Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins ...frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD) simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP) and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP), one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d) each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future.
Toll-like receptor TLR5 recognizes a conserved domain, termed D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori. Highly virulent H. pylori strains ...possess a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. Here, we show that one of the H. pylori T4SS components, protein CagL, can act as a flagellin-independent TLR5 activator. CagL contains a D1-like motif that mediates adherence to TLR5
epithelial cells, TLR5 activation, and downstream signaling in vitro. TLR5 expression is associated with H. pylori infection and gastric lesions in human biopsies. Using Tlr5-knockout and wild-type mice, we show that TLR5 is important for efficient control of H. pylori infection. Our results indicate that CagL, by activating TLR5, may modulate immune responses to H. pylori.
Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in ...utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in immunodeficient hosts. When present before infection, both neutralizing and non-neutralizing anti-gB exhibited protective capacity.
The CD83 molecule has been identified to be expressed on numerous activated immune cells, including B and T lymphocytes, monocytes, dendritic cells, microglia, and neutrophils. Both isoforms of CD83, ...the membrane-bound as well as its soluble form are topic of intensive research investigations. Several studies revealed that CD83 is not a typical co-stimulatory molecule, but rather plays a critical role in controlling and resolving immune responses. Moreover, CD83 is an essential factor during the differentiation of T and B lymphocytes, and the development and maintenance of tolerance. The identification of its interaction partners as well as signaling pathways have been an enigma for the last decades. Here, we report the latest data on the expression, structure, and the signaling partners of CD83. In addition, we review the regulatory functions of CD83, including its striking modulatory potential to maintain the balance between tolerance versus inflammation during homeostasis or pathologies. These immunomodulatory properties of CD83 emphasize its exceptional therapeutic potential, which has been documented in specific preclinical disease models.
The histamine H1 receptor (H1R) is a G protein-coupled receptor (GPCR) and represents a main target in the treatment of allergic reactions as well as inflammatory reactions and depressions. Although ...the overall effect of antagonists on H1 function has been extensively investigated, rather little is known about the potential modulatory effect of ions or sequence variants on antagonist binding. We investigated the dynamics of a phosphate ion present in the crystal structure and of a sodium ion, for which we determined the position in the allosteric pocket by metadynamics simulations. Both types of ions exhibit significant dynamics within their binding site; however, some key contacts remain stable over the simulation time, which might be exploited to develop more potent drugs targeting these sites. The dynamics of the ions is almost unaffected by the presence or absence of doxepin, as also reflected in their small effect (less than 1 kcal·mol−1) on doxepin binding affinity. We also examined the effect of four H1R sequence variants observed in the human population on doxepin binding. These variants cause a reduction in doxepin affinity of up to 2.5 kcal·mol−1, indicating that personalized medical treatments that take into account individual mutation patterns could increase precision in the dosage of GPCR-targeting drugs.
Despite numerous studies investigating histamine and its receptors, the impact of histamine protonation states on binding to the histamine H1-receptor (H1R) has remained elusive. Therefore, we ...assessed the influence of different histamine tautomers (τ-tautomer, π-tautomer) and charge states (mono- vs. dicationic) on the interaction with the ternary histamine-H1R-Gq complex. In atomistic molecular dynamics simulations, the τ-tautomer formed stable interactions with the receptor, while the π-tautomer induced a rotation of the histamine ring by 180° and formed only weaker hydrogen bonding interactions. This suggests that the τ-tautomer is more relevant for stabilization of the active ternary histamine-H1R-Gq complex. In addition to the two monocationic tautomers, the binding of dicationic histamine was investigated, whose interaction with the H1R had been observed in a previous experimental study. Our simulations showed that the dication is less compatible with the ternary histamine-H1R-Gq complex and rather induces an inactive conformation in the absence of the Gq protein. Our data thus indicate that the charge state of histamine critically affects its interactions with the H1R. Ultimately these findings might have implications for the future development of new ligands that stabilize distinct H1R activation states.
Pontocerebellar hypoplasia (PCH) describes a group of rare heterogeneous neurodegenerative diseases with prenatal onset. Here we describe eight children with PCH from four unrelated families ...harboring the homozygous MINPP1 (NM_004897.4) variants; c.75_94del, p.(Leu27Argfs*39), c.851 C > A, p.(Ala284Asp), c.1210 C > T, p.(Arg404*), and c.992 T > G, p.(Ile331Ser). The homozygous p.(Leu27Argfs*39) change is predicted to result in a complete absence of MINPP1. The p.(Arg404*) would likely lead to a nonsense mediated decay, or alternatively, a loss of several secondary structure elements impairing protein folding. The missense p.(Ala284Asp) affects a buried, hydrophobic residue within the globular domain. The introduction of aspartic acid is energetically highly unfavorable and therefore predicted to cause a significant reduction in protein stability. The missense p.(Ile331Ser) affects the tight hydrophobic interactions of the isoleucine by the disruption of the polar side chain of serine, destabilizing the structure of MINPP1. The overlap of the above-mentioned genotypes and phenotypes is highly improbable by chance. MINPP1 is the only enzyme that hydrolyses inositol phosphates in the endoplasmic reticulum lumen and several studies support its role in stress induced apoptosis. The pathomechanism explaining the disease mechanism remains unknown, however several others genes of the inositol phosphatase metabolism (e.g., INPP5K, FIG4, INPP5E, ITPR1) are correlated with phenotypes of neurodevelopmental disorders. Taken together, we present MINPP1 as a novel autosomal recessive pontocerebellar hypoplasia gene.
Summary
Two‐pore channels (TPC) have been established as components of calcium signalling networks in plants and animals. In plants, TPC1 in the vacuolar membrane is gated open upon binding of ...calcium in a voltage‐dependent manner. Here, we analyzed the molecular mechanism of the Ca2+‐dependent activity of TPC1 from Arabidopsis thaliana, using site‐directed mutagenesis of its two canonical EF‐hands. Wild‐type TPC1 and TPC1‐D335A with a mutated first Ca2+ ligand in EF‐hand 1 produced channels that retained their voltage‐ and Ca2+‐dependent gating characteristics, but were less sensitive at Ca2+ concentrations <200 μm. Additional mutation of the first Ca2+ ligand in EF‐hand 2 resulted in silent TPC1‐D335A/D376A channels. Similarly, the single mutant TPC1‐D376A could not be activated up to 1 mm Ca2+, indicating that the second EF‐hand is essential for the Ca2+‐dependent channel gating. Molecular modeling suggests that EF‐hand 1 displays a low‐affinity Ca2+/Mg2+‐binding site, while EF‐hand 2 represents a high‐affinity Ca2+‐binding site. Together, our data prove that EF‐hand 2 is responsible for the Ca2+‐receptor characteristics of TPC1, while EF‐hand 1 is a structural site required to enable channel responses at physiological changes in Ca2+ concentration.
Severe acute respiratory syndrome coronavirus (SARS-CoV), an enveloped single-stranded positive-sense RNA virus, is a member of the genus
Betacoronavirus
, family Coronaviridae. The SARS-CoV envelope ...protein E is a small (∼8.4 kDa) channel-forming membrane protein whose sequence is highly conserved between SARS-CoV and SARS-CoV-2. As a viroporin, it is involved in various aspects of the virus life cycle including assembly, budding, envelope formation, virus release, and inflammasome activation. Here, SARS-CoV E protein was recombinantly expressed in HEK293 cells and channel activity and the effects of viroporin inhibitors studied using patch-clamp electrophysiology and a cell viability assay. We introduced a membrane-directing signal peptide to ensure transfer of recombinant E protein to the plasma membrane. E protein expression induced transmembrane currents that were blocked by various inhibitors. In an ion-reduced buffer system, currents were proton-dependent and blocked by viroporin inhibitors rimantadine and amantadine. I-V relationships of recombinant E protein were not pH-dependent in a classical buffer system with high extracellular Na
+
and high intracellular K
+
. E-protein mediated currents were inhibited by amantadine and rimantadine, as well as 5-(N,N-hexamethylene)amiloride (HMA). We tested a total of 10 flavonoids, finding inhibitory activity of varying potency. Epigallocatechin and quercetin were most effective, with IC
50
values of 1.5 ± 0.1 and 3.7 ± 0.2 nM, respectively, similar to the potency of rimantadine (IC
50
= 1.7 ± 0.6 nM). Patch-clamp results were independently verified using a modified cell viability assay for viroporin inhibitors. These results contribute to the development of novel antiviral drugs that suppress virus activity and proliferation.
SARS-CoV-2 has caused a worldwide pandemic since December 2019 and the search for pharmaceutical targets against COVID-19 remains an important challenge. Here, we studied the envelope protein E of ...SARS-CoV and SARS-CoV-2, a highly conserved 75-76 amino acid viroporin that is crucial for virus assembly and release. E protein channels were recombinantly expressed in HEK293 cells, a membrane-directing signal peptide ensured transfer to the plasma membrane.
Viroporin channel activity of both E proteins was investigated using patch-clamp electrophysiology in combination with a cell viability assay. We verified inhibition by classical viroporin inhibitors amantadine, rimantadine and 5-(N,N-hexamethylene)-amiloride, and tested four ivermectin derivatives.
Classical inhibitors showed potent activity in patch-clamp recordings and viability assays. In contrast, ivermectin and milbemycin inhibited the E channel in patch-clamp recordings but displayed only moderate activity on the E protein in the cell viability assay, which is also sensitive to general cytotoxic activity of the tested compounds. Nemadectin and ivermectin aglycon were inactive. All ivermectin derivatives were cytotoxic at concentrations > 5 µM, i.e. below the level required for E protein inhibition.
This study demonstrates direct inhibition of the SARS-CoV-2 E protein by classical viroporin inhibitors. Ivermectin and milbemycin inhibit the E protein channel but their cytotoxicity argues against clinical application.
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