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1.
Nat Commun ; 12(1): 7069, 2021 12 03.
Article in English | MEDLINE | ID: mdl-34862384

ABSTRACT

Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional mechanism where antibody-mediated "disruption" of stable high-affinity macromolecular complexes can potentially enhance therapeutic efficacy. However, this mechanism is not well understood or utilized therapeutically. Here, we investigate and engineer the weak disruptive activity of an existing therapeutic antibody, omalizumab, which targets IgE antibodies to block the allergic response. We develop a yeast display approach to select for and engineer antibody disruptive efficiency and generate potent omalizumab variants that dissociate receptor-bound IgE. We determine a low resolution cryo-EM structure of a transient disruption intermediate containing the IgE-Fc, its partially dissociated receptor and an antibody inhibitor. Our results provide a conceptual framework for engineering disruptive inhibitors for other targets, insights into the failure in clinical trials of the previous high affinity omalizumab HAE variant and anti-IgE antibodies that safely and rapidly disarm allergic effector cells.


Subject(s)
Immunoglobulin E/metabolism , Omalizumab/pharmacology , Protein Engineering , Receptors, IgE/metabolism , Animals , Cell Membrane , Cryoelectron Microscopy , Crystallography, X-Ray , Healthy Volunteers , Humans , Immunoglobulin E/ultrastructure , Ligands , Mice , Mice, Transgenic , Omalizumab/genetics , Omalizumab/therapeutic use , Primary Cell Culture , Receptors, IgE/ultrastructure , Sf9 Cells , Spodoptera
2.
J Struct Biol ; 212(3): 107649, 2020 12 01.
Article in English | MEDLINE | ID: mdl-33075486

ABSTRACT

HpAC1, a protein from Hippeastrum hybrid cultivars, was previously suggested to be a plant adenylyl cyclase. We describe a structural and enzymatic characterization of HpAC1. A crystal structure of HpAC1 in complex with a non-hydrolyzable GTP analog confirms a generic CYTH architecture, comprising a ß-barrel with an internal substrate site. The structure reveals significant active site differences to AC proteins with CYTH fold, however, and we find that HpAC1 lacks measurable AC activity. Instead, HpAC1 has substantial triphosphatase activity, indicating this protective activity or a related activity as the protein's physiological function.


Subject(s)
Adenylyl Cyclases/chemistry , Amaryllidaceae/chemistry , Plant Proteins/chemistry , Catalytic Domain/physiology , Crystallography, X-Ray/methods
3.
Nat Commun ; 11(1): 165, 2020 01 08.
Article in English | MEDLINE | ID: mdl-31913280

ABSTRACT

Targeting of immunoglobulin E (IgE) represents an interesting approach for the treatment of allergic disorders. A high-affinity monoclonal anti-IgE antibody, ligelizumab, has recently been developed to overcome some of the limitations associated with the clinical use of the therapeutic anti-IgE antibody, omalizumab. Here, we determine the molecular binding profile and functional modes-of-action of ligelizumab. We solve the crystal structure of ligelizumab bound to IgE, and report epitope differences between ligelizumab and omalizumab that contribute to their qualitatively distinct IgE-receptor inhibition profiles. While ligelizumab shows superior inhibition of IgE binding to FcεRI, basophil activation, IgE production by B cells and passive systemic anaphylaxis in an in vivo mouse model, ligelizumab is less potent in inhibiting IgE:CD23 interactions than omalizumab. Our data thus provide a structural and mechanistic foundation for understanding the efficient suppression of FcεRI-dependent allergic reactions by ligelizumab in vitro as well as in vivo.


Subject(s)
Anti-Allergic Agents/administration & dosage , Antibodies, Anti-Idiotypic/administration & dosage , Antibodies, Monoclonal, Humanized/administration & dosage , Hypersensitivity/drug therapy , Omalizumab/administration & dosage , Animals , Anti-Allergic Agents/chemistry , Antibodies, Anti-Idiotypic/chemistry , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Basophils/drug effects , Basophils/immunology , Humans , Hypersensitivity/immunology , Immunoglobulin E/chemistry , Immunoglobulin E/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Omalizumab/chemistry , Receptors, IgE/immunology
4.
Nat Chem Biol ; 12(10): 838-44, 2016 10.
Article in English | MEDLINE | ID: mdl-27547922

ABSTRACT

The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.


Subject(s)
Adenylyl Cyclase Inhibitors/pharmacology , Adenylyl Cyclases/metabolism , Pyrimidines/pharmacology , Thiophenes/pharmacology , Adenylyl Cyclase Inhibitors/chemistry , Adenylyl Cyclases/chemistry , Allosteric Regulation/drug effects , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Pyrimidines/chemistry , Solubility , Structure-Activity Relationship , Thiophenes/chemistry
5.
J Biol Chem ; 291(18): 9776-84, 2016 Apr 29.
Article in English | MEDLINE | ID: mdl-26961873

ABSTRACT

The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs.


Subject(s)
Adenosine Triphosphate/chemistry , Adenylyl Cyclases/chemistry , Bicarbonates/chemistry , Bithionol/chemistry , Allosteric Regulation , Catalytic Domain , Crystallography, X-Ray , Humans
6.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 11): 2297-308, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26527146

ABSTRACT

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.


Subject(s)
Anthranilate Synthase/antagonists & inhibitors , Anthranilate Synthase/chemistry , Mycobacterium tuberculosis/enzymology , Tryptophan/metabolism , Tuberculosis/microbiology , Anthranilate Synthase/metabolism , Biosynthetic Pathways , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/pharmacology , Humans , Models, Molecular , Mycobacterium tuberculosis/metabolism , Protein Conformation , Protein Multimerization , Protein Subunits/antagonists & inhibitors , Protein Subunits/chemistry , Protein Subunits/metabolism
7.
J Biol Chem ; 290(20): 12731-43, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25802331

ABSTRACT

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.


Subject(s)
Adenosine Triphosphate , Bacterial Proteins , Cyclic AMP , Heat-Shock Proteins , Mycobacterium , Second Messenger Systems/physiology , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cyclic AMP/chemistry , Cyclic AMP/metabolism , Genome, Bacterial , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Mycobacterium/chemistry , Mycobacterium/genetics , Mycobacterium/metabolism
8.
FEBS J ; 281(18): 4151-64, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25040695

ABSTRACT

UNLABELLED: The ubiquitous second messenger cAMP regulates a wide array of functions, from bacterial transcription to mammalian memory. It is synthesized by six evolutionarily distinct adenylyl cyclase (AC) families. In mammals, there are two AC types: nine transmembrane ACs (tmACs) and one soluble AC (sAC). Both AC types belong to the widespread cyclase class III, which has members in numerous organisms from archaeons to mammals. Class III also contains all known guanylyl cyclases (GCs), which synthesize the cAMP-related messenger cGMP in many eukaryotes and possibly some prokaryotes. Among mammalian ACs, sAC is uniquely regulated by bicarbonate, and has been proposed to be more closely related to a bacterial AC subfamily than to mammalian ACs, on the basis of sequence comparisons. Here, we used crystal structures of human sAC catalytic domains to analyze its relationships with other class III ACs and GCs, and to study its substrate selection mechanisms. Structural comparisons revealed a similarity within an sAC-like subfamily but no family-specific structure elements, and an unexpected sAC similarity to eukaryotic GCs and a potential bacterial GC. We further solved novel crystal structures of sAC catalytic domains in complex with a substrate analog, unprocessed ATP substrate, and product after soaking with ATP or GTP. The structures show a novel ATP-binding conformation, and suggest mechanisms for substrate association and recognition. Our results could explain the limited substrate specificity of sAC, suggest how specificity is increased in other cyclases, and indicate evolutionary relationships among class III enzymes, with sAC being close to a putative 'ancestor' cyclase. DATABASE: Coordinates and structure factors for the novel sAC-cat structures described have been deposited with the Worldwide PDB (www.pdb.org): ApCpp soak (entry 4usu), ATP + Ca(2+) soak (entry 4usv), GTP + Mg(2+) soak (entry 4ust), ATP soak (entry 4usw).


Subject(s)
Adenosine Triphosphate/chemistry , Adenylyl Cyclases/chemistry , Guanosine Triphosphate/chemistry , Adenosine Triphosphate/analogs & derivatives , Amino Acid Sequence , Animals , Biocatalysis , Catalytic Domain , Cells, Cultured , Crystallography, X-Ray , Evolution, Molecular , Humans , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Solubility , Substrate Specificity
9.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 4): 467-9, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24699740

ABSTRACT

The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1-10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ∼2.0 Šresolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04 Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies.


Subject(s)
Adenylyl Cyclases/chemistry , Adenylyl Cyclases/isolation & purification , Crystallography, X-Ray/methods , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Binding Sites , Catalytic Domain , Cloning, Molecular , Crystallization , Humans , Models, Molecular , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
10.
Proc Natl Acad Sci U S A ; 111(10): 3727-32, 2014 Mar 11.
Article in English | MEDLINE | ID: mdl-24567411

ABSTRACT

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3(-)); it thereby serves as a cellular sensor for HCO3(-), carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3(-) binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3(-) activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3(-) sensing and a basis for targeting this system with drugs.


Subject(s)
Adenylyl Cyclases/chemistry , Enzyme Activation/physiology , Models, Molecular , Protein Conformation , Signal Transduction/genetics , Sodium Bicarbonate/metabolism , Catalysis , Cloning, Molecular , Crystallization , Enzyme Activation/genetics , Humans , Protein Binding
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