Loops Of Class Research Articles

Unique features of different classes of G-protein-coupled receptors revealed from sequence coevolutionary and structural analysis.

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and represent the primary targets of about one third of currently marketed drugs. Despite the critical importance, experimental structures have been determined for only a limited portion of GPCRs and functional mechanisms of GPCRs remain poorly understood. Here, we have constructed novel sequence coevolutionary models of the A and B classes of GPCRs and compared them with residue contact frequency maps generated with available experimental structures. Significant portions of structural residue contacts were successfully detected in the sequence-based covariational models. "Exception" residue contacts predicted from sequence coevolutionary models but not available structures added missing links that were important for GPCR activation and allosteric modulation. Moreover, we identified distinct residue contacts involving different sets of functional motifs for GPCR activation, such as the Na+ pocket, CWxP, DRY, PIF, and NPxxY motifs in the class A and the HETx and PxxG motifs in the class B. Finally, we systematically uncovered critical residue contacts tuned by allosteric modulation in the two classes of GPCRs, including those from the activation motifs and particularly the extracellular and intracellular loops in class A GPCRs. These findings provide a promising framework for rational design of ligands to regulate GPCR activation and allosteric modulation.

Continuous Choreographies as Limiting Solutions of N-body Type Problems with Weak Interaction

We consider the limit $N\to +\infty$ of $N$-body type problems with weak interaction, equal masses and $-\sigma$-homogeneous potential, $0<\sigma<1$. We obtain the integro-differential equation that the motions must satisfy, with limit choreographic solutions corresponding to travelling waves of this equation. Such equation is the Euler-Lagrange equation of a corresponding limiting action functional. Our main result is that the circle is the absolute minimizer of the action functional among zero mean (travelling wave) loops of class $H^1$.

Abstract PR02: Targeting PI3K: The PIP2 binding site

Abstract Structural biology has yielded many significant insights into inhibitor binding and isoform selectivity, directing the design of many important clinical candidates. To date, structural studies have focused on the ATP-binding site and ATP-competitive inhibitors. We have recently determined the structure of PI3Kα; in complex with the soluble lipid substrate mimetic, diC4-PIP2. The substrate binds in a positively charged pocket, defined by the activation and P-loops of the kinase domain, and the iSH2 domain of p85. The positively charged residues responsible for binding PIP2 that are present on the activation loop of Class I PI3K are missing in Class II and III PI3K. This explains why these two classes have lower affinity for PIP2. The complex structure provides insights into the catalysis and regulation of PI3K. Key interactions between the activation loop and nSH2 domain may modulate active and inactive conformations of the enzyme upon the binding of phosphorylated receptor tyrosine kinases. In addition, a second lipid substrate was identified in the structure. It binds in a hydrophobic pocket between the Adaptor Binding Domain (ABD), kinase domain and iSH2 domain of p85. Fluorescence quenching studies confirm the ability of PI3K to bind an additional PIP2 molecule. This abstract is also being presented as Poster B17. Citation Format: Michelle Miller, Oleg Schmidt-Kittler, David M. Bolduc, Evan T. Brower, Daniele Chaves-Moreira, Marc Allaire, Kenneth W. Kinzler, Ian G. Jennings, Philip E. Thompson, Philip A. Cole, L. Mario Amzel, Bert Vogelstein, Sandra B. Gabelli. Targeting PI3K: The PIP2 binding site. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR02.

The functional role of Cys3-Cys4 loop in hydrophobin HGFI.

Hydrophobins are a large group of low-molecular weight proteins. These proteins are highly surface-active and can form amphipathic membranes by self-assembling at hydrophobic-hydrophilic interfaces. Based on physical properties and hydropathy profiles, hydrophobins are divided into two classes. Upon the analysis of amino acid sequences and higher structures, some models suggest that the Cys3-Cys4 loop regions in class I and II hydrophobins can exhibit remarkable difference in their alignment and conformation, and have a critical role in the rodlets structure formation. To examine the requirement for the Cys3-Cys4 loop in class I hydrophobins, we used protein fusion technology to obtain a mutant protein HGFI-AR by replacing the amino acids between Cys3 and Cys4 of the class I hydrophobin HGFI from Grifola frondosa with those ones between Cys3 and Cys4 of the class II hydrophobin HFBI from Trichoderma reesei. The gene of the mutant protein HGFI-AR was successfully expressed in Pichia pastoris. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the purified HGFI-AR could form amphipathic membranes by self-assembling at mica and hydrophobic polystyrene surfaces. This property enabled them to alter the surface wettabilities of polystyrene and mica and change the elemental composition of siliconized glass. In comparison to recombinant class I hydrophobin HGFI (rHGFI), the membranes formed on hydrophobic surfaces by HGFI-AR were not robust enough to resist 1% hot SDS washing. Atomic force microscopy (AFM) measurements indicated that unlike rHGFI, no rodlet structure was observed on the mutant protein HGFI-AR coated mica surface. In addition, when compared to rHGFI, no secondary structural change was detected by Circular Dichroism (CD) spectroscopy after HGFI-AR self-assembled at the water-air interface. HGFI-AR could not either be deemed responsible for the fluorescence intensity increase of Thioflavin T (THT) and the Congo Red (CR) absorption spectra shift (after the THT(CR)/HGFI-AR mixed aqueous solution was drastically vortexed). Remarkably, replacement of the Cys3-Cys4 loop could impair the rodlet formation of the class I hydrophobin HGFI. So, it could be speculated that the Cys3-Cys4 loop plays an important role in conformation and functionality, when the class I hydrophobin HGFI self-assembles at hydrophobic-hydrophilic interfaces.

A Comparative Study of Dermatoglyphics in Individuals with Normal Occlusions and Malocclusions

Dermatoglyphics is the study on edpidermal ridges on the palmar and plantar surfaces of the feet and hand. Embryological development of orodental structures and these dermal patterns occur during the same period. The environmentally influenced genetic predisposition is found in different types of malocclusions. This malocclusion should also exhibit different dermal patterns which are unique for each class. The present study was conducted to compare the Dermatoglyphics parameters of individuals with normal occlusions and various classes of malocclusions. The participants were divided into four groups; Group 1: class I ideal; Group 2: class I malocclusionl; Group 3: class II Div 1 and Div 2; Group IV: class III. The finger and palmar prints were obtained and they were analyzed for each type of pattern. The Total Finger Fridge Count (TFRC), a-b ridge count and the angle "atd" (angle formed by connecting lines from digital triradius 'a' to the axial triradius and from this triradius to the digital triradius 'd') were associated with particular groups. The data was entered and analyzed by using Students t-test and ANOVA. Particular predictive occurrence of patterns was not found to be associated with each group. However, some of the finger patterns, such as increase in twinned loops in class II malocclusions and absence of radial loops in class III malocclusions were found to be statistically significant (p<0.001). The parameters associated with palmar prints had no statistical significance. Further studies with more samples in each group should be carried out.

ISW Treatment for the Skeletal Class III with Anterior Crossbite and Minor Facial Asymmetry

ABSTRACT Skeletal class III with anterior crossbite is a very common orthodontic problem in Taiwan. Early diagnosis and treatment are very important to achieve effective correction. This article reports the treatment outcomes using improved superelastic TiNi alloy wire (ISW; developed by Tokyo Medical and Dental University) for a case of skeletal class III malocclusion with anterior crossbite and lower anterior crowding combined minor facial asymmetry. ISW allowed relief of crowding to facilitate correction for this case. In comparison with the traditional treatment with SSW multiple L loops of class III patient, ISW multiloop edgewise archwire (MEAW) can provide a more efficient and easier way for the patient. After 16 months of treatment, a desirable outcome was achieved and patient was satisfied with the treatment procedure and result. How to cite this article Tseng CH, Jian-Hong YU. ISW Treatment for the Skeletal Class III with Anterior Crossbite and Minor Facial Asymmetry. Int J Experiment Dent Sci 2013;2(1):53-59.

Hydrolysis Spectrum Extension of CMY-2-Like β-Lactamases Resulting from Structural Alteration in the Y-X-N Loop

The Citrobacter freundii isolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) β-lactamase. The chromosome-borne bla(AmpC-CHA) gene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded the Escherichia coli TOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resulting E. coli TOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 β-lactamase, which is structurally related to the chromosome-borne cephalosporinase of C. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficient E. coli strain. Moreover, it exhibited increased catalytic efficiency (k(cat)/K(m)) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (k(cat)), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A β-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.

Probing the Self-Assembly and the Accompanying Structural Changes of Hydrophobin SC3 on a Hydrophobic Surface by Mass Spectrometry

The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate α-helical state to the stable end form called the β-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the α-helical state. The β-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39–S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of α-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the β-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly.

The Second Intracellular Loop of Metabotropic Glutamate Receptors Recognizes C Termini of G-protein α-Subunits

Heptahelical receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G-protein. In the G-protein alpha subunit, the C-terminal 5 residues, the N terminus, and the alpha N-beta 1 and alpha 4-alpha 5 loops play important roles. On the receptor side, both the second and third (i2 and i3) intracellular loops as well as the C-terminal tail probably contact these different regions of the G-protein. It is now accepted that the C terminus of the alpha subunit binds in a cavity formed by the i2 and i3 loops. Among the various G-protein-coupled receptors (GPCRs), class III receptors that include metabotropic glutamate (mGlu) receptors greatly differ from the rhodopsin-like GPCRs, but the contact zone between these receptors and the G-protein is less understood. The C terminus of the alpha subunit has been shown to play a pivotal role in the selective recognition of class III GPCRs. Indeed, the mGlu2 and mGlu4 and -8 receptors can discriminate between alpha subunits that differ at the level of their C-terminal end only (such as Gqo and Gqz). Here, we examine the role of the i2 loop of mGluRs in the selective recognition of this region of the alpha subunit. To that aim, we analyzed the coupling properties of mGlu2 and mGlu4 or -8 receptors and chimeras containing the i2 loop of the converse receptor to G-protein alpha subunits that only differ by their C termini (Gqo,Gqz, and their point mutants). Our data demonstrate that the central portion of the i2 loop is responsible for the selective recognition of the C-terminal end of the alpha subunit, especially the residue on position -4. These data are consistent with the proposal that the C-terminal end of the G-protein alpha subunit interacts with residues in a cavity formed by the i2 and i3 loops in class III GPCRs, as reported for class I GPCRs.

Microtubule Alterations and Mutations Induced by Desoxyepothilone B: Implications for Drug-Target Interactions

Epothilones, like paclitaxel, bind to β-tubulin and stabilize microtubules. We selected a series of four leukemia sublines that display increasing levels of resistance to the epothilone analog desoxyepothilone B (dEpoB). The dEpoB cells selected in 30–140 nM were ∼15-fold cross-resistant to paclitaxel, while 300 nM selected cells were 467-fold resistant to this agent. The dEpoB-selected cells are hypersensitive to microtubule destabilizing agents, and express increased levels of class III β-tubulin and MAP4. A novel class I β-tubulin mutation, A231T, that affects microtubule stability but does not alter paclitaxel binding, was identified. The 300 nM selected cells acquired a second mutation, Q292E, situated near the M loop of class I β-tubulin. These cells fail to undergo drug-induced tubulin polymerization due to dramatically reduced drug binding. The dEpoB-resistant leukemia cells provide novel insights into microtubule dynamics and, in particular, drug-target interactions.

Mutations in specific I-Ak α2 and β2 domain residues affect surface expression

A previous investigation demonstrated that several mutations in class II dimer-of-dimers contact residues interfere with antigen presentation by transfectants but not with plasma membrane expression of the mutant class II. In the present study we examined other class II mutations in this region that did inhibit plasma membrane expression of mutant class II molecules. Molecules containing both mutations H alpha 181D in the alpha(2) domain and E beta 170K in the beta(2) domain exhibited low plasma membrane expression, but molecules with only one of these mutations were expressed normally. The mutant class II molecules were transported to organelles that were accessible to a fluid-phase protein, hen egg lysozyme (HEL). Culture of transfectants with lysozyme enhanced the amount of class II compact dimer (alpha beta plus peptide; CD), and this was especially marked for the class II mutant H alpha 181D/E beta 170K and for other molecules possessing both mutations. Formation of class II CD was not paralleled by an increase in class II surface expression. Thus the joint mutation of H alpha 181 and E beta 170 has two effects. In the absence o high concentrations of exogenous peptide, it prevents efficient CD formation, possibly by affecting invariant chain (Ii) proteolysis and/or the stability of the class II after Ii/CLIP is removed. At high peptide concentrations supplied by exogenous HEL, the mutations allow CD formation, but not expression of class II on the plasma membrane. Molecular modeling of the possible interaction of class II and Ii suggests that the mutant amino acids H alpha 181D and E beta 170K, besides affecting the overall stability of class II, might also interact with Ii via two loops in class II's alpha(2) and beta(2) domains respectively.

Identification of a CD4 binding site on the beta 2 domain of HLA-DR molecules.

The CD4 and CD8 molecules are transmembrane glycoproteins expressed by functionally distinct subsets of mature T cells. CD4+ and CD8+ T cells recognize antigens on major histocompatibility complex (MHC) class II-bearing and class I-bearing target cells respectively. The ability of monoclonal antibodies against CD4 and CD8 to block antigen recognition by T cells, as well as cell-cell adhesion assays, indicate that CD4 and CD8 bind to nonpolymorphic determinants of class II or class I MHC. Here we demonstrate that soluble recombinant HLA-DR4 molecules from insect cells and HLA-DR-derived peptides bind to immobilized recombinant soluble CD4. CD4 binds recombinant soluble DR4 heterodimers, as well as the soluble DR4-beta chain alone. Furthermore, two out of twelve DR4-beta peptides could interact specifically with CD4. These findings show that CD4 interacts with a region of MHC class II molecules analogous to a previously identified loop in class I MHC proteins that binds CD8 (refs 8, 9).