Traditional Biomolecular Structure Determination by NMR Spectroscopy Allows for Major Errors

One of the major goals of structural genomics projects is to determine the three-dimensional structure of representative members of as many different fold families as possible. Comparative modeling is expected to fill the remaining gaps by providing structural models of homologs of the experimentally determined proteins. However, for such an approach to be successful it is essential that the quality of the experimentally determined structures is adequate. In an attempt to build a homology model for the protein dynein light chain 2A (DLC2A) we found two potential templates, both experimentally determined nuclear magnetic resonance (NMR) structures originating from structural genomics efforts. Despite their high sequence identity (96%), the folds of the two structures are markedly different. This urged us to perform in-depth analyses of both structure ensembles and the deposited experimental data, the results of which clearly identify one of the two models as largely incorrect. Next, we analyzed the quality of a large set of recent NMR-derived structure ensembles originating from both structural genomics projects and individual structure determination groups. Unfortunately, a visual inspection of structures exhibiting lower quality scores than DLC2A reveals that the seriously flawed DLC2A structure is not an isolated incident. Overall, our results illustrate that the quality of NMR structures cannot be reliably evaluated using only traditional experimental input data and overall quality indicators as a reference and clearly demonstrate the urgent need for a tight integration of more sophisticated structure validation tools in NMR structure determination projects. In contrast to common methodologies where structures are typically evaluated as a whole, such tools should preferentially operate on a per-residue basis

The Structural Basis of Calcium Dependent Inactivation of the Transient Receptor Potential Vanilloid 5 Channel.

The Transient Receptor Potential Vanilloid Channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca2+-binding protein Calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterisation of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while conferring conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with, and support the novel model for Ca2+/CaM-dependent inactivation of TRPV channels as proposed by Bate et al. (Biochemistry, 2018, in press)

Early mobilisation in critically ill COVID-19 patients: a subanalysis of the ESICM-initiated UNITE-COVID observational study

Background Early mobilisation (EM) is an intervention that may improve the outcome of critically ill patients. There is limited data on EM in COVID-19 patients and its use during the first pandemic wave. Methods This is a pre-planned subanalysis of the ESICM UNITE-COVID, an international multicenter observational study involving critically ill COVID-19 patients in the ICU between February 15th and May 15th, 2020. We analysed variables associated with the initiation of EM (within 72 h of ICU admission) and explored the impact of EM on mortality, ICU and hospital length of stay, as well as discharge location. Statistical analyses were done using (generalised) linear mixed-effect models and ANOVAs. Results Mobilisation data from 4190 patients from 280 ICUs in 45 countries were analysed. 1114 (26.6%) of these patients received mobilisation within 72 h after ICU admission; 3076 (73.4%) did not. In our analysis of factors associated with EM, mechanical ventilation at admission (OR 0.29; 95% CI 0.25, 0.35; p = 0.001), higher age (OR 0.99; 95% CI 0.98, 1.00; p ≤ 0.001), pre-existing asthma (OR 0.84; 95% CI 0.73, 0.98; p = 0.028), and pre-existing kidney disease (OR 0.84; 95% CI 0.71, 0.99; p = 0.036) were negatively associated with the initiation of EM. EM was associated with a higher chance of being discharged home (OR 1.31; 95% CI 1.08, 1.58; p = 0.007) but was not associated with length of stay in ICU (adj. difference 0.91 days; 95% CI − 0.47, 1.37, p = 0.34) and hospital (adj. difference 1.4 days; 95% CI − 0.62, 2.35, p = 0.24) or mortality (OR 0.88; 95% CI 0.7, 1.09, p = 0.24) when adjusted for covariates. Conclusions Our findings demonstrate that a quarter of COVID-19 patients received EM. There was no association found between EM in COVID-19 patients' ICU and hospital length of stay or mortality. However, EM in COVID-19 patients was associated with increased odds of being discharged home rather than to a care facility. Trial registration ClinicalTrials.gov: NCT04836065 (retrospectively registered April 8th 2021)

Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide

The twin-arginine translocation (Tat) pathway is a protein targeting system present in many prokaryotes. The physiological role of the Tat pathway is the transmembrane translocation of fully-folded proteins, which are targeted by N-terminal signal peptides bearing conserved SRRxFLK twin-arginine amino acid motifs. In Escherichia coli the majority of Tat targeted proteins bind redox cofactors and it is important that only mature, cofactor-loaded precursors are presented for export. Cellular processes have been unearthed that sequence these events, for example the signal peptide of the periplasmic nitrate reductase (NapA) is bound by a cytoplasmic chaperone (NapD) that is thought to regulate assembly and export of the enzyme. In this work, genetic, biophysical and structural approaches were taken to dissect the interaction between NapD and the NapA signal peptide. A NapD binding epitope was identified towards the N-terminus of the signal peptide, which overlapped significantly with the twin-arginine targeting motif. NMR spectroscopy revealed that the signal peptide adopted a a-helical conformation when bound by NapD, and substitution of single residues within the NapA signal peptide was sufficient to disrupt the interaction. This work provides an increased level of understanding of signal peptide function on the bacterial Tat pathway

The Structural Basis of Calcium-Dependent Inactivation of the Transient Receptor Potential Vanilloid 5 Channel

Accession Codes Chemical shifts and restraints were submitted to the BMRB (accession number 34161), and the ensemble of 20 conformers were submitted to the wwPDB (accession number 5OEO).The file associated with this record is under embargo until 12 months after publication, in accordance with the publisher's self-archiving policy. The full text may be available through the publisher links provided above.The transient receptor potential vanilloid channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca 2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca 2+ binding protein calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterization of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca 2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while it confers conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with and support the novel model for Ca 2+ /CaM-dependent inactivation of TRPV channels as proposed by Bate and co-workers [ Bate, N., et al. (2018) Biochemistry, (57), DOI: 10.1021/acs.biochem.7b01286 ].G.W.V. acknowledges funding during various stages of this project by the Dutch Organization of Scientific Research (NWO; 700.55.443 and 700.57.101), BBSRC (BB/J007897/1), and MRC (MR/L000555/1 and MR/P00038X/1).Peer-reviewedPost-prin

<i>[I_uni,I_ave]</i> Plot for 1TGQ Calculated Using the QUEEN Program [36]

<p>Long-range restraints (blue filled circles) and the 1TGQ_sim restraints (red filled circles) are indicated. Restraints that are among the 30 most unique and most important (those above the dashed gray line) and that involve residues in either the α2 or β3 region (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020009#pcbi-0020009-g001" target="_blank">Figure 1</a>A) are indicated by black boxes.</p

Five Different per-Residue Structural Quality Indicators

<div><p>(A) Packing quality <i>Z</i>-score.</p><p>(B) Ramachandran plot appearance <i>Z</i>-score.</p><p>(C) Rotamer normality <i>Z</i>-score.</p><p>(D) Backbone normality score. The values listed on the <i>y</i>-axis indicate the number of times the local backbone (defined by the current residue plus or minus two residues) was found in WHAT IF's internal database (with a cut-off on the number of hits at 80).</p><p>(E) Sum of the NOE violations. Scores for the refined 1Y4O ensemble are shown in green; those for the refined 1TGQ ensemble are shown in orange. Secondary structure of the 1Y4O ensemble is indicated using colored boxes: α-helices are shown in blue, β-strands are shown in red.</p></div

Examples of Observed Structural Anomalies

<div><p>(A) An arginine side chain protruding the hydrophobic core of the second PDZ domain of PTP-Bas [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020009#pcbi-0020009-b039" target="_blank">39</a>].</p><p>(B) The corresponding arginine in the highly homologous second PDZ domain of PTP-BL [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020009#pcbi-0020009-b037" target="_blank">37</a>] is solvent exposed.</p><p>(C) The C-terminal region of DR1885 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020009#pcbi-0020009-b042" target="_blank">42</a>] (residues 120 to 149 are color-coded from yellow to red) forms a knot-like structure in the apo-form of DR1885.</p><p>(D) In the copper bound form of DR1885, the C-terminus wraps around the protein, instead of traversing through it. For each of the four structure ensembles, only the first, and presumably best, model is shown.</p></div

Structure Quality <i>Z</i>-Scores for a Large Set of Recent NMR Structures

<p>The quality scores of 620 NMR ensembles released from the PDB after January 1, 2003, are shown. For comparison, the dataset is separated in structures solved as part of structural genomics projects (orange) and structures originating from individual research groups (green). For each quality indicator, the average <i>Z</i>-score is indicated with a filled black circle. The black horizontal markers indicate (from top to bottom) the 90th, 75th, 50th (the median), 25th, and 10th percentiles of the data points for each quality indicator. The distribution of the outliers outside the markers is indicated using colored data points. The quality scores of the original and refined 1TGQ ensemble (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020009#pcbi-0020009-t001" target="_blank">Table 1</a>) are indicated by red and blue crosses, respectively. The backbone normality score of 1TGQ is identical for the original and refined ensemble.</p