ABSTRACT
Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1''-phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macro domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182-355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2(1), with unit-cell parameters a = 37.5, b = 55.6, c = 108.9 angstroms, beta = 91.4 degrees, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8 angstroms.
Subject(s)
RNA-Dependent RNA Polymerase/chemistry , Severe acute respiratory syndrome-related coronavirus/enzymology , Viral Nonstructural Proteins/chemistry , Cloning, Molecular , Crystallization , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/isolation & purification , RNA-Dependent RNA Polymerase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/isolation & purification , Viral Nonstructural Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/isolation & purification , Viral Proteins/metabolism , X-Ray DiffractionABSTRACT
The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4(1)32 or P4(3)32, with unit-cell parameters a = b = c = 166.8 angstroms. Diffraction data were collected to a maximum resolution of 2.7 angstroms.
Subject(s)
RNA-Dependent RNA Polymerase/chemistry , Severe acute respiratory syndrome-related coronavirus/chemistry , Viral Nonstructural Proteins/chemistry , Cloning, Molecular , Crystallization , Endoribonucleases , Escherichia coli , Macromolecular Substances/chemistry , Macromolecular Substances/isolation & purification , Peptide Fragments/chemistry , Protein Conformation , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/isolation & purification , X-Ray DiffractionABSTRACT
The aetiologic agent of the recent epidemics of Severe Acute Respiratory Syndrome (SARS) is a positive-stranded RNA virus (SARS-CoV) belonging to the Coronaviridae family and its genome differs substantially from those of other known coronaviruses. SARS-CoV is transmissible mainly by the respiratory route and to date there is no vaccine and no prophylactic or therapeutic treatments against this agent. A SARS-CoV whole-genome approach has been developed aimed at determining the crystal structure of all of its proteins or domains. These studies are expected to greatly facilitate drug design. The genomes of coronaviruses are between 27 and 31.5 kbp in length, the largest of the known RNA viruses, and encode 20-30 mature proteins. The functions of many of these polypeptides, including the Nsp9-Nsp10 replicase-cleavage products, are still unknown. Here, the cloning, Escherichia coli expression, purification and crystallization of the SARS-CoV Nsp9 protein, the first SARS-CoV protein to be crystallized, are reported. Nsp9 crystals diffract to 2.8 A resolution and belong to space group P6(1/5)22, with unit-cell parameters a = b = 89.7, c = 136.7 A. With two molecules in the asymmetric unit, the solvent content is 60% (V(M) = 3.1 A(3) Da(-1)).
