Differential voltage-dependent modulation of the ACh-gated K+ current by adenosine and acetylcholine

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the ‘relaxation’ property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.

Somatic genetics of CDR3 control TCR V-domain rotational probability and germline CDR2 scanning of polymorphic MHC

The mechanism which adapts the T-cell antigen receptor (TCR) within a given major histocompatibility complex (MHC/HLA) genotype is essential for protection against pathogens. Historically attributed to relative affinity, genetically vast TCRs are surprisingly focused towards a micromolar affinity for their respective peptide (p) plus MHC (pMHC) ligands. Thus, the somatic diversity of the TCR with respect to MHC-restriction, and (ultimately) to pathogens, remains enigmatic. Here, we derive a triple integral solution (from fixed geometry) for any given V domain in TCR bound to pMHC. Solved complexes involving HLA-DR and HLA-DQ, where genetic linkage to the TCR is most profound, were examined in detail. Certain V domains displayed rare geometry within this panel—specifying a restricted rotational probability/volumetric density (dV). Remarkably, hydrogen (H) bond charge-relays distinguished these structures from the others; suggesting that CDR3 binding chemistry dictates CDR2 contacts on the opposite MHC-II alpha helix. Together, these data suggest that TCR recapitulate dV and specialise target pMHC recognition, i.e., a dynamics alternative to a relative TCR-affinity based mechanism.

Mechanism of the small ATP-independent chaperone Spy is substrate specific

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy’s action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.

16α-hydroxyestrone and its receptor complex: high affinity antigen for antibodies from prostate cancer patients

Background and objectives: Elevated levels of 16α-hydroxyestrone (16α-OHE1) have been linked to increased risk of prostate cancer (PC) and estrogen receptor (ER) had been expressed in prostate tissue but the combined effect of 16α-OHE1 and ER (α) is lacking. We investigated the binding specificity of antibodies from PC with 16α-OHE1-ER complex in the sera from PC patients. Materials and methods: The antibodies in the serum from 60 PC patients and 40 control subjects were evaluated from ELISA (direct binding and competition) and quantitative precipitin titration. Competition ELISA was also used to estimates 16α-OHE1 concentration and 2-hydroxyestrone (2-OHE1)/16α-OHE1 ratio in PC patients. Results: Antibodies from PC patients demonstrate high binding to 16α-OHE1-ER in comparison to ER (p < 0.05) or 16α-OHE1 (p < 0.001). The relative affinity of PC IgG was found to be high for 16α-OHE1-ER (1.19 × 10−7 M) as compared to ER (1.45 × 10−6 M) or 16α-OHE1 (1.13 × 10−6 M). Conclusion: High affinity of PC IgG with 16α-OHE1-ER might explain the possible antigenic role and 16α-OHE1-ER acted as high affinity antigen for antibodies from PC. The interaction between 16α-OHE1 and ER makes a complex in the prostate tissues and this may generate antibodies against this complex in the cancer patients.

MRBLE-pep measurements reveal accurate binding affinities for B56, a PP2A regulatory subunit

Signal transduction pathways rely on dynamic interactions between protein globular domains and short linear motifs (SLiMs). The weak affinities of these interactions are essential to allow fast rewiring of signaling pathways and downstream responses, but pose technical challenges for interaction detection and measurement. We recently developed a technique (MRBLE-pep) that leverages spectrally encoded hydrogel beads to measure binding affinities between a single protein and 48 different peptide sequences in a single small volume. In prior work, we applied it to map the binding specificity landscape between calcineurin and the PxIxIT SLiM (Nguyen et al. 2019). Here, using peptide sequences known to bind the PP2A regulatory subunit B56, we systematically compare affinities measured by MRBLE-pep or isothermal calorimetry (ITC) and confirm that MRBLE-pep accurately quantifies relative affinity over a wide dynamic range while using a fraction of the material required for traditional methods such as ITC.

Millisecond-scale molecular dynamics simulation of spike RBD structure reveals evolutionary adaption of SARS-CoV-2 to stably bind ACE2

The Receptor Binding Domain (RBD) of the SARS-CoV-2 surface spike (S) protein interacts with host angiotensin converting enzyme 2 (ACE2) to gain entry to host cells and initiate infection 1-3. Detailed, accurate understanding of key interactions between S RBD and ACE2 provides critical information that may be leveraged in the development of strategies for the prevention and treatment of COVID-19. Utilizing the published sequences and cryo-EM structures of both the viral S RBD and ACE2 4,5, we performed in silico molecular dynamics (MD) simulations of free S RBD and of its interaction with ACE2 over the exceptionally long durations of 2.9 and 2 milliseconds, respectively, to elucidate the nature and relative affinity of S RBD surface residues for the ACE2 binding region. Our findings reveal that free S RBD has assumed an optimized ACE2 binding-ready conformation, incurring little entropic penalty for binding, an evolutionary adaptation that contributes to its high affinity for the receptor 6. We further identified high probability molecular binding interactions that inform both vaccine design and therapeutic development, which may include recombinant ACE2-based spike decoys 7 and/or allosteric S RBD-ACE2 binding inhibitors 8,9 to prevent or arrest infection and thus disease.

Synthesis and Glycosidase Inhibition Properties of Calix[8]arene-Based Iminosugar Click Clusters

A set of 6- to 24-valent clusters was constructed with terminal deoxynojirimycin (DNJ) inhibitory heads through C6 or C9 linkers by way of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions between mono- or trivalent azido-armed iminosugars and calix[8]arene scaffolds differing in their valency and their rigidity but not in their size. The power of multivalency to upgrade the inhibition potency of the weak DNJ inhibitor (monovalent DNJ Ki being at 322 and 188 µM for C6 or C9 linkers, respectively) was evaluated on the model glycosidase Jack Bean α-mannosidase (JBα-man). Although for the clusters with the shorter C6 linker the rigidity of the scaffold was essential, these parameters had no influence for clusters with C9 chains: all of them showed rather good relative affinity enhancements per inhibitory epitopes between 70 and 160 highlighting the sound combination of the calix[8]arene core and the long alkyl arms. Preliminary docking studies were performed to get insights into the preferred binding modes.

The affinity of copper(ii) ions towards l-amino acids in the solid-state: a simple route towards mixed complexes

Competitive milling was successfully employed to determine the relative affinity of Cu(ii) ions towards selected l-amino acids (Asn, Gln, His, Phe, Pro, and Trp). Described process opens a simple route towards mixed coordination compounds.

Somatic genetics of CDR3 control TCR V-domain rotational probability effecting germline CDR2 scanning of polymorphic MHC

The mechanism which adapts the T-cell antigen receptor (TCR) within a given major histocompatibility complex (MHC; HLA, in humans) genotype is essential for protection against pathogens. Historically attributed to relative affinity, genetically vast TCRs are surprisingly focused towards a micromolar affinity for their respective peptide (p) plus MHC (pMHC) ligands. Thus, the somatic diversity of the TCR with respect to MHC restriction, and (ultimately) to pathogens, remains enigmatic. Here, we derive a triple integral equation (from fixed geometry) for any given V-domain in TCR bound to pMHC. We examine solved complexes involving HLA-DR and HLA-DQ, where genetic linkage to the TCR is most profound. Certain V-beta domains displayed rare geometry within this panel—specifying a very low (highly-restricted) rotational probability/volumetric density (dV). Remarkably, hydrogen (H)-bond charge-relays distinguished these structures from the others; suggesting that CDR3 binding chemistry dictates CDR2 contacts on the respective MHC-II alpha-helix.