In Situ Spectroscopic Detection of Large-Scale Reorientations of Transmembrane Helices During Influenza A M2 Channel Opening.

Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in-depth mechanistic understanding and, with that, methods that enable investigations under in situ conditions. Here, we apply surface-enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid-supported membrane, to shed light on the mechanics of its viroporin function. M2 is a paradigm of pH-activated proton channels and controls the proton flux into the viral interior during viral infection. We use SEIRA to track the large-scale reorientation of M2's transmembrane α-helices in situ during pH-activated channel opening. We quantify this event as a helical tilt from 26° to 40° by correlating the experimental results with solid-state nuclear magnetic resonance-informed computational spectroscopy. This mechanical motion is impeded upon addition of the inhibitor rimantadine, giving a direct spectroscopic marker to test antiviral activity. The presented approach provides a spectroscopic tool to quantify large-scale structural changes and to track the function and inhibition of the growing number of viroporins from pathogenic viruses in future studies.

The opening dynamics of the lateral gate regulates the activity of rhomboid proteases.

Rhomboid proteases hydrolyze substrate helices within the lipid bilayer to release soluble domains from the membrane. Here, we investigate the mechanism of activity regulation for this unique but wide-spread protein family. In the model rhomboid GlpG, a lateral gate formed by transmembrane helices TM2 and TM5 was previously proposed to allow access of the hydrophobic substrate to the shielded hydrophilic active site. In our study, we modified the gate region and either immobilized the gate by introducing a maleimide-maleimide (M2M) crosslink or weakened the TM2/TM5 interaction network through mutations. We used solid-state nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations, and molecular docking to investigate the resulting effects on structure and dynamics on the atomic level. We find that variants with increased dynamics at TM5 also exhibit enhanced activity, whereas introduction of a crosslink close to the active site strongly reduces activity. Our study therefore establishes a strong link between the opening dynamics of the lateral gate in rhomboid proteases and their enzymatic activity.

Accurate Determination of Motional Amplitudes in Biomolecules by Solid-State NMR.

Protein dynamics are an intrinsically important factor when considering a protein's biological function. Understanding these motions is often limited through the use of static structure determination methods, namely, X-ray crystallography and cryo-EM. Molecular simulations have allowed for the prediction of global and local motions of proteins from these static structures. Nevertheless, determining local dynamics at residue-specific resolution through direct measurement remains crucial. Solid-state nuclear magnetic resonance (NMR) is a powerful tool for studying dynamics in rigid or membrane-bound biomolecules without prior structural knowledge with the help of relaxation parameters such as T 1 and T 1ρ. However, these provide only a combined result of amplitude and correlation times in the nanosecond-millisecond frequency range. Thus, direct and independent determination of the amplitude of motions might considerably improve the accuracy of dynamics studies. In an ideal situation, the use of cross-polarization would be the optimal method for measuring the dipolar couplings between chemically bound heterologous nuclei. This would unambiguously provide the amplitude of motion per residue. In practice, however, the inhomogeneity of the applied radio-frequency fields across the sample leads to significant errors. Here, we present a novel method to eliminate this issue through including the radio-frequency distribution map in the analysis. This allows for direct and accurate measurement of residue-specific amplitudes of motion. Our approach has been applied to the cytoskeletal protein BacA in filamentous form, as well as to the intramembrane protease GlpG in lipid bilayers.

Direct Detection of Bound Ammonium Ions in the Selectivity Filter of Ion Channels by Solid-State NMR.

The flow of ions across cell membranes facilitated by ion channels is an important function for all living cells. Despite the huge amount of structural data provided by crystallography, elucidating the exact interactions between the selectivity filter atoms and bound ions is challenging. Here, we detect bound 15N-labeled ammonium ions as a mimic for potassium ions in ion channels using solid-state NMR under near-native conditions. The non-selective ion channel NaK showed two ammonium peaks corresponding to its two ion binding sites, while its potassium-selective mutant NaK2K that has a signature potassium-selective selectivity filter with four ion binding sites gave rise to four ammonium peaks. Ions bound in specific ion binding sites were identified based on magnetization transfer between the ions and carbon atoms in the selectivity filters. Magnetization transfer between bound ions and water molecules revealed that only one out of four ions in the selectivity filter of NaK2K is in close contact with water, which is in agreement with the direct knock-on ion conduction mechanism where ions are conducted through the channel by means of direct interactions without water molecules in between. Interestingly, the potassium-selective ion channels investigated here (NaK2K and, additionally, KcsA-Kv1.3) showed remarkably different chemical shifts for their bound ions, despite having identical amino acid sequences and crystal structures of their selectivity filters. Molecular dynamics simulations show similar ion binding and conduction behavior between ammonium and potassium ions and identify the origin of the differences between the investigated potassium channels.

A combination of solid-state NMR and MD simulations reveals the binding mode of a rhomboid protease inhibitor.

Intramembrane proteolysis plays a fundamental role in many biological and pathological processes. Intramembrane proteases thus represent promising pharmacological targets, but few selective inhibitors have been identified. This is in contrast to their soluble counterparts, which are inhibited by many common drugs, and is in part explained by the inherent difficulty to characterize the binding of drug-like molecules to membrane proteins at atomic resolution. Here, we investigated the binding of two different inhibitors to the bacterial rhomboid protease GlpG, an intramembrane protease characterized by a Ser-His catalytic dyad, using solid-state NMR spectroscopy. H/D exchange of deuterated GlpG can reveal the binding position while chemical shift perturbations additionally indicate the allosteric effects of ligand binding. Finally, we determined the exact binding mode of a rhomboid protease-inhibitor using a combination of solid-state NMR and molecular dynamics simulations. We believe this approach can be widely adopted to study the structure and binding of other poorly characterized membrane protein-ligand complexes in a native-like environment and under physiological conditions.

Dynamic interactions of CbiN and CbiM trigger activity of a cobalt energy-coupling-factor transporter.

Energy-coupling factor (ECF) transporters for uptake of vitamins and transition-metal ions into prokaryotic cells share a common architecture consisting of a substrate-specific integral membrane protein (S), a transmembrane coupling protein (T) and two cytoplasmic ATP-binding-cassette-family ATPases. S components rotate within the membrane to expose their binding pockets alternately to the exterior and the cytoplasm. In contrast to vitamin transporters, metal-specific systems rely on additional proteins with essential but poorly understood functions. CbiN, a membrane protein composed of two transmembrane helices tethered by an extracytoplasmic loop of 37 amino-acid residues represents the auxiliary component that temporarily interacts with the CbiMQO2 Co2+ transporter. CbiN was previously shown to induce significant Co2+ transport activity in the absence of CbiQO2 in cells producing the S component CbiM plus CbiN or a Cbi(MN) fusion. Here we analyzed the mode of interaction between the two protein domains. Any deletion in the CbiN loop abolished transport activity. In silico predicted protein-protein contacts between segments of the CbiN loop and loops in CbiM were confirmed by cysteine-scanning mutagenesis and crosslinking. Likewise, an ordered structure of the CbiN loop was observed by electron paramagnetic resonance analysis after site-directed spin labeling. The N-terminal loop of CbiM containing three of four metal ligands was partially immobilized in wild-type Cbi(MN) but completely immobile in inactive variants with CbiN loop deletions. Decreased dynamics of the inactive form was also detected by solid-state nuclear magnetic resonance of isotope-labeled protein in proteoliposomes. In conclusion, CbiM-CbiN loop-loop interactions facilitate metal insertion into the binding pocket.

Structure and Dynamics of the Rhomboid Protease GlpG in Liposomes Studied by Solid-State NMR.

Rhomboid proteases are intramembrane proteases that hydrolyze substrate peptide bonds within the lipid bilayer and are important for a wide range of biological processes. The bacterial intramembrane protease GlpG is one of the model systems for structural investigations of the rhomboid family. Two different models of substrate gating have been proposed, based on crystal structures of GlpG in detergent micelles. Here, we present a detailed investigation of enzymatically active GlpG in a native-like lipid environment using solid-state NMR spectroscopy. Proton-detected experiments confirm the presence of water molecules in the catalytic cavity. A secondary chemical shift analysis indicates a previously unobserved kink in the central part of the gating helix TM5. Dynamics measurements revealed a dynamic hotspot of GlpG at the N-terminal part of TM5 and the adjacent loop L4, indicating that this region is important for gating. In addition, relaxation dispersion experiments suggest that TM5 is in conformational exchange between an open and a closed conformation.

The conduction pathway of potassium channels is water free under physiological conditions.

Ion conduction through potassium channels is a fundamental process of life. On the basis of crystallographic data, it was originally proposed that potassium ions and water molecules are transported through the selectivity filter in an alternating arrangement, suggesting a "water-mediated" knock-on mechanism. Later on, this view was challenged by results from molecular dynamics simulations that revealed a "direct" knock-on mechanism where ions are in direct contact. Using solid-state nuclear magnetic resonance techniques tailored to characterize the interaction between water molecules and the ion channel, we show here that the selectivity filter of a potassium channel is free of water under physiological conditions. Our results are fully consistent with the direct knock-on mechanism of ion conduction but contradict the previously proposed water-mediated knock-on mechanism.

Characterization of H/D exchange in type 1 pili by proton-detected solid-state NMR and molecular dynamics simulations.

Uropathogenic Escherichia coli invades and colonizes hosts by attaching to cells using adhesive pili on the bacterial surface. Although many biophysical techniques have been used to study the structure and mechanical properties of pili, many important details are still unknown. Here we use proton-detected solid-state NMR experiments to investigate solvent accessibility and structural dynamics. Deuterium back-exchange at labile sites of the perdeuterated, fully proton back-exchanged pili was conducted to investigate hydrogen/deuterium (H/D) exchange patterns of backbone amide protons in pre-assembled pili. We found distinct H/D exchange patterns in lateral and axial intermolecular interfaces in pili. Amide protons protected from H/D exchange in pili are mainly located in the core region of the monomeric subunit and in the lateral intermolecular interface, whereas the axial intermolecular interface and the exterior region of pili are highly exposed to H/D exchange. Additionally, we performed molecular dynamics simulations of the type 1 pilus rod and estimated the probability of H/D exchange based on hydrogen bond dynamics. The comparison of the experimental observables and simulation data provides insights into stability and mechanical properties of pili.

A stochastic model of myeloid cell lineages in hematopoiesis and pathway mutations in acute myeloid leukemia.

A model for hematopoiesis is presented that explicitly includes the erythrocyte, granulocyte, and thrombocyte lineages and their common precursors. A small number of stem cells proliferate and differentiate through different compartments to produce the vast number of blood cells needed every day. Growth factors regulate the proliferation of cells dependent on the current demand. We provide a steady state analysis of the model and rough parameter estimates. Furthermore, we extend the model to include mutations that alter the replicative capacity of cells and introduce differentiation blocks. With these mutations the model develops signs of acute myeloid leukemia.

Protein-Protein Interfaces Probed by Methyl Labeling and Proton-Detected Solid-State NMR Spectroscopy.

Proton detection and fast magic-angle spinning have advanced biological solid-state NMR, allowing for the backbone assignment of complex protein assemblies with high sensitivity and resolution. However, so far no method has been proposed to detect intermolecular interfaces in these assemblies by proton detection. Herein, we introduce a concept based on methyl labeling that allows for the assignment of these moieties and for the study of protein-protein interfaces at atomic resolution.

Key steps in unconventional secretion of fibroblast growth factor 2 reconstituted with purified components.

FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.

Bacteriophage Tail-Tube Assembly Studied by Proton-Detected 4D Solid-State NMR.

Obtaining unambiguous resonance assignments remains a major bottleneck in solid-state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three-dimensional (3D) spectra are used. Here, we present a proton-detected 4D solid-state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non-uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH, (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail-tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.

Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR.

Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA-adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes. A distinction of the respective pathways depends largely on the hydrophobicity of the recognition sequence. Hydrophobic transmembrane domains stabilize SRP binding, whereas less hydrophobic signal sequences, typical for periplasmic and outer membrane proteins, stimulate SecA binding and disfavor SRP interactions. In this context, the formation of helical structures of signal peptides within the ribosome was considered to be an important factor. We applied dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance to investigate the conformational states of the disulfide oxidoreductase A (DsbA) signal peptide stalled within the exit tunnel of the ribosome. Our results suggest that the nascent chain comprising the DsbA signal sequence adopts an extended structure in the ribosome with only minor populations of helical structure.

Increased expression of (pro)renin receptor does not cause hypertension or cardiac and renal fibrosis in mice.

Binding of renin and prorenin to the (pro)renin receptor (PRR) increases their enzymatic activity and upregulates the expression of pro-fibrotic genes in vitro. Expression of PRR is increased in the heart and kidney of hypertensive and diabetic animals, but its causative role in organ damage is still unclear. To determine whether increased expression of PRR is sufficient to induce cardiac or renal injury, we generated a mouse that constitutively overexpresses PRR by knocking-in the Atp6ap2/PRR gene in the hprt locus under the control of a CMV immediate early enhancer/chicken beta-actin promoter. Mice were backcrossed in the C57Bl/6 and FVB/N strain and studied at the age of 12 months. In spite of a 25- to 80-fold renal and up to 400-fold cardiac increase in Atp6ap2/PRR expression, we found no differences in systolic blood pressure or albuminuria between wild-type and PRR overexpressing littermates. Histological examination did not show any renal or cardiac fibrosis in mutant mice. This was supported by real-time PCR analysis of inflammatory markers as well as of pro-fibrotic genes in the kidney and collagen in cardiac tissue. To determine whether the concomitant increase of renin would trigger fibrosis, we treated PRR overexpressing mice with the angiotensin receptor-1 blocker losartan over a period of 6 weeks. Renin expression increased eightfold in the kidney but no renal injury could be detected. In conclusion, our results suggest no major role for PRR in organ damage per se or related to its function as a receptor of renin.

Myeloperoxidase deficiency ameliorates progression of chronic kidney disease in mice.

Myeloperoxidase (MPO) is an enzyme expressed in neutrophils and monocytes/macrophages. Beside its well-defined role in innate immune defence, it may also be responsible for tissue damage. To identify the role of MPO in the progression of chronic kidney disease (CKD), we investigated CKD in a model of renal ablation in MPO knockout and wild-type mice. CKD was induced by 5/6 nephrectomy. Mice were followed for 10 wk to evaluate the impact of MPO deficiency on renal morbidity. Renal ablation induced CKD in wild-type mice with increased plasma levels of MPO compared with controls. No difference was found between MPO-deficient and wild-type mice regarding albuminuria 1 wk after renal ablation, indicating similar acute responses to renal ablation. Over the next 10 wk, however, MPO-deficient mice developed significantly less albuminuria and glomerular injury than wild-type mice. This was accompanied by a significantly lower renal mRNA expression of the fibrosis marker genes plasminogen activator inhibitor-I, collagen type III, and collagen type IV as well as matrix metalloproteinase-2 and matrix metalloproteinase-9. MPO-deficient mice also developed less renal inflammation after renal ablation, as indicated by a lower infiltration of CD3-positive T cells and F4/80-positive monocytes/macrophages compared with wild-type mice. In vitro chemotaxis of monocyte/macrophages isolated from MPO-deficient mice was impaired compared with wild-type mice. No significant differences were observed for mortality and blood pressure after renal ablation. In conclusion, these results demonstrate that MPO deficiency ameliorates renal injury in the renal ablation model of CKD in mice.

Susceptibility to HLA-DM protein is determined by a dynamic conformation of major histocompatibility complex class II molecule bound with peptide.

HLA-DM mediates the exchange of peptides loaded onto MHCII molecules during antigen presentation by a mechanism that remains unclear and controversial. Here, we investigated the sequence and structural determinants of HLA-DM interaction. Peptides interacting nonoptimally in the P1 pocket exhibited low MHCII binding affinity and kinetic instability and were highly susceptible to HLA-DM-mediated peptide exchange. These changes were accompanied by conformational alterations detected by surface plasmon resonance, SDS resistance assay, antibody binding assay, gel filtration, dynamic light scattering, small angle x-ray scattering, and NMR spectroscopy. Surprisingly, all of those changes could be reversed by substitution of the P9 pocket anchor residue. Moreover, MHCII mutations outside the P1 pocket and the HLA-DM interaction site increased HLA-DM susceptibility. These results indicate that a dynamic MHCII conformational determinant rather than P1 pocket occupancy is the key factor determining susceptibility to HLA-DM-mediated peptide exchange and provide a molecular mechanism for HLA-DM to efficiently target unstable MHCII-peptide complexes for editing and exchange those for more stable ones.

Lectin OS-9 delivers mutant neuroserpin to endoplasmic reticulum associated degradation in familial encephalopathy with neuroserpin inclusion bodies.

A feature of neurodegenerative diseases is the intraneuronal accumulation of misfolded proteins. In familial encephalopathy with neuroserpin inclusion bodies (FENIB), mutations in neuroserpin lead to accumulation of neuroserpin polymers within the endoplasmic reticulum (ER) of neurons. Cell culture based studies have shown that ER-associated degradation (ERAD) is involved in clearance of mutant neuroserpin. Here, we investigate how mutant neuroserpin is delivered to ERAD using cell culture and a murine model of FENIB. We show that the ER-lectin OS-9 but not XTP3-B is involved in ERAD of mutant neuroserpin. OS-9 binds mutant neuroserpin and the removal of glycosylation sites leads to increased neuroserpin protein load whereas overexpression of OS-9 decreases mutant neuroserpin. In FENIB mice, OS-9 but not XTP3-B is differently expressed and impairment of ERAD by partial inhibition of the ubiquitin proteasome system leads to increased neuroserpin protein load. These findings show that OS-9 delivers mutant neuroserpin to ERAD by recognition of glycan side chains and provide the first in vivo proof of involvement of ERAD in degradation of mutant neuroserpin.

Deficiency of the interleukin 17/23 axis accelerates renal injury in mice with deoxycorticosterone acetate+angiotensin ii-induced hypertension.

T cells participate in angiotensin II (Ang II)-induced hypertension. However, the specific subsets of T cells that are important in the end-organ damage are unknown. T-helper 17 cells are a recently identified subset that produces interleukin 17 (IL-17) and requires interleukin 23 (IL-23) for expansion. To evaluate the role of the T-helper 17 immune response in hypertensive renal and cardiac end-organ damage, hypertension was induced with deoxycorticosterone acetate (DOCA)+Ang II in wild-type (n=39) and IL-17-deficient (n=31) mice. The injury was evaluated at day 4 and day 14. To inactivate the IL-17/IL-23 axis at a different point, DOCA+Ang II hypertension was also induced in IL-23p19-deficient mice. Renal infiltration by T-helper 17 cells was increased in hypertensive wild-type mice. Systolic blood pressure did not differ between hypertensive IL-17-deficient and wild-type mice. Three days after induction of hypertension, a significantly higher albuminuria was found in IL-17-deficient than in wild-type mice (196±64 versus 58±16 mg/mg albumin/creatinine). Histology revealed significantly more glomerular injury (1.04±0.06 versus 0.67±0.05) and renal infiltration of γδ T cells in IL-17-deficient than in wild-type mice after 14 days. Similarly, significantly higher albuminuria, glomerular injury, and γδ T cell infiltration were found in IL-23p19-deficient mice with DOCA+Ang II-induced hypertension. DOCA+Ang II also induced cardiac damage as assessed by heart weight, cardiac fibrosis, as well as expression of fetal genes and matrix components, but no significant differences were found among IL-17(-/-), IL-23p19(-/-), and wild-type mice. IL-17/IL-23 deficiency accelerates DOCA+Ang II-induced albuminuria and hypertensive renal but not cardiac end-organ damage.

Differential diagnosis of membranous nephropathy with autoantibodies to phospholipase A2 receptor 1.

Membranous nephropathy (MN) accounts for most cases of the nephrotic syndrome in adults. Recently, studies on the underlying pathomechanisms led to the identification of the podocyte M-type receptor for secretory phospholipase A2 (PLA2R1) as a target antigen of circulating autoantibodies. Autoantibodies to PLA2R1 may not only play a role in the development of primary MN, but also serve as a marker for diagnosis, disease activity and therapy monitoring. Antibody detection is crucial to discriminate between patients with primary MN and those with a secondary form of the disease, as both forms require different diagnostic approaches and treatment strategies. Standardized test systems based on recombinant PLA2R1 allow for the sensitive and specific analysis of anti-PLA2R1 autoantibodies. Further research into pathogenic mechanisms and other disease markers can pave the way for improved patient care.