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PDBsum entry 2rhb
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Viral protein
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PDB id
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2rhb
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References listed in PDB file
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Key reference
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Title
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Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease nsp15.
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Authors
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K.Bhardwaj,
S.Palaninathan,
J.M.Alcantara,
L.Li yi,
L.Guarino,
J.C.Sacchettini,
C.C.Kao.
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Ref.
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J Biol Chem, 2008,
283,
3655-3664.
[DOI no: ]
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PubMed id
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Abstract
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The severe acute respiratory syndrome (SARS) coronavirus encodes several
RNA-processing enzymes that are unusual for RNA viruses, including Nsp15
(nonstructural protein 15), a hexameric endoribonuclease that preferentially
cleaves 3' of uridines. We solved the structure of a catalytically inactive
mutant version of Nsp15, which was crystallized as a hexamer. The structure
contains unreported flexibility in the active site of each subunit.
Substitutions in the active site residues serine 293 and proline 343 allowed
Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15
able to cleave at purines as well as pyrimidines. Mutations that targeted the
residues involved in subunit interactions generally resulted in the formation of
catalytically inactive monomers. The RNA-binding residues were mapped by a
method linking reversible cross-linking, RNA affinity purification, and peptide
fingerprinting. Alanine substitution of several residues in the RNA-contacting
portion of Nsp15 did not affect hexamer formation but decreased the affinity of
RNA binding and reduced endonuclease activity. This suggests a model for Nsp15
hexamer interaction with RNA.
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Figure 1.
FIGURE 1. Structure of SARS-CoV Nsp15. A, surface
representation of a subunit showing three domains. Active site
residues are colored by element and indicated by an arrow. B,
arrangement of six subunits from top view of hexamer.
N-terminal, middle, and C-terminal domains are indicated as N,
M, and C, respectively. The arrows indicate the positions of
catalytic sites in top trimer. C, side view of hexamer showing
arrangement and N- to N-terminal interaction of the top trimer
(T1) with bottom trimer (T2). Six subunits are labeled as a-c
and are colored as follows: T1a, pink; T1b, blue; T1c, cyan;
T2a, red; T2b, green; T2c, golden. This color scheme is used
throughout the figures. Catalytic residues are colored yellow.
D, worm diagram drawn based on b-factor. Worm thickness is
directly proportional to flexibility, i.e. the thickest region
indicates most flexible. E, overlap of the catalytic residues
within the active sites of the six subunits. The structures are
anchored by the backbond of His^249.
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Figure 2.
FIGURE 2. Subunit interaction. A, wire diagram of subunit
T1a and its interaction with other subunits. Contact regions are
boxed and labeled (boxes B-E). The inset shows molecular
arrangement and atomic distances between E3 and L2 (left) and
the gel filtration profile of the E3A mutant of Nsp15 (20). H,
T, and M denote positions corresponding to elution volume of a
hexamer, trimer, and monomer of Nsp15, respectively. B-E,
contacting residues and the calculated distances between them
(left). Atomic distances are calculated using Chimera. Gel
filtration chromatography elution profiles of the indicated
mutant proteins (right). The values below the mutant name refer
to the cleavage rate relative to WT in parentheses.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2008,
283,
3655-3664)
copyright 2008.
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