High-affinity State Research Articles

Insight into the Tumor Suppression Mechanism from the Structure of Human Polypyrimidine Splicing Factor (PSF/SFPQ) Complexed with a 30mer RNA from Murine Virus-like 30S Transcript-1.

Human polypyrimidine-binding splicing factor (PSF/SFPQ) is a tumor suppressor protein that regulates the gene expression of several proto-oncogenes and binds to the 5'-polyuridine negative-sense template (5'-PUN) of some RNA viruses. The activity of PSF is negatively regulated by long-noncoding RNAs, human metastasis associated in lung adenocarcinoma transcript-1 and murine virus-like 30S transcript-1 (VL30-1). PSF is a 707-amino acid protein that has a DNA-binding domain and two RNA recognition motifs (RRMs). Although the structure of the apo-truncated PSF is known, how PSF recognizes RNA remains elusive. Here, we report the 2.8 Å and 3.5 Å resolution crystal structures of a biologically active truncated construct of PSF (sPSF, consisting of residues 214-598) alone and in a complex with a 30mer fragment of VL30-1 RNA, respectively. The structure of the complex reveals how the 30mer RNA is recognized at two U-specific induced-fit binding pockets, located at the previously unrecognized domain-swapped, inter-subunit RRM1 (of the first subunit)-RRM2 (of the second subunit) interfaces that do not exist in the apo structure. Thus, the sPSF dimer appears to have two conformations in solution: one in a low-affinity state for RNA binding, as seen in the apo-structure, and the other in a high-affinity state for RNA binding, as seen in the sPSF-RNA complex. PSF undergoes an all or nothing transition between having two or no RNA-binding pockets. We predict that the RNA binds with a high degree of positive cooperativity. These structures provide an insight into a new regulatory mechanism that is likely involved in promoting malignancies and other human diseases.

Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide-bound state of eIF4A regulate eIF4F binding to RNA

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXβ2.

SUMMARYAllosteric activation and silencing of leukocyte β2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXβ2 ligand-binding domain—the αX I-domain—corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH− and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXβ2. This work highlights how intrinsically flexible pH− and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

S136: CONSTITUTIVE VLA-4 ACTIVATION IN CHRONIC LYMPHOCYTIC LEUKEMIA. THE OTHER SIDE OF BCR AUTONOMOUS SIGNALING.

Background: In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator and key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its ligands. In CLL, beside the canonical antigen (Ag)-dependent mechanism, BCR signaling has been recently demonstrated to occur via an autonomous Ag-independent manner. Aims: To investigate the role of autonomous BCR signaling on constitutive VLA-4 activation state in CLL. Methods: VLA-4 activation/affinity was determined by flow cytometry (FC) using the conformation-sensitive anti-CD29 mAb HUTS21 and/or by “real-time” (FC) measuring the binding of the VLA-4 ligand LDV-FITC as reported (Tissino et al, J Exp Med, 2018) in: i) 1,984 consecutive CLL all with IGHV gene mutations/BCR features available; ii) sequential samples (0, 14, 30, 60 90 days) from CLL patients (n=26) treated in vivo with ibrutinib (IB) in real-world and from a clinical trial (NCT02827617). HUTS21 staining was also performed in the presence of: plasma depletion/replacement, soluble (s) VCAM-1, fibronectin (FN) and blocking anti-CD49d (HP1/2) mAbs. ELISA assays were used to quantify sVCAM-1 in plasma samples (n=122). BCR signaling were investigated by Ca++ influx assay in a 4-OH tamoxifen (4-OHT)-inducible murine TKO cell model (Dühren-von Minden M et al, Nature, 2012). Results: Out of 1,984 CLL, 1,070 (54%) expressed CD49d (cutoff 30%) and, among them, 250/1,070 (23%) were HUTS21+ (cutoff 20%), indicating an activated VLA-4 conformation. HUTS21 staining was: i) impaired by depletion of plasma from whole blood samples, and reconstituted by specific plasma components (sVCAM-1, FN); ii) impaired by pre-incubation with anti-CD49d HP1/2 blocking mAbs before addition of plasma, sVCAM-1 and FN. sVCAM-1 was higher in CD49d+ vs CD49d- CLL (p<0.0001); among CD49d+ cases, sVCAM-1 was lower in HUTS21+ (i.e. VLA-4 activated) cases (p=0.0096), suggesting ligand sequestration by activated surface VLA-4. CLL with mutated IGHV expressed higher levels of activated VLA-4 compared to unmutated IGHV CLL (p=0.001). Higher levels of activated VLA-4 were found in CLL using the IGHV3 and IGHV4 families, compared to cases using the IGHV1 family (p=0.043 and p=0.004). Finally, analysis of BCR stereotypy highlighted higher VLA-4 activation levels in CLL from subset#2 compared to CLL from subset#1 (p=0.02). To validate these data, murine TKO cells, expressing high VLA-4 levels, were transfected with different BCRs derived from 4 CLL with high level of constitutively activated VLA-4 (TKO-high) and 4 CLL with low level of constitutively activated VLA-4 (TKO-low). Compared to TKO-low cells, TKO-high cells showed a higher autonomous Ca++ influx (p=0.03), and consistently higher VLA-4 affinity (p=0.01). IB treatment impaired both BCR autonomous signaling and VLA-4 affinity. Notably, anti-IgM stimulation induced high Ca++ influx and high VLA-4 affinity state in both TKO-high and TKO-low, irrespective of IB treatment. According to TKO data, a decreased constitutive VLA-4 activation was observed in CLL cells collected at pre-treatment and at day 14, 30, 60 and 90 from patients on IB, confirming an IB-dependent impairment of VLA-4 activation via BCR signal. Summary/Conclusion: The presence of a constitutively activated form of VLA-4 is observed in a fraction of CD49d+ CLL, due to a continuous VLA-4 inside-out stimulation derived from autonomous BCR signaling.

Yeast Trk1 Potassium Transporter Gradually Changes Its Affinity in Response to Both External and Internal Signals.

Yeasts need a high intracellular concentration of potassium to grow. The main K+ uptake system in Saccharomyces cerevisiae is the Trk1 transporter, a complex protein with four MPM helical membrane motifs. Trk1 has been shown to exist in low- or high-affinity modes, which reflect the availability of potassium in the environment. However, when and how the affinity changes, and whether the potassium availability is the only signal for the affinity switch, remains unknown. Here, we characterize the Trk1 kinetic parameters under various conditions and find that Trk1’s KT and Vmax change gradually. This gliding adjustment is rapid and precisely reflects the changes in the intracellular potassium content and membrane potential. A detailed characterization of the specific mutations in the P-helices of the MPM segments reveals that the presence of proline in the P-helix of the second and third MPM domain (F820P and L949P) does not affect the function of Trk1 in general, but rather specifically prevents the transporter’s transition to a high-affinity state. The analogous mutations in the two remaining MPM domains (L81P and L1115P) result in a mislocalized and inactive protein, highlighting the importance of the first and fourth P-helices in proper Trk1 folding and activity at the plasma membrane.

Structural Basis of Arrestin Selectivity for Active Phosphorylated G Protein-Coupled Receptors.

Arrestins are a small family of proteins that bind G protein-coupled receptors (GPCRs). Arrestin binds to active phosphorylated GPCRs with higher affinity than to all other functional forms of the receptor, including inactive phosphorylated and active unphosphorylated. The selectivity of arrestins suggests that they must have two sensors, which detect receptor-attached phosphates and the active receptor conformation independently. Simultaneous engagement of both sensors enables arrestin transition into a high-affinity receptor-binding state. This transition involves a global conformational rearrangement that brings additional elements of the arrestin molecule, including the middle loop, in contact with a GPCR, thereby stabilizing the complex. Here, we review structural and mutagenesis data that identify these two sensors and additional receptor-binding elements within the arrestin molecule. While most data were obtained with the arrestin-1-rhodopsin pair, the evidence suggests that all arrestins use similar mechanisms to achieve preferential binding to active phosphorylated GPCRs.

Structure of New Binary and Ternary DNA Polymerase Complexes From Bacteriophage RB69.

DNA polymerase plays a critical role in passing the genetic information of any living organism to its offspring. DNA polymerase from enterobacteria phage RB69 (RB69pol) has both polymerization and exonuclease activities and has been extensively studied as a model system for B-family DNA polymerases. Many binary and ternary complex structures of RB69pol are known, and they all contain a single polymerase-primer/template (P/T) DNA complex. Here, we report a crystal structure of the exonuclease-deficient RB69pol with the P/T duplex in a dimeric form at a resolution of 2.2 Å. The structure includes one new closed ternary complex with a single divalent metal ion bound and one new open binary complex in the pre-insertion state with a vacant dNTP-binding pocket. These complexes suggest that initial binding of the correct dNTP in the open state is much weaker than expected and that initial binding of the second divalent metal ion in the closed state is also much weaker than measured. Additional conformational changes are required to convert these complexes to high-affinity states. Thus, the measured affinities for the correct incoming dNTP and divalent metal ions are average values from many conformationally distinctive states. Our structure provides new insights into the order of the complex assembly involving two divalent metal ions. The biological relevance of specific interactions observed between one RB69pol and the P/T duplex bound to the second RB69pol observed within this dimeric complex is discussed.

Arming of iPSC-Derived NK Cells Expressing a Novel CD64 Fusion Receptor with Therapeutic Antibodies Represents a Novel Off-the-Shelf, Antigen-Targeting Strategy for Cancer

Natural killer (NK) cells are innate lymphocytes that target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and that recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Human NK cells exclusively mediate ADCC through the IgG Fc receptor, CD16A, and studies have demonstrated that increasing the binding affinity between CD16A and therapeutic monoclonal antibodies (mAbs), mediated by the high-affinity 158V polymorphism, can augment clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we sought to arm iPSC-derived NK (iNK) cells expressing a high-affinity recombinant FcγR with various mAbs as unique tumor-targeting strategy for various malignancies.As a member of the FcγR family, CD64 (FcγRI) possesses the highest affinity and can uniquely facilitate antibody preabsorption but it is normally expressed by myeloid cells. To leverage CD64 in NK cells, we developed a novel FcγR recombinant fusion comprising the extracellular region of CD64 with the transmembrane and intracellular regions of other NK cell activating receptors, including CD16A (CD64/16A) (figure 1A). The recombinant CD64/16A engineered into a clonal master induced pluripotent stem cell (iPSC) line for mass production of off-the-shelf iPSC-derived CD64/16A NK (iNK-CD64/16A) cells, can be armed with mAbs, including various combinations thereof to enable multi-antigen targeting and to address tumor heterogeneity (figures 1B and 2).To determine optimal binding and FcR saturation of iNK-CD64/16 cells, rituximab (anti-CD20 therapeutic mAb) was added in a two-hour preabsorbtion assay (figure 3A). Using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against Raji cells, a Burkitt Lymphoma cell line, when the iNKs were preabsorbed and armed with rituximab (figure 3B). Considering the high-affinity state of CD64, we examined the effects of free IgG on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis, ADCC was evaluated in the presence of purified human IgG. Despite the high levels of excess IgG, iNK-CD64/16A cells mediated efficient ADCC when rituximab was either added to the assay (figure 4A) or preabsorbed to the cells (figure 4B), demonstrating that saturating levels of free IgG did not prevent ADCC in either setting. To determine the ability of preabsorbed and armed iNK-CD64/16 cells to retain rituximab and perform serial killing, we performed a sequential killing assay using an IncuCyte® Live Cell Analysis where preabsorbed iNK-CD64/16A cells were thawed and co-cultured with or without Raji cells for 48 hours, followed by a second round of co-culture. As shown in figure 5, iNK-CD64/16A cells armed with rituximab retain ADCC capacity and perform serial killing for an extended time.To establish that iNK-CD64/16A cells can be armed with assorted therapeutic mAbs to target other tumor-associated antigens, we next determined the ability of iNK-CD64/16A cells preabsorbed and armed with anti-HER2 mAb, trastuzumab, to target the adenocarcinoma ovarian cancer cell line SKOV-3. Indeed iNK-CD64/16A cells armed with preabsorbed trastuzumab were able to effectively kill SKOV-3 cells via in vitro ADCC by IncuCyte® Live Cell Analysis (figure 6). We next investigated in vivo ADCC using NSG mice implanted with 3x10 5 SKOV-3 cells expressing firefly luciferase intraperitoneally (IP). 10 million iNK-CD64/16A with or without preabsorbed trastuzumab were injected IP (figure 7A), and a significant reduction in tumor volume in animals treated with iNK-CD64/16A cells armed with trastuzumab compared to unarmed iNK-CD64/16A cells (figure 7B).Collectively, our data show that iNK-CD64/16A cells can be armed with various therapeutic mAbs through a unique preabsorption strategy to mediate a potent and durable ADCC activity. The versatility of mAb-armed iNK-CD64/16A cells is being further investigated in various preclinical models to further elucidate the potential of this approach to overcome antigen escape and address tumor heterogeneity. [Display omitted] DisclosuresLee: Fate Therapeutics, Inc: Current Employment. Chu: Fate Therapeutics: Current Employment. Rogers: Fate Therapeutics: Current Employment. Bjordahl: Fate Therapeutics: Current Employment. Hosking: Fate Therapeutics: Current Employment. Shirinbak: Fate Therapeutics, Inc.: Current Employment. Miller: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees. Valamehr: Fate Therapeutics, Inc.: Current Employment. Walcheck: Fate Therapeutics: Research Funding.

Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the β3–β4 loop to favor a low-affinity drug-binding state

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.

Integrin-based mechanosensing through conformational deformation

Conversion of integrins from low to high affinity states, termed activation, is important in biological processes, including immunity, hemostasis, angiogenesis, and embryonic development. Integrin activation is regulated by large-scale conformational transitions from closed, low affinity states to open, high affinity states. Although it has been suggested that substrate stiffness shifts the conformational equilibrium of integrin and governs its unbinding, here, we address the role of integrin conformational activation in cellular mechanosensing. Comparison of wild-type versus activating mutants of integrin αVβ3 show that activating mutants shift cell spreading, focal adhesion kinase activation, traction stress, and force on talin toward high stiffness values at lower stiffness. Although all activated integrin mutants showed equivalent binding affinity for soluble ligands, the β3 S243E mutant showed the strongest shift in mechanical responses. To understand this behavior, we used coarse-grained computational models derived from molecular level information. The models predicted that wild-type integrin αVβ3 displaces under force and that activating mutations shift the required force toward lower values, with S243E showing the strongest effect. Cellular stiffness sensing thus correlates with computed effects of force on integrin conformation. Together, these data identify a role for force-induced integrin conformational deformation in cellular mechanosensing.

The Non-Anhydrous, Minimally Basic Synthesis of the Dopamine D2 Agonist [18F]MCL-524.

The dopamine D2 agonist MCL-524 is selective for the D2 receptor in the high-affinity state (D2high), and, therefore, the PET analogue, [18F]MCL-524, may facilitate the elucidation of the role of D2high in disorders such as schizophrenia. However, the previously reported synthesis of [18F]MCL-524 proved difficult to replicate and was lacking experimental details. We therefore developed a new synthesis of [18F]MCL-524 using a "non-anhydrous, minimally basic" (NAMB) approach. In this method, [18F]F- is eluted from a small (10-12 mg) trap-and-release column with tetraethylammonium tosylate (2.37 mg) in 7:3 MeCN:H2O (0.1 mL), rather than the basic carbonate or bicarbonate solution that is most often used for [18F]F- recovery. The tosylated precursor (1 mg) in 0.9 mL anhydrous acetonitrile was added directly to the eluate, without azeotropic drying, and the solution was heated (150 °C/15 min). The catechol was then deprotected with the Lewis acid In(OTf)3 (10 equiv.; 150 °C/20 min). In contrast to deprotection with protic acids, Lewis-acid-based deprotection facilitated the efficient removal of byproducts by HPLC and eliminated the need for SPE extraction prior to HPLC purification. Using the NAMB approach, [18F]MCL-524 was obtained in 5-9% RCY (decay-corrected, n = 3), confirming the utility of this improved method for the multistep synthesis of [18F]MCL-524 and suggesting that it may applicable to the synthesis of other 18F-labeled radiotracers.

NRT1.1 Dual-Affinity Nitrate Transport/Signalling and its Roles in Plant Abiotic Stress Resistance.

NRT1.1 is the first nitrate transport protein cloned in plants and has both high- and low-affinity functions. It imports and senses nitrate, which is modulated by the phosphorylation on Thr101 (T101). Structural studies have revealed that the phosphorylation of T101 either induces dimer decoupling or increases structural flexibility within the membrane, thereby switching the NRT1.1 protein from a low- to high-affinity state. Further studies on the adaptive regulation of NRT1.1 in fluctuating nitrate conditions have shown that, at low nitrate concentrations, nitrate binding only at the high-affinity monomer initiates NRT1.1 dimer decoupling and priming of the T101 site for phosphorylation activated by CIPK23, which functions as a high-affinity nitrate transceptor. However, nitrate binding in both monomers retains the unmodified NRT1.1, maintaining the low-affinity mode. This NRT1.1-mediated nitrate signalling and transport may provide a key to improving the efficiency of plant nitrogen use. However, recent studies have revealed that NRT1.1 is extensively involved in plant tolerance of several adverse environmental conditions. In this context, we summarise the recent progress in the molecular mechanisms of NRT1.1 dual-affinity nitrate transport/signalling and focus on its expected and unexpected roles in plant abiotic stress resistance and their regulation processes.

Unique Transmembrane Domain Interactions Differentially Modulate Platelet αIIbβ3 and αvβ3 Function

Lateral transmembrane (TM) helix-helix associations play essential roles in membrane-protein folding, assembly, and signal transduction. Thus, dissociation of the TM helices of resting integrins is central to integrin activation. Atomic-level models have revealed that a small residue-X3-small residue motif G-x3-G mediates the interaction of αIIb TM helix with β3, whereas a reciprocating large residue-small residue motif V-x3-I-x3-G mediates the interaction of the β3 helix with αIIb. Platelets contain a second β3 integrin αvβ3. Like αIIβ3, αvβ3 on resting platelets is inactive until platelets are stimulated by agonists such as ADP and thrombin after which it is able to interact with immobilized ligands such as osteopontin (OPN) and vitronectin. It is noteworthy that besides a V-x3-I-x3-G motif, the β3 TM domain contains the small residue-X3-small residue motif, S-x3-A and a small residue-X3-small residue motif in the β7 TM domain mediates its association with α4. Accordingly, we asked whether the motifs mediating the interaction of the β3 TM domain with the TM domains of αIIb and αv are necessarily the same. To address this question, we scanned the TM helix of intact β3 with leucine and alanine replacements, stably co-expressed the β3 mutants with wild-type human αv in CHO cells, and used optical trap-based force spectroscopy to measure the constitutive interaction of αvβ3 with either immobilized OPN or the activation-dependent αvβ3-specific monoclonal antibody WOW-1 (a kind gift from Dr. Sanford Shattil). The latter method measures the probability of receptor-ligand binding and the forces required to rupture single receptor-ligand bonds. Initially, we measured the interaction of wild-type (WT) αvβ3 in its low-affinity state with OPN. The majority of measured rupture forces were in the range of 0-40 pN with a cumulative probability of detecting meaningful rupture forces >10 pN of only 1.1±0.7%. However, when αvβ3 was shifted to its high-affinity state by 1 mM Mn2+, the range of rupture forces increased up to 100 pN with a peak in the force spectrum at 45-50 pN and a cumulative probability of detecting rupture forces >10 pN of 3.1±1.3%. We then scanned the β3 TM domain with leucine and alanine residues and found a periodic increase in the cumulative probability of constitutive OPN binding that was comparable to Mn2+-stimulated WT binding and with probability peaks at S699L, A703L, and I707L. To confirm the latter result, we replaced OPN with WOW-1. Again, there was a periodic increase in the cumulative probability of constitutive WOW-1 binding with peaks at S669L, V700A, A703L, I704L, I707L, and G708L. Thus, in contrast to αIIbβ3, these data indicate that the β3 TM domain associates with the αv TM domain via its S-x3-A motif. To investigate the structure of the αvβ3 TM complex, we built a molecular model for the αvβ3 TM and cytoplasmic domains from an αIIbβ3 NMR model by threading the aligned αv sequence onto αIIb. The model was then subjected to cycles of minimization and rotamer optimization using the Rosetta modeling suite. The resulting structures deviated from the TM domain interface used by αIIbβ3, suggesting it is not favorable for αvβ3. Next, to investigate the preferred interface of the αvβ3 TM helices, we used the lowest energy model to build an all-atom water-lipid-protein system and then conducted unbiased molecular dynamics simulations. For the majority of these simulations, the helices were tightly associated and maintained a relatively uniform conformation. While the dimer complex was stable, the orientation of the αv and β3 TM helices rapidly equilibrated to the small residue-X3-small residue heterodimer interface. No additional forces were applied to this system to bias the formation of helical geometries. Strikingly, the αvβ3 interface observed throughout the simulation correlated well with the helical face suggested by the optical force spectroscopy experiments. In conclusion, we found that compared to αIIbβ3, there is a clear shift in the helical register and periodicity of the αvβ3 TM heterodimer such that both the αv and β3 helices interacted via small residue-X3-small residue motifs. Subsequent molecular modeling and molecular dynamics simulations were consistent with the results of the scanning mutagenesis experiments. Thus, these studies provide insight into how unique but homologous integrins regulate signal activation across diverse biological processes. DisclosuresNo relevant conflicts of interest to declare.

The Hsp70-Chaperone Machines in Bacteria.

The ATP-dependent Hsp70s are evolutionary conserved molecular chaperones that constitute central hubs of the cellular protein quality surveillance network. None of the other main chaperone families (Tig, GroELS, HtpG, IbpA/B, ClpB) have been assigned with a comparable range of functions. Through a multitude of functions Hsp70s are involved in many cellular control circuits for maintaining protein homeostasis and have been recognized as key factors for cell survival. Three mechanistic properties of Hsp70s are the basis for their high versatility. First, Hsp70s bind to short degenerate sequence motifs within their client proteins. Second, Hsp70 chaperones switch in a nucleotide-controlled manner between a state of low affinity for client proteins and a state of high affinity for clients. Third, Hsp70s are targeted to their clients by a large number of cochaperones of the J-domain protein (JDP) family and the lifetime of the Hsp70-client complex is regulated by nucleotide exchange factors (NEF). In this review I will discuss advances in the understanding of the molecular mechanism of the Hsp70 chaperone machinery focusing mostly on the bacterial Hsp70 DnaK and will compare the two other prokaryotic Hsp70s HscA and HscC with DnaK.

Different Alterations of Agonist and Antagonist Binding to 5-HT1A Receptor in a Rat Model of Parkinson's Disease and Levodopa-Induced Dyskinesia: A MicroPET Study.

The gold-standard treatment for Parkinson's disease is L-DOPA, which in the long term often leads to levodopa-induced dyskinesia. Serotonergic neurons are partially responsible for this, by converting L-DOPA into dopamine leading to its uncontrolled release as a "false neurotransmitter". The stimulation of 5-HT1A receptors can reduce involuntary movements but this mechanism is poorly understood. This study aimed to investigate the functionality of 5-HT1A receptors using positron emission tomography in hemiparkinsonian rats with or without dyskinesia induced by 3-weeks daily treatment with L-DOPA. Imaging sessions were performed "off" L-DOPA. Each rat underwent a positron emission tomography scan with [18F]F13640, a 5-HT1AR agonist which labels receptors in a high affinity state for agonists, or with [18F]MPPF, a 5-HT1AR antagonist which labels all the receptors. There were decreases of [18F]MPPF binding in hemiparkinsonian rats in cortical areas. In dyskinetic animals, changes were slighter but also found in other regions. In hemiparkinsonian rats, [18F]F13640 uptake was decreased bilaterally in the globus pallidus and thalamus. On the non-lesioned side, binding was increased in the insula, the hippocampus and the amygdala. In dyskinetic animals, [18F]F13640 binding was strongly increased in cortical and limbic areas, especially in the non-lesioned side. These data suggest that agonist and antagonist 5-HT1A receptor-binding sites are differently modified in Parkinson's disease and levodopa-induced dyskinesia. In particular, these observations suggest a substantial involvement of the functional state of 5-HT1AR in levodopa-induced dyskinesia and emphasize the need to characterize this state using agonist radiotracers in physiological and pathological conditions.

Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.

Deciphering the Mechanism of Inhibition of SERCA1a by Sarcolipin Using Molecular Simulations.

SERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well-understood. To decipher this mechanism, we have performed normal mode analysis in the all-atom model, with the SERCA1a-SLN complex, or the isolated SERCA1a, embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation at the atomic level for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304, and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 Å away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+ low-affinity E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight into the conformational transition between the E2 and E1 states.

Receptor-Arrestin Interactions: The GPCR Perspective.

Arrestins are a small family of four proteins in most vertebrates that bind hundreds of different G protein-coupled receptors (GPCRs). Arrestin binding to a GPCR has at least three functions: precluding further receptor coupling to G proteins, facilitating receptor internalization, and initiating distinct arrestin-mediated signaling. The molecular mechanism of arrestin–GPCR interactions has been extensively studied and discussed from the “arrestin perspective”, focusing on the roles of arrestin elements in receptor binding. Here, we discuss this phenomenon from the “receptor perspective”, focusing on the receptor elements involved in arrestin binding and emphasizing existing gaps in our knowledge that need to be filled. It is vitally important to understand the role of receptor elements in arrestin activation and how the interaction of each of these elements with arrestin contributes to the latter’s transition to the high-affinity binding state. A more precise knowledge of the molecular mechanisms of arrestin activation is needed to enable the construction of arrestin mutants with desired functional characteristics.

The Cycling of Intracellular Calcium Released in Response to Fluid Shear Stress Is Critical for Migration-Associated Actin Reorganization in Eosinophils.

The magnitude of eosinophil mobilization into respiratory tissues drives the severity of inflammation in several airway diseases. In classical models of leukocyte extravasation, surface integrins undergo conformational switches to high-affinity states via chemokine binding activation. Recently, we learned that eosinophil integrins possess mechanosensitive properties that detect fluid shear stress, which alone was sufficient to induce activation. This mechanical stimulus triggered intracellular calcium release and hallmark migration-associated cytoskeletal reorganization including flattening for increased cell–substratum contact area and pseudopodia formation. The present study utilized confocal fluorescence microscopy to investigate the effects of pharmacological inhibitors to calcium signaling and actin polymerization pathways on shear stress-induced migration in vitro. Morphological changes (cell elongation, membrane protrusions) succeeded the calcium flux in untreated eosinophils within 2 min, suggesting that calcium signaling was upstream of actin cytoskeleton rearrangement. The inhibition of ryanodine receptors and endomembrane Ca2+-ATPases corroborated this idea, indicated by a significant increase in time between the calcium spike and actin polymerization. The impact of the temporal link is evident as the capacity of treated eosinophils to move across fibronectin-coated surfaces was significantly hampered relative to untreated eosinophils. Furthermore, we determined that the nature of cellular motility in response to fluid shear stress was nondirectional.

The ligand-bound state of a G protein-coupled receptor stabilizes the interaction of functional cholesterol molecules

AbstractCholesterol is a major component of mammalian plasma membranes that not only affects the physical properties of the lipid bilayer but also is the function of many membrane proteins including G protein-coupled receptors. The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined. Using the ion channel-coupled receptor technology, we developed a functional assay of cholesterol modulation of G protein-coupled receptors that is independent of intracellular signaling pathways and operational in living cells. Using this assay, we discovered a stable binding of cholesterol molecules to the receptor when it adopts an orthosteric ligand-bound state. This stable interaction preserves the cholesterol-dependent activity of the receptor in cholesterol-depleted membranes. This mechanism was confirmed using time-resolved FRET experiments on WT OXTR expressed in CHO cells. Consequently, a positive cross-regulation sequentially occurs in OXTR between cholesterol and orthosteric ligands.