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1.
J Struct Biol ; 199(1): 84-86, 2017 07.
Article in English | MEDLINE | ID: mdl-28433496

ABSTRACT

We present here a new CryoEM grid boxes storage system designed to simplify sample labeling, tracking and retrieval. The system is based on the crystal pucks widely used by the X-ray crystallographic community for storage and shipping of crystals. This system is suitable for any cryoEM laboratory, but especially for large facilities that will need accurate tracking of large numbers of samples coming from different sources.


Subject(s)
Cryoelectron Microscopy/methods , Specimen Handling/methods , Automation, Laboratory , Preservation, Biological/methods , Research Design , Specimen Handling/standards
2.
Nat Struct Mol Biol ; 22(12): 953-8, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26595420

ABSTRACT

Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility.


Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Antineoplastic Agents/chemistry , Immunoglobulin G/chemistry , Programmed Cell Death 1 Receptor/immunology , Crystallography, X-Ray , Humans , Protein Conformation , Protein Structure, Tertiary
3.
Protein Eng Des Sel ; 21(7): 425-33, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18456871

ABSTRACT

The nuclear xenobiotic receptor PXR is a ligand-inducible transcription factor regulating drug-metabolizing enzymes and transporters and a master switch mediating potentially adverse drug-drug interactions. In addition to binding a coactivator protein such as SRC-1, the C-terminal ligand-binding domain (LBD) is solely responsible for ligand recognition and thus the ligand-dependent downstream effects. In an effort to facilitate structural studies of PXR to understand and abolish the interactions between PXR and its ligands, several recombinant PXR/SRC-1 constructs were designed and evaluated for expression, stability and activity. Expression strategies employing either dual expression or translationally coupled bicistronic expression were found to be unsuitable for producing stable PXR in a stochiometric complex with a peptide derived from SRC-1 (SRC-1p). A single polypeptide chain encompassing PXR and SRC-1p tethered with a peptidyl linker was designed to promote intramolecular complex formation. This tethered protein was overexpressed as a soluble protein and required no additional SRC-1p for further stabilization. X-ray crystal structures in the presence and absence of the known PXR agonist SR-12813 were determined to high resolution. In addition, a circular dichroism-based binding assay was developed to allow rapid evaluation of PXR ligand affinity, making this tethered protein a convenient and effective reagent for the rational attenuation of drug-induced PXR-mediated metabolism.


Subject(s)
Histone Acetyltransferases/genetics , Receptors, Steroid/genetics , Transcription Factors/genetics , Crystallization , Crystallography, X-Ray , Hepatocytes/metabolism , Hot Temperature , Humans , Models, Molecular , Nuclear Receptor Coactivator 1 , Pregnane X Receptor , Protein Denaturation , Protein Engineering/methods
4.
J Synchrotron Radiat ; 15(Pt 3): 204-7, 2008 May.
Article in English | MEDLINE | ID: mdl-18421139

ABSTRACT

The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease, which is currently in clinical trials.


Subject(s)
Hepacivirus/enzymology , Proline/analogs & derivatives , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Models, Molecular , Molecular Structure , Proline/chemistry
5.
J Med Chem ; 50(10): 2310-8, 2007 May 17.
Article in English | MEDLINE | ID: mdl-17444623

ABSTRACT

The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contribution to the binding energy arises from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets [the numbering of the subsites is as defined in Berger, A.; Schechter, I. Philos. Trans. R. Soc. London, Ser. B 1970, 257, 249-264]. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease that is currently in clinical trials.


Subject(s)
Antiviral Agents/chemical synthesis , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/chemistry , Hepacivirus/enzymology , Proline/analogs & derivatives , Serine Proteinase Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , Antiviral Agents/chemistry , Binding Sites , Crystallography, X-Ray , Intracellular Signaling Peptides and Proteins , Models, Molecular , Proline/chemical synthesis , Proline/chemistry , Protein Conformation , Stereoisomerism , Structure-Activity Relationship
6.
Protein Expr Purif ; 38(2): 292-301, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15555945

ABSTRACT

Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in several insect cells. The pro-CAT was cloned into baculovirus under the regulation of the polyhedron promoter and using either the honeybee mellitin or ADAM33 signal sequence. Sf9 or Hi5 cells infected with these recombinant viruses expressed the majority of the protein unprocessed and as inclusion bodies ( approximately 10 mg/L). On the other hand, similar constructs could be expressed, processed, and secreted by Drosophila S2 cells using a variety of constitutive (actin, pAc5.1) or inducible (metallothionein, PMT) promoters and leader sequences (e.g., native and BiP). Higher expression level of 10-fold was observed for the inducible system resulting in an average yield of 20 mg/L after purification. The majority of the catalytic domain purified from the Drosophila conditioned media remained associated with the pro-domain after several chromatography steps. An induction cocktail containing cadmium chloride and zinc chloride was subsequently developed for the PMT system as an alternative to using cupric sulfate or cadmium chloride as single inducers. The novel induction cocktail resulted in an increased ratio of secreted catalytic to pro-domain, and yielded milligram amounts of highly purified protease. The availability of this modified expression system facilitated purification of the wild type and several glycosylation mutants, one of which (N231Q) crystallized recently for X-ray structure determination [J. Mol. Biol. 335 (2003) 129].


Subject(s)
Metalloendopeptidases/biosynthesis , Metalloendopeptidases/genetics , ADAM Proteins , Animals , Cadmium Chloride/chemistry , Catalysis , Cell Line , Cloning, Molecular , Copper Sulfate/chemistry , Drosophila , Gene Expression Regulation, Enzymologic , Genetic Vectors/genetics , Glycosylation , Humans , Metalloendopeptidases/isolation & purification , Mutation , Promoter Regions, Genetic , Protein Structure, Tertiary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Zinc/chemistry
7.
J Mol Biol ; 335(1): 129-37, 2004 Jan 02.
Article in English | MEDLINE | ID: mdl-14659745

ABSTRACT

Adam33 is a putative asthma susceptibility gene encoding for a membrane-anchored metalloprotease belonging to the ADAM family. The ADAMs (a disintegrin and metalloprotease) are a family of glycoproteins implicated in cell-cell interactions, cell fusion, and cell signaling. We have determined the crystal structure of the Adam33 catalytic domain in complex with the inhibitor marimastat and the inhibitor-free form. The structures reveal the polypeptide fold and active site environment resembling that of other metalloproteases. The substrate-binding site contains unique features that allow the structure-based design of specific inhibitors of this enzyme.


Subject(s)
Catalytic Domain , Crystallography, X-Ray , Metalloendopeptidases/chemistry , ADAM Proteins , Amino Acid Sequence , Enzyme Inhibitors/chemistry , Humans , Hydroxamic Acids/chemistry , Metalloendopeptidases/genetics , Models, Molecular , Molecular Structure , Mutation , Protein Binding , Protein Structure, Tertiary , Sequence Alignment
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