Structure and dynamics of a constitutively active neurotensin receptor

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist

The Role of Active and Passive Smoking in Chronic Obstructive Pulmonary Disease and Systemic Inflammation: A 12-year Prospective Study in China

Background: There is no consensus on the cause and effect of systemic chronic inflammation (SCI) regarding chronic obstructive pulmonary disease (COPD). The impact of second-hand smoke (SHS) on COPD has reached inconsistent conclusions. Methods: The China Kadoorie Biobank cohort was followed up from the 2004–08 baseline survey to 31 December 2018. Among the selected 445,523 participants in the final analysis, Cox and linear regressions were performed to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of tobacco exposure with COPD risk and baseline levels of log-transformed inflammatory factors [βs (95% CIs)], respectively. Results: Participants were followed up for a median of 12.1 years and 11,825 incident COPD events were documented. Ever-smokers were associated with a higher risk of COPD than non-smokers with non-weekly SHS exposure. A younger age to start smoking, a greater amount of daily tobacco consumption, and deeper inhalation were associated with increased risk of COPD and correlated with elevated levels of plasma high-sensitivity C-reactive protein (hs-CRP, all Ptrend < 0.001) even two years before COPD onset. Among former smokers, COPD risk declined with longer smoking cessation (Ptrend < 0.001) and those quitting smoking for over ten years presented no difference in COPD risk and hs-CRP level from non-smokers [HR (95% CI) = 1.05 (0.89, 1.25), β (95% CI) = 0.17 (− 0.09, 0.43)]. Among non-smokers, weekly SHS exposure was associated with a slightly higher COPD risk [HR (95% CI) = 1.06 (1.01, 1.12)]. Conclusions: Incremental exposure to tobacco smoke was related to elevated SCI level before COPD onset, then an increase in COPD susceptibility. Quitting smoking as early as possible is suggested as a practical approach to reducing COPD risk in smokers. Given the high prevalence of both COPD and SHS exposure, the risk associated with SHS exposure deserves attention

Human Neutralizing Antibodies Target a Conserved Lateral Patch on H7N9 Hemagglutinin Head

Avian influenza A virus H7N9 causes severe human infections with \u3e30% fatality. Currently, there is no H7N9-specific prevention or treatment for humans. Here, from a 2013 H7N9 convalescent case in Hong Kong, we isolate four hemagglutinin (HA)-reactive monoclonal antibodies (mAbs), with three directed to the globular head domain (HA1) and one to the stalk domain (HA2). Two clonally related HA1-directed mAbs, H7.HK1 and H7.HK2, potently neutralize H7N9 and protect female mice from lethal H7N9/AH1 challenge. Cryo-EM structures reveal that H7.HK1 and H7.HK2 bind to a β14-centered surface and disrupt the 220-loop that makes hydrophobic contacts with sialic acid on an adjacent protomer, thereby blocking viral entry. Sequence analysis indicates the lateral patch targeted by H7.HK1 and H7.HK2 to be conserved among influenza subtypes. Both H7.HK1 and H7.HK2 retain HA1 binding and neutralization capacity to later H7N9 isolates from 2016-2017, consistent with structural data showing that the antigenic mutations during this timeframe occur at their epitope peripheries. The HA2-directed mAb H7.HK4 lacks neutralizing activity but when used in combination with H7.HK2 moderately augments female mouse protection. Overall, our data reveal antibodies to a conserved lateral HA1 supersite that confer neutralization, and when combined with a HA2-directed non-neutralizing mAb, augment protection

NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1

Proposed surfaces of interaction.

<p><b>A.</b> Surface depiction of the two binding interfaces of alpha45 that could interact with the Bbox1 domain and PP2A simultaneously. <b>B.</b> Ribbon representation of the A- (scaffolding (PR65), colored gray) and C- (catalytic, colored yellow) subunits of PP2A, pdb accession code 3DW8. PR65 adopts a helix-turn-helix HEAT repeat structure. All glutamic and aspartic acids are shown in red on the A(PR65)-subunit. Located near the N-terminus of PR65 is a large negative patch, depicted by surface representation (colored red) that could accommodate the positively charged surface of alpha45. This is also the same surface on which the B- (regulatory, B55) subunit of PP2A binds. The alpha4 N-terminal binding site on PP2Ac is shown in orange.</p

Secondary structure analysis of alpha45 in aqueous solution by NMR.

<p><b>A.</b> HSQC spectrum of alpha45 in aqueous solution reveals that the NH signals fall within 1 ppm of each other, suggesting a labile structure. The NH assignments were identified using the 3D NMR data. M* represents one of the three amino acids that is the result from TEV cleavage; this residue is not part of alpha4 sequence. <b>B.</b> Strip plots taken from the 2D ¹H-¹H projections from the ¹⁵N planes of the 3D ¹H-¹⁵N NOESY-HSQC spectrum showing NOE correlations between the NH_(i) to intra-residue and preceding Hα atoms (top panels) and sequential NH to NH atoms (bottom panels) for residues predicted to be helix II. An attempt to show NH-NH_(i,i±1) NOE correlations is indicated by lines, but the NOEs are weak, ambiguous and mostly missing. <b>C(i).</b> Analysis of the Cα and Hα atom chemical shift index (CSI) of alpha45 in aqueous solution. Upfield shifted Cα and simultaneous downfield shifted Hα values are indicative of α-helices. For the peptide in aqueous solution, helix I cannot be definitively characterized because while the Hα values are upfield shifted, the Cα values are closer to zero compared to those of helix II. <b>C(ii).</b> The phi (Φ, blue line) and psi (Ψ, red line) values and the order parameter (S², green line) are plotted for each amino acid. These values were predicted by TALOS+ based on the chemical shift data. Residues adopting helical structure will have similar Φ and Ψ values. The higher the S² values, the less mobile it is for that amino acid.</p

Structure of alpha45.

<p><b>A.</b> Strip plots taken from the 2D ¹H-¹H projections of the ¹³C planes of the 3D-¹H-¹³C-edited HSQC-NOESY show long range NOEs between residues Val267 with Leu245 and Thr246, and between Thr246 and Tyr271, which were used in the tertiary structural calculation of alpha45. All intra-residue NOEs are labeled on the figure and arrows indicate inter-residues NOEs. In this modified version of the ¹³C-edited NOESY spectrum, the diagonal auto-peaks were suppressed. <b>B.</b> Superposition of the backbone Cα, C, N atoms for residues 243 to 277 of fourteen structures of alpha45 calculated based on NMR restraints acquired in 1% SDS. The backbone atoms of the two α-helices are colored red (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028877#pone-0028877-t001" target="_blank">Table 1</a>). <b>C.</b> Ribbon representation of the structure of alpha45 in 1% SDS. For clarity, two orientations of the structure are shown using the same color scheme noted above.</p

NMR Structure Determination Statistics.

a<p> <i>Helix I in SDS includes residues 243 to 254.</i></p>b<p> <i>Helix I in H₂O includes 247–253.</i></p>c<p> <i>Helix II in SDS includes residues 264 to 277.</i></p>d<p> <i>Helix II in H₂O includes residues 266 to 276.</i></p>e<p> <i>Value averaged for 14 structures.</i></p

Paramagnetic effect on alpha45.

<p><b>A.</b> Superposition of the ¹H-¹⁵N HSQC spectra of alpha45 in 1% SDS (black) and in 1% SDS with 4× excess MnCl₂ (red). Amino acids that are solvent exposed and/or accessible to Mn²⁺ had their ¹H-¹⁵N signals broadened. Those amino acids are labeled. <b>B.</b> Ribbon representation of alpha45 shows the location of the basic residues that were most protected from the paramagnetic effects in 1% SDS. The regions colored in green represent the amino acids that were protected from paramagnetic effect only in SDS but not in aqueous solution. The residues whose NH signals were affected by Mn²⁺ in both SDS and aqueous solution are shown in red and found to be located on the outer surface of helix II <b>C.</b> Two views of the electrostatic map of alpha45. The blue represents basic patches and red indicates acid patches. The orientation of the left image is the same as that shown in ‘B’. The map was generated by PBEQ-solver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028877#pone.0028877-Jo1" target="_blank">[59]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028877#pone.0028877-Im1" target="_blank">[60]</a>.</p