ABSTRACT
Hyperlipidemia predisposes individuals to cardiometabolic diseases, the most common cause of global mortality. Microsomal triglyceride transfer protein (MTP) transfers multiple lipids and is essential for the assembly of apolipoprotein B-containing lipoproteins. MTP inhibition lowers plasma lipids but causes lipid retention in the liver and intestine. Previous studies suggested two lipid transfer domains in MTP and that specific inhibition of triglyceride (TG) and not phospholipid (PL) transfer can lower plasma lipids without significant tissue lipid accumulation. However, how MTP transfers different lipids and the domains involved in these activities are unknown. Here, we tested a hypothesis that two different ß-sandwich domains in MTP transfer TG and PL. Mutagenesis of charged amino acids in ß2-sandwich had no effect on PL transfer activity indicating that they are not critical. In contrast, amino acids with bulky hydrophobic side chains in ß1-sandwich were critical for both TG and PL transfer activities. Substitutions of these residues with smaller hydrophobic side chains or positive charges reduced, whereas negatively charged side chains severely attenuated MTP lipid transfer activities. These studies point to a common lipid transfer domain for TG and PL in MTP that is enriched with bulky hydrophobic amino acids. Furthermore, we observed a strong correlation in different MTP mutants with respect to loss of both the lipid transfer activities, again implicating a common binding site for TG and PL in MTP. We propose that targeting of areas other than the identified common lipid transfer domain might reduce plasma lipids without causing cellular lipid retention.
Subject(s)
Carrier Proteins , Hydrophobic and Hydrophilic Interactions , Phospholipids , Triglycerides , Humans , Amino Acids/chemistry , Amino Acids/genetics , Amino Acids/metabolism , Apolipoproteins B/chemistry , Apolipoproteins B/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Phospholipids/blood , Phospholipids/metabolism , Triglycerides/blood , Triglycerides/metabolism , Protein Domains , Mutation , Structure-Activity Relationship , Binding SitesABSTRACT
Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.
Subject(s)
Carrier Proteins/genetics , Lipids/genetics , Lipoproteins/genetics , Triglycerides/genetics , Animals , Fatty Liver/genetics , Fatty Liver/pathology , Gastrointestinal Tract/metabolism , Humans , Immunoprecipitation , Lipid Droplets/metabolism , Lipoproteins/metabolism , Mutation, Missense/genetics , Point Mutation/genetics , Protein Transport/genetics , Triglycerides/metabolism , Zebrafish/geneticsABSTRACT
The major cat allergen Fel d 1 is one of the most common and potent causes of animal related allergy. Medical treatment of cat allergy has relied on immunotherapy carried out with cat dander extract. This approach has been problematic, mainly due to inconsistent levels of the major allergen in the produced extracts. Recombinant DNA technology has been proposed as an alternative method to produce more consistent pharmaceuticals for immunotherapy and diagnostics of allergy. Current approaches to produce recombinant Fel d 1 (recFel d 1) in the cytoplasm of Escherichia coli have however resulted in protein folding deficiencies and insoluble inclusion body formation, requiring elaborate in vitro processing to acquire folded material. In this study, we introduce an efficient method for cytoplasmic production of recFel d 1 that utilizes eukaryotic folding factors to aid recFel d 1 to fold and be produced in the soluble fraction of E. coli. The solubly expressed recFel d 1 is shown by biophysical in vitro experiments to contain structural disulfides, is extremely stable, and has a sensitivity for methionine sulfoxidation. The latter is discussed in the context of functional relevance.
Subject(s)
Allergens , Glycoproteins , Protein Folding , Allergens/biosynthesis , Allergens/chemistry , Allergens/genetics , Allergens/isolation & purification , Animals , Cats , Glycoproteins/biosynthesis , Glycoproteins/chemistry , Glycoproteins/genetics , Glycoproteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Intercellular cell adhesion molecule-5 (ICAM-5) is a member of the ICAM subfamily that is exclusively expressed in the telencephalon region of the brain. The crystal structure of the four most N-terminal glycosylated domains (D1-D4) of ICAM-5 was determined in three different space groups and the D1-D5 fragment was modelled. The structures showed a curved molecule with two pronounced interdomain bends between D2 and D3 and between D3 and D4, as well as some interdomain flexibility. In contrast to ICAM-1, ICAM-5 has patches of positive and negative electrostatic charge at D1-D2 and at D3-D5, respectively. ICAM-5 can mediate homotypic interactions. In the crystals, several charge-based intermolecular interactions between the N-terminal and C-terminal moieties of the ICAM-5 molecules were observed, which defined an interacting surface in the D1-D4 fragment. One of the crystal lattices has a molecular assembly that could represent the homophilic ICAM-5 cell adhesion complex in neurons.
Subject(s)
Cell Adhesion Molecules/chemistry , Nerve Tissue Proteins/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Humans , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino Acid , Static ElectricityABSTRACT
Global health challenges such as the coronavirus pandemic warrant the urgent need for a system that allows efficient production of diagnostic and therapeutic interventions. Antibody treatments against SARS-CoV-2 were developed with an unprecedented pace and this enormous progress was achieved mainly through recombinant protein production technologies combined with expeditious screening approaches. A heterologous protein production system that allows efficient soluble production of therapeutic antibody candidates against rapidly evolving variants of deadly pathogens is an important step in preparedness towards future pandemic challenges. Here, we report cost and time-effective soluble production of SARS-CoV-2 receptor binding domain (RBD) variants as well as an array of neutralizing antibody fragments (Fabs) based on Casirivimab and Imdevimab using the CyDisCo system in the cytoplasm of E. coli. We also report variants of the two Fabs with higher binding affinity against SARS-CoV-2 RBD and suggest this cytoplasmic production of disulfide containing antigens and antibodies can be broadly applied towards addressing future global public health threats.
Subject(s)
Antibodies, Neutralizing , COVID-19 , Humans , SARS-CoV-2 , Escherichia coli/metabolism , Antibodies, Viral , Cytoplasm/metabolismABSTRACT
Small GTPase Rab6 regulates vesicle trafficking at the level of Golgi via recruitment of numerous and unrelated effectors. The crystal structure of Rab6a(GTP) in complex with a 378-residue internal fragment of the effector Rab6IP1 was solved at 3.2 angstroms resolution. This Rab6IP1 region encompasses an all alpha-helical RUN domain followed in tandem by a PLAT domain that adopts a beta sandwich fold. The structure reveals that the first and last alpha helices of the RUN domain mediate binding to switch I, switch II, and the interswitch region of Rab6. It represents the largest Rab-effector complex determined to date. Comparisons with the recent structure of Rab6 in complex with an unrelated effector, human golgin GCC185, reveals significant conformational changes in the conserved hydrophobic triad of Rab6. Flexibility in the switch and interswitch regions of Rab6 mediates recognition of compositionally distinct alpha-helical coiled coils, thereby contributing to Rab6 promiscuity in effector recruitment.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Golgi Apparatus/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Base Sequence , DNA Primers , Models, Molecular , Protein Conformation , Two-Hybrid System TechniquesABSTRACT
The small GTPase Rab6 regulates vesicle trafficking at the level of Golgi. Recently, the crystal structures of Rab6 in complexes with two unrelated effectors have been determined. The structure of Rab6a-GTP in complex with a 378-residue internal fragment of the effector Rab6IP1 (Rab6-interacting protein 1) has been solved. In addition, the structure of Rab6 with the golgin, GCC185, has also been determined. In both complexes, two alpha-helices from the effector mediate binding to switch I, switch II and the interswitch region of Rab6. Comparisons of the complexes reveal significant conformational changes in the conserved hydrophobic triad of Rab6. Thus conformational flexibility in the triad mediates recognition of compositionally distinct alpha-helical coiled coils, providing a rationale for the promiscuity of Rab6 in effector recruitment.
Subject(s)
Protein Conformation , rab GTP-Binding Proteins/chemistry , Golgi Apparatus/metabolism , Guanine Nucleotide Exchange Factors , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.
Subject(s)
CD13 Antigens/antagonists & inhibitors , Coronavirus Infections/pathology , Coronavirus Infections/virology , Coronavirus/physiology , Neoplasms/pathology , Allosteric Regulation/drug effects , Animals , Antineoplastic Agents/pharmacology , Biocatalysis/drug effects , CD13 Antigens/chemistry , CD13 Antigens/metabolism , CHO Cells , Catalytic Domain , Cell Proliferation/drug effects , Coronavirus/drug effects , Cricetinae , Cricetulus , Disulfides/metabolism , Humans , Models, Molecular , Protein Domains , Sus scrofaABSTRACT
Plasmepsin IV from Plasmodium falciparum (PM IV) is a promising target for the development of novel antimalarial drugs. Here, the crystal structure of the truncated zymogen of PM IV (pPM IV), consisting of the mature enzyme plus a prosegment of 47 residues, has been determined at 1.5â Å resolution. pPM IV presents the fold previously described for studied proplasmepsins, displaying closer similarities to proplasmepin IV from P. vivax (pPvPM) than to the other two proplasmepsins from P. falciparum. The study and comparison of the pPM IV structure with the proplasmepsin structures described previously provide information about the similarities and differences in the inactivation-activation mechanisms among the plasmepsin zymogens.
Subject(s)
Aspartic Acid Endopeptidases/chemistry , Enzyme Precursors/chemistry , Plasmodium falciparum/chemistry , Protozoan Proteins/chemistry , Amino Acid Sequence , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Molecular , Plasmids/chemistry , Plasmids/metabolism , Plasmodium falciparum/enzymology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
2-Aminoquinazolin-4(3H)-ones were identified as a novel class of malaria digestive vacuole plasmepsin inhibitors by using NMR-based fragment screening against Plm II. Initial fragment hit optimization led to a submicromolar inhibitor, which was cocrystallized with Plm II to produce an X-ray structure of the complex. The structure showed that 2-aminoquinazolin-4(3H)-ones bind to the open flap conformation of the enzyme and provided clues to target the flap pocket. Further improvement in potency was achieved via introduction of hydrophobic substituents occupying the flap pocket. Most of the 2-aminoquinazolin-4(3H)-one based inhibitors show a similar activity against digestive Plms I, II, and IV and >10-fold selectivity versus CatD, although varying the flap pocket substituent led to one Plm IV selective inhibitor. In cell-based assays, the compounds show growth inhibition of Plasmodium falciparum 3D7 with IC50 â¼ 1 µM. Together, these results suggest 2-aminoquinazolin-4(3H)-ones as perspective leads for future development of an antimalarial agent.
Subject(s)
Antimalarials/chemical synthesis , Antimalarials/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Quinazolines/chemical synthesis , Quinazolines/pharmacology , 3T3 Cells , Animals , Cell Survival/drug effects , Crystallography, X-Ray , Malaria/drug therapy , Malaria/parasitology , Mice , Models, Molecular , Plasmodium falciparum/drug effects , Structure-Activity RelationshipABSTRACT
Plasmepsin II (PMII) is one of the ten plasmepsins (PMs) identified in the genome of Plasmodium falciparum, the causative agent of the most severe and deadliest form of malaria. Owing to the emergence of P. falciparum strains that are resistant to current antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is a constant pressure to find new and lasting chemotherapeutic drug therapies. Previously, the crystal structure of PMII in complex with NU655, a potent antimalarial hydroxyethylamine-based inhibitor, and the design of new compounds based on it have been reported. In the current study, two of these newly designed hydroxyethylamine-based inhibitors, PG418 and PG394, were cocrystallized with PMII and their structures were solved, analyzed and compared with that of the PMII-NU655 complex. Structural analysis of the PMII-PG418 complex revealed that the flap loop can adopt a fully closed conformation, stabilized by interactions with the inhibitor, and a fully open conformation, causing an overall expansion in the active-site cavity, which in turn causes unstable binding of the inhibitor. PG418 also stabilizes the flexible loop Gln275-Met286 of another monomer in the asymmetric unit of PMII, which is disordered in the PMII-NU655 complex structure. The crystal structure of PMII in complex with the inhibitor PG418 demonstrates the conformational flexibility of the active-site cavity of the plasmepsins. The interactions of the different moieties in the P1' position of PG418 and PG394 with Thr217 have to be taken into account in the design of new potent plasmepsin inhibitors.
Subject(s)
Aspartic Acid Endopeptidases/chemistry , Benzamides/pharmacology , Enzyme Inhibitors/pharmacology , Ethanolamines/pharmacology , Plasmodium falciparum/metabolism , Propanolamines/pharmacology , Protozoan Proteins/chemistry , Antimalarials/chemistry , Antimalarials/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Catalytic Domain , Cathepsins/antagonists & inhibitors , Crystallography, X-Ray , Dimerization , Enzyme Inhibitors/chemistry , Humans , Ligands , Plasmodium falciparum/drug effects , Protein Structure, Secondary , Protozoan Proteins/antagonists & inhibitorsABSTRACT
Inhibitors of human mast cell tryptase (EC 3.4.21.59) have therapeutic potential for treating allergic or inflammatory disorders. We have investigated transition-state mimetics possessing a heterocycle-activated ketone group and identified in particular benzothiazole ketone (2S)-6 (RWJ-56423) as a potent, reversible, low-molecular-weight tryptase inhibitor with a K(i) value of 10 nM. A single-crystal X-ray analysis of the sulfate salt of (2S)-6 confirmed the stereochemistry. Analogues 12 and 15-17 are also potent tryptase inhibitors. Although RWJ-56423 potently inhibits trypsin (K(i) = 8.1 nM), it is selective vs other serine proteases, such as kallikrein, plasmin, and thrombin. We obtained an X-ray structure of (2S)-6 complexed with bovine trypsin (1.9-A resolution), which depicts inter alia a hemiketal involving Ser-189, and hydrogen bonds with His-57 and Gln-192. Aerosol administration of 6 (2R,2S; RWJ-58643) to allergic sheep effectively antagonized antigen-induced asthmatic responses, with 70-75% blockade of the early response and complete ablation of the late response and airway hyperresponsiveness.
Subject(s)
Anti-Asthmatic Agents/chemical synthesis , Dipeptides/chemistry , Ketones/chemical synthesis , Pyrrolidines/chemical synthesis , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/chemical synthesis , Thiazoles/chemical synthesis , Aerosols , Animals , Anti-Asthmatic Agents/chemistry , Anti-Asthmatic Agents/pharmacology , Asthma/drug therapy , Asthma/immunology , Asthma/physiopathology , Benzothiazoles , Cattle , Crystallography, X-Ray , Humans , Ketones/chemistry , Ketones/pharmacology , Kinetics , Mast Cells/enzymology , Models, Molecular , Protein Binding , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Sheep , Stereoisomerism , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacology , Trypsin/chemistry , TryptasesABSTRACT
The fructose-based sugar sulphamate RWJ-37497, a potent analogue of the widely used anti-epileptic drug topiramate, possesses anti-convulsant and carbonic anhydrase-inhibitory activities. We have studied the binding interactions of RWJ-37497 in the active site of human carbonic anhydrase II by X-ray crystallography. The atomic positions of the enzyme inhibitor complex were refined at a resolution of 2.1 A (1 A=0.1 nm) to the final crystallographic R and R(free) values of 0.18 and 0.23, respectively. The inhibitor co-ordinates to the active-site zinc ion through its oxygen atom and the ionized nitrogen atom of the sulphamate group by replacing the metal-bound water molecules, although the sulphamoyl oxygen atom provides a rather lengthy co-ordination. The 4,5-cyclic sulphate group is positioned in a hydrophobic pocket of the active site, making contacts with the residues Phe-131, Leu-198, Pro-201 and Pro-202. Since the ligand was found to be intact, concerns about RWJ-37947 irreversibly alkylating the enzyme through its 4,5-cyclic sulphate group were dispelled.
Subject(s)
Carbonic Anhydrase II/chemistry , Sulfonic Acids/chemistry , Binding Sites , Crystallography, X-Ray , Electrons , Fructose/analogs & derivatives , Fructose/pharmacology , Humans , Ligands , Models, Molecular , Oxygen/chemistry , Protein Binding , Sulfates , Sulfonic Acids/pharmacologyABSTRACT
The serine protease cathepsin G (EC 3.4.21.20; Cat G), which is stored in the azurophilic granules of neutrophils (polymorphonuclear leukocytes) and released on degranulation, has been implicated in various pathological conditions associated with inflammation. By employing high-throughput screening, we identified beta-ketophosphonic acid 1 as a moderate inhibitor of Cat G (IC(50) = 4.1 microM). We were fortunate to obtain a cocrystal of 1 with Cat G and solve its structure by X-ray crystallography (3.5 A). Structural details from the X-ray analysis of 1.Cat G served as a platform for optimization of this lead compound by structure-based drug design. With the aid of molecular modeling, substituents were attached to the 3-position of the 2-naphthyl ring of 1, which occupies the S1 pocket of Cat G, to provide an extension into the hydrophobic S3 region. Thus, we arrived at analogue 7 with an 80-fold potency improvement over 1 (IC(50) = 53 nM). From these results, it is evident that the beta-ketophosphonic acid unit can form the basis for a novel class of serine protease inhibitors.