Scientific Report 2008
Structural Biology and Structural Genomics
With Nuclear Magnetic Resonance Spectroscopy
K. Wüthrich, W. Augustyniak, A.
Chatterjee, M. Geralt, R. Horst, M. Johnson, B. Pedrini, W.J. Placzek, J.K.
Rhee, P. Serrano, P. Stanczak
We are developing
methods to improve the efficiency and reliability of nuclear magnetic resonance
(NMR) structure determination of proteins in solution and are applying the methods
to target proteins selected within the framework of the Joint Center for Structural
Genomics. In addition, as part of the research of the Center for Functional and
Structural Proteomics of SARS-CoV (FSPS; http://visp.scripps.edu/SARS/default.aspx),
we are characterizing the proteome of the coronavirus (SARS-CoV) that causes severe
acute respiratory syndrome (SARS).
SARS-COV Structural Genomics
Although a 2003 SARS pandemic was contained
by public health measures, no vaccine or effective treatment for SARS is available,
and the basic mechanisms of coronavirus infections are not yet understood. SARS-CoV
contains a 29-kb positive-stranded RNA genome. About two-thirds of the genome is
devoted to encoding a replicase polyprotein, which is cleaved by 2 viral proteases
to release the mature nonstructural proteins. These proteins are responsible for
the enzymatic functions that allow the virus to replicate in infected cells and
therefore are potential targets for drug development. The FSPS project was started
with the expectation that structure-based functional studies will reveal new functional
features that are not detectable when only the amino acid sequence is known.
Structure Determination of
the Nonstructural Protein 3c
The region of the SARS-CoV nonstructural
protein 3 (nsp3) that spans residues 366—722 is a functional domain termed
the SARS-unique domain (SUD) because it is not present in other known coronaviruses.
Expression in Escherichia coli indicated that SUD does not form a single
globular structure, and NMR studies showed that it consists of at least 3 distinct
structural domains, which may provide for multiple functions (Fig. 1). A central
globular domain, SUD-M (M stands for middle), spans residues 527—651. The NMR
structure of this protein shows a macrodomain fold with similarity to that of proteins
that bind the important regulatory molecule ADP-ribose in eukaryotic cells (Fig.
Structural coverage of nsp3. The horizontal black line represents the polypeptide
segment 1—1318; the initially annotated functional domains are indicated above
the line. Globular domain structures determined so far are shown in ribbon representation,
along with color-coded information on the structure determination method used and
the new, structure-based functional annotation. In between the globular domains,
blue lines represent flexibly disordered segments as determined by NMR spectroscopy,
and black lines indicate unstructured segments implicated by the absence of x-ray
diffraction in protein crystals. Regions of the protein with unknown structures
are colored green; these regions extend to the C terminus of nsp3 at residue 1922.
A, Ensemble of 20 conformers representing the solution structure of the protein
domain SUD-M (see also Fig. 1). The conformers were superimposed for minimal root-mean-square
deviation of the backbone N, Cα,
atoms of the residues 527—651. Selected sequence positions relative to the
intact nsp3 (Fig. 1) are indicated by numbers. Helical secondary structures are
are green, and segments with no regular secondary structure are gray. B, Surface
view of SUD-M in the same orientation as in A, with the regions affected by the
binding of single-stranded polyadenosine RNA in magenta to highlight the probable
Structure-based attempts to determine
the function of SUD-M started with a search of the Protein Data Bank for structural
homologs of SUD-M. The closest 3-dimensional structural homolog was the protein
nsp3b, which is located immediately N-terminal to the SUD region in the SARS-CoV
proteome and functions as an ADP-ribose-1′′-phosphatase.
This finding was a surprise, because the sequence identity between the 2 domains
is only 6%. Tests for binding of a variety of different ligands, based on NMR chemical-shift
perturbation measurements, revealed that SUD-M recognizes single-stranded polyadenosine
RNA. A possible function suggested by this observation is in viral genome replication
or transcription, which may involve the recognition of polyadenylated tails of viral
RNA by one or more viral proteins. SUD-M might thus be a potential
target for the development of antiviral drugs that disrupt viral replication.
the basis of phylogenetic and bioinformatics analyses, nsp3 was initially predicted
to consist of 7 functional domains: nsp3a—nsp3g. The NMR structure determination
of SUD-M was just one of many steps toward elucidating the structure of the much
larger nsp3 polypeptide. The overall structural characterization of the region nsp3a—nsp3e
now provides a detailed picture of the domain organization (Fig. 1), and new functions
have already been identified.
As illustrated in Figure 1, using NMR
spectroscopy and x-ray crystallography with polypeptide constructs of variable lengths
makes it possible not only to determine the structures of the individual segmentally
arranged globular domains but also to characterize the intervening linker regions.
With this strategy, which was adapted from target selection in structural genomics
projects, we can obtain a comprehensive view of the multidomain protein, which shows
that the protein has overall a predominantly extended shape. The structural information
thus obtained enabled us to immediately predict different functions of nsp3. We
are following up our structural findings with biomedical and physiologic studies.
Almeida, M.S., Johnson, M.A., Herrmann,
T., Geralt, M., Wüthrich, K.
fold in the nuclear magnetic resonance structure of the replicase nonstructural
protein 1 from the severe acute respiratory syndrome coronavirus. J. Virol. 81:3151,
Chatterjee, A., Johnson, M.A., Serrano,
P., Pedrini, B., Wüthrich, K. NMR
assignment of the domain 513-651 from the SARS-CoV nonstructural protein nsp3. Biomol.
NMR Assign. 1:191, 2007.
Horst, R., Fenton, W.A., Englander,
W.S., Wüthrich, K., Horwich A.L.
Folding trajectories of human dihydrofolate reductase inside the GroEL GroES chaperonin
cavity and free in solution. Proc. Natl. Acad. Sci. U. S. A. 104:20788, 2007.
Johnson, M.A., Southworth, M.W., Herrmann,
T., Brace, L., Perler, F.B., Wüthrich, K.
NMR structure of a KlbA intein precursor from Methanococcus jannaschii. Protein
Sci. 16:1316, 2007.
Johnson, M.A., Southworth, M.W., Perler,
F.B., Wüthrich K. NMR
assignment of a KlbA intein precursor from Methanococcus jannaschii. Biomol.
NMR Assign. 1:19, 2007.
Pedrini, B., Placzek,
W.J., Koculi, E., Alimenti, C., LaTerza, A., Luporini, P., Wüthrich, K.
Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of
the pheromone En-6 from the Antarctic ciliate Euplotes nobilii. J.
Mol. Biol. 372:277, 2007.
Placzek, W.J., Almeida, M.S., Wüthrich,
K. NMR structure and functional
characterization of a human cancer-related nucleoside triphosphatase. J. Mol. Biol.
Placzek, W.J., Etezady-Esfarjani,
T., Herrmann, T., Pedrini, B., Peti, W., Alimenti, C., Luporini, P., Wüthrich,
K. Cold-adapted signal proteins:
NMR structures of pheromones from the Antarctic ciliate Euplotes nobilii.
IUBMB Life 59:578, 2007.
Serrano, P., Johnson, M.A., Almeida,
M.S., Horst, R., Herrmann, T., Joseph, J.S., Neuman, B.W., Subramanian, V., Saikatendu,
K.S., Buchmeier, M.J., Stevens, R.C., Kuhn, P., Wüthrich, K. Nuclear
magnetic resonance structure of the N-terminal domain of nonstructural protein 3
from the severe acute respiratory syndrome coronavirus. J. Virol. 81:12049, 2007.