Structural analysis of light chain-driven bispecific antibodies targeting CD47 and PD-L1.

In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in which the light chain (LC) drives antigen binding and specificity. To better understand epitope-paratope interactions in this context, we determined the X-ray crystallographic structures of an antigen binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1. These Fabs contain a κ-LC and a λ-LC, respectively, which are paired with an identical heavy chain (HC). The structural analysis of these complexes revealed the dominant contribution of the LCs to antigen binding, but also that the common HC provides some contacts in both CD47 and PD-L1 Fab complexes. The anti-CD47 Fab was affinity optimized by diversifying complementary-determining regions of the LC followed by phage display selections. Using homology modeling, the contributions of the amino acid modification to the affinity increase were analyzed. Our results demonstrate that, despite a less prominent role in natural antibodies, the LC can mediate high affinity binding to different antigens and neutralize their biological function. Importantly, Fabs containing a common variable heavy (VH) domain enable the generation of bispecific antibodies retaining a truly native structure, maximizing their therapeutic potential.

Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors.

Plants and animals use cell surface receptors to sense and interpret environmental signals. In legume symbiosis with nitrogen-fixing bacteria, the specific recognition of bacterial lipochitooligosaccharide (LCO) signals by single-pass transmembrane receptor kinases determines compatibility. Here, we determine the structural basis for LCO perception from the crystal structures of two lysin motif receptor ectodomains and identify a hydrophobic patch in the binding site essential for LCO recognition and symbiotic function. We show that the receptor monitors the composition of the amphiphilic LCO molecules and uses kinetic proofreading to control receptor activation and signaling specificity. We demonstrate engineering of the LCO binding site to fine-tune ligand selectivity and correct binding kinetics required for activation of symbiotic signaling in plants. Finally, the hydrophobic patch is found to be a conserved structural signature in this class of LCO receptors across legumes that can be used for in silico predictions. Our results provide insights into the mechanism of cell-surface receptor activation by kinetic proofreading of ligands and highlight the potential in receptor engineering to capture benefits in plant-microbe interactions.

Full-length human GLP-1 receptor structure without orthosteric ligands.

Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family.

Intraoperative Ultrasound: A Tool to Support Tissue-Sparing Curative Pancreatic Resection in Focal Congenital Hyperinsulinism.

Background: Focal congenital hyperinsulinism (CHI) may be cured by resection of the focal, but often non-palpable, pancreatic lesion. The surgical challenge is to minimize removal of normal pancreatic tissue. Aim: To evaluate the results of intraoperative ultrasound-guided, tissue-sparing pancreatic resection in CHI patients at an international expert center. Methods: Retrospective study of CHI patients treated at Odense University Hospital, Denmark, between January 2010 and March 2017. Results: Of 62 consecutive patients with persistent CHI, 24 (39%) had focal CHI by histology after surgery. All patients had a paternal ABCC8 or KCNJ11 mutation and a focal lesion by 18F-DOPA-PET/CT. Intraoperative ultrasound localized the focal lesion in 16/20 patients (sensitivity 0.80), including one ectopic lesion in the duodenal wall. Intraoperative ultrasound showed no focal lesion in 11/11 patients with diffuse CH (specificity 1.0). The positive predictive value for focal histology was 1.0, negative predictive value 0.73. Tissue-sparing pancreatic resection (focal lesion enucleation, local resection of tail or uncinate process) was performed in 67% (n = 16). In 11/12 having tissue-sparing resection and intraoperative ultrasound, the location of the focal lesion was exactly identified. Eight patients had resection of the pancreatic head or head/body, four with Roux-en-Y, three with pancreatico-gastrostomy and one without reconstruction. None had severe complications to surgery. Cure of hypoglycaemia was seen in all patients after one (n = 21) or two (n = 3) pancreatic resections. Conclusion: In focal CHI, tissue-sparing pancreatic resection was possible in 67%. Intraoperative ultrasound was a helpful supplement to the mandatory use of genetics, preoperative 18F-DOPA-PET/CT and intraoperative frozen sections.

Results from the 5-year SQ grass sublingual immunotherapy tablet asthma prevention (GAP) trial in children with grass pollen allergy.

BACKGROUND: Allergy immunotherapy targets the immunological cause of allergic rhinoconjunctivitis and allergic asthma and has the potential to alter the natural course of allergic disease. OBJECTIVE: The primary objective was to investigate the effect of the SQ grass sublingual immunotherapy tablet compared with placebo on the risk of developing asthma. METHODS: A total of 812 children (5-12 years), with a clinically relevant history of grass pollen allergic rhinoconjunctivitis and no medical history or signs of asthma, were included in the randomized, double-blind, placebo-controlled trial, comprising 3 years of treatment and 2 years of follow-up. RESULTS: There was no difference in time to onset of asthma, defined by prespecified asthma criteria relying on documented reversible impairment of lung function (primary endpoint). Treatment with the SQ grass sublingual immunotherapy tablet significantly reduced the risk of experiencing asthma symptoms or using asthma medication at the end of trial (odds ratio = 0.66, P < .036), during the 2-year posttreatment follow-up, and during the entire 5-year trial period. Also, grass allergic rhinoconjunctivitis symptoms were 22% to 30% reduced (P < .005 for all 5 years). At the end of the trial, the use of allergic rhinoconjunctivitis pharmacotherapy was significantly less (27% relative difference to placebo, P < .001). Total IgE, grass pollen-specific IgE, and skin prick test reactivity to grass pollen were all reduced compared to placebo. CONCLUSIONS: Treatment with the SQ grass sublingual immunotherapy tablet reduced the risk of experiencing asthma symptoms and using asthma medication, and had a positive, long-term clinical effect on rhinoconjunctivitis symptoms and medication use but did not show an effect on the time to onset of asthma.

Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex.

Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na(+),K(+)-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na(+),K(+)-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg(2+) is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K(+) in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.

Na(+),K(+)-ATPase isoform selectivity for digitalis-like compounds is determined by two amino acids in the first extracellular loop.

Digitalis-like compounds (DLCs) comprise a diverse group of molecules characterized by a cis-trans-cis ring-fused steroid core linked to a lactone. They have been used in the treatment of different medical problems including heart failure, where their inotropic effect on heart muscle is attributed to potent Na(+),K(+)-ATPase inhibition. Their application as drugs, however, has declined in recent past years due to their small safety margin. Since human Na(+),K(+)-ATPase is represented by four different isoforms expressed in a tissue-specific manner, one of the possibilities to improve the therapeutic index of DLCs is to exploit and amend their isoform selectivity. Here, we aimed to reveal the determinants of selectivity of the ubiquitously expressed α1 isoform and the more restricted α2 isoform toward several well-known DLCs and their hydrogenated forms. Using baculovirus to express various mutants of the α2 isoform, we were able to link residues Met(119) and Ser(124) to differences in affinity between the α1 and α2 isoforms to ouabain, dihydro-ouabain, digoxin, and dihydro-digoxin. We speculate that the interactions between these amino acids and DLCs affect the initial binding of these DLCs. Also, we observed isoform selectivity for DLCs containing no sugar groups.

Crystals of Na(+)/K(+)-ATPase with bound cisplatin.

Cisplatin is the most widely used chemotherapeutics for cancer treatment, however, its administration is connected to inevitable adverse effects. Previous studies suggested that cisplatin is able to inhibit Na(+)/K(+)-ATPase (NKA), the enzyme responsible for maintaining electrochemical potential and sodium gradient across the plasma membrane. Here we report a crystallographic analysis of cisplatin bound to NKA in the ouabain bound E2P form. Despite a moderate resolution (7.4 Å and 7.9 Å), the anomalous scattering from platinum and a model representation from a recently published structure enabled localization of seven cisplatin binding sites by anomalous difference Fourier maps. Comparison with NKA structures in the E1P conformation suggested two possible inhibitory mechanisms for cisplatin. Binding to Met151 can block the N-terminal pathway for transported cations, while binding to Met171 can hinder the interaction of cytoplasmic domains during the catalytic cycle.

Crystal structure of the high-affinity Na+K+-ATPase-ouabain complex with Mg2+ bound in the cation binding site.

The Na(+),K(+)-ATPase maintains electrochemical gradients for Na(+) and K(+) that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na(+),K(+)-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na(+),K(+)-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the ß-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg(2+), and crystallographic studies indicate that Rb(+) and Mn(2+), but not Na(+), bind to this site. Comparison with the low-affinity [K2]E2-MgF(x)-ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742-13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg(2+). K(+) binding at the same site unwinds a turn of αM4, dragging residues Ile318-Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K(+)-Mg(2+) competition and explain the well-known antagonistic effect of K(+) on CTS binding.

Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase.

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 Å resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks.

Cyclopiazonic acid is complexed to a divalent metal ion when bound to the sarcoplasmic reticulum Ca2+-ATPase.

We have determined the structure of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in an E2.P(i)-like form stabilized as a complex with MgF(4)(2-), an ATP analog, adenosine 5'-(beta,gamma-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5A resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca(2+)-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na(+),K(+)-ATPase reveals that the Phe(93) side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na(+),K(+)-ATPase.

A tomato ER-type Ca2+-ATPase, LCA1, has a low thapsigargin-sensitivity and can transport manganese.

Recombinant Ca(2+)-ATPase from tomato (i.e. LCA1 for Lycopersicon esculentum [Since the identification and naming of LCA1, the scientific name for the tomato has been changed to Solanum lycopersicum.] Ca-ATPase) was heterologously expressed in yeast for structure-function characterization. We investigate the differences between plant and animal Ca pumps utilizing comparisons between chicken and rabbit SERCA-type pumps with Arabidopsis (ECA1) and tomato plant (LCA1) Ca(2+)-ATPases. Enzyme function was confirmed by the ability of each Ca(2+)-ATPase to rescue K616 growth on EGTA-containing agar and directly via in vitro ATP hydrolysis. We found LCA1 to be approximately 300-fold less sensitive to thapsigargin than animal SERCAs, whereas ECA1 was thapsigargin-resistant. LCA1 showed typical pharmacological sensitivities to cyclopiazonic acid, vanadate, and eosin, consistent with it being a P(IIA)-type Ca(2+)-ATPase. Possible amino acid changes responsible for the reduced plant thapsigargin-sensitivity are discussed. We found that LCA1 also complemented K616 yeast growth in the presence of Mn(2+), consistent with moving Mn(2+) into the secretory pathway and functionally compensating for the lack of secretory pathway Ca-ATPases (SPCAs) in plants.