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1.
J Virol ; 89(1): 165-80, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25320291

ABSTRACT

UNLABELLED: The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication. IMPORTANCE: The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A(S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5BΔ21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors.


Subject(s)
Hepacivirus/enzymology , Hepacivirus/physiology , Host-Pathogen Interactions , Replication Protein A/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication , Humans , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Binding , RNA, Viral/metabolism , Sequence Deletion , Viral Nonstructural Proteins/genetics
2.
Structure ; 18(9): 1083-93, 2010 Sep 08.
Article in English | MEDLINE | ID: mdl-20826335

ABSTRACT

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.


Subject(s)
Azepines/chemistry , Imidazoles/chemistry , Piperazines/chemistry , Quinazolines/chemistry , Receptors, Calcitonin Gene-Related Peptide/chemistry , Azepines/pharmacology , Binding Sites , Calcitonin Gene-Related Peptide/chemistry , Calcitonin Gene-Related Peptide/metabolism , Calcitonin Gene-Related Peptide Receptor Antagonists , Calcitonin Receptor-Like Protein/chemistry , Calcitonin Receptor-Like Protein/metabolism , Crystallography, X-Ray , Imidazoles/pharmacology , Piperazines/pharmacology , Protein Structure, Tertiary , Quinazolines/pharmacology , Receptors, Calcitonin Gene-Related Peptide/metabolism
3.
Biochemistry ; 49(9): 1862-72, 2010 Mar 09.
Article in English | MEDLINE | ID: mdl-20099900

ABSTRACT

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of two membrane proteins: calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a class B G-protein-coupled receptor (GPCR), possessing a characteristic large amino-terminal extracellular domain (ECD) important for ligand recognition and binding. Dimerization of CLR with RAMP1 provides specificity for CGRP versus related agonists. Here we report the expression, purification, and refolding of a soluble form of the CGRP receptor comprising a heterodimer of the CLR and RAMP1 ECDs. The extracellular protein domains corresponding to residues 23-133 of CLR and residues 26-117 of RAMP1 were shown to be sufficient for formation of a stable, monodisperse complex. The binding affinity of the purified ECD complex for the CGRP peptide was significantly lower than that of the native receptor (IC(50) of 12 microM for the purified ECD complex vs 233 pM for membrane-bound CGRP receptor), indicating that other regions of CLR and/or RAMP1 are important for peptide agonist binding. However, high-affinity binding to known potent and specific nonpeptide antagonists of the CGRP receptor, including olcegepant and telcagepant (K(D) < 0.02 muM), as well as N-terminally truncated peptides and peptide analogues (140 nM to 1.62 microM) was observed.


Subject(s)
Extracellular Space/chemistry , Protein Folding , Receptors, Calcitonin Gene-Related Peptide/chemistry , Receptors, Calcitonin/chemistry , Amino Acid Sequence , Binding, Competitive , Calcitonin Receptor-Like Protein , Cell Line, Tumor , Crystallography, X-Ray , Dimerization , Extracellular Space/metabolism , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Ligands , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Magnetic Resonance Spectroscopy , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Proteins , Receptors, Calcitonin/metabolism , Receptors, Calcitonin Gene-Related Peptide/biosynthesis , Receptors, Calcitonin Gene-Related Peptide/genetics , Receptors, Calcitonin Gene-Related Peptide/isolation & purification , Solubility
4.
Curr Protein Pept Sci ; 8(5): 439-45, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17979759

ABSTRACT

Structure-based drug design (SBDD) has played an integral role in the development of highly specific, potent protease inhibitors resulting in a number of drugs in clinical trials and on the market. Possessing biochemical assays and structural information on both the target protease and homologous family members helps ensure compound selectivity. We have redesigned the path from clone to protein eliminating many of the traditional bottlenecks associated with protein production to ensure a constant supply to feed many diverse protease drug discovery programs. The process was initiated with the design of a multi-system vector, capable of expression in both eukaryotic and prokaryotic hosts; this vector also facilitated high-throughput cloning, expression and purification. When combined into an expression screen, supplemented with salvage screens for detergent extraction and refolding, a route for protein production was established rapidly. Using this process-orientated approach we have successfully expressed and purified all mechanistic classes of active human and viral proteases for enzymatic assays and crystallization studies. While exploiting recent developments in high-throughput biochemistry, we still employ classical biophysical techniques such as light-scattering and analytical ultracentrifugation, to ensure the highest quality protein enters crystallization trials. We have drawn on examples from our own research programs to illustrate how these strategies have been successfully used in the production of proteases for SBDD.


Subject(s)
Drug Design , Peptide Hydrolases/chemistry , Animals , Humans , Models, Molecular , Peptide Hydrolases/biosynthesis , Peptide Hydrolases/genetics , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Binding , Protein Folding , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
5.
J Biol Chem ; 277(45): 42419-22, 2002 Nov 08.
Article in English | MEDLINE | ID: mdl-12237287

ABSTRACT

Aurora-2 is a key member of a closely related subgroup of serine/threonine kinases that plays important roles in the completion of essential mitotic events. Aurora-2 is oncogenic and amplified in various human cancers and could be an important therapeutic target for inhibitory molecules that would disrupt the cell cycle and block proliferation. We report the first crystal structure of Aurora-2 kinase in complex with adenosine. Analysis of residues in the active site suggests differences with structurally and biologically related protein kinases. The activation loop, which contains residues specific to the Aurora family of kinases, has a unique conformation. These results provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of the Aurora kinases.


Subject(s)
Protein Serine-Threonine Kinases/chemistry , Aurora Kinases , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Neoplasms/enzymology , Protein Conformation , Protein Structure, Secondary , Recombinant Proteins/chemistry , Sequence Deletion
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