resistance protein.

120 THE DIAGNOSIS OF PAEDIATRIC VOCAL CORD DYSFUNCTION (VCD)

VCD in children is a poorly recognised entity, easily confused with asthma. Thirty-seven children with a diagnosis of VCD made by rhinolaryngoscopy were identified. Eight of 29 patients diagnosed with both VCD and asthma were evaluated during attacks of VCD (defined by rhinolaryngoscopy) or asthma (defined by a positive methacholine challenge). We examined retrospectively the value of clinical assessment, spirometry and blood gas analysis in differentiating between episodes of VCD and asthma in these patients. Laryngeal stridor was the most reliable clinical feature that distinguished VCD from asthma, occurring in 6/8 episodes of VCD and 1/8 episodes of asthma. The presence or localisation of expiratory wheezing was not helpful. Spirometric data did not reliably distinguish VCD from asthma, although inspiratory loop truncation occurred during 4/8 episodes of VCD and not during, asthma. The mean arterial oxygen level, though lower in asthma than in VCD, did not reliably differentiate between both conditions. However, the calculated alveolar-arterial (A-a) gradient was narrow during all episodes of VCD (mean = 6.75 mm Hg) whereas it was widened during all episodes of asthma mean = 20.3 mm Hg). We conclude that VCD occurs in children and frequently coexists with asthma. Episodes of VCD and asthma are not reliably distinguished in children by clinical and spirometric evaluation. Rhinolaryngoscopy is therefore necessary to establish a diagnosis of VCD. Subsequent episodes of VCD may be reliably distinguished from asthma by a normal A-a gradient.