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Scientific Report 2008

Molecular Biology

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. 2).

Fig. 1. 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.

Fig. 2. 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, and C′ atoms of the residues 527—651. Selected sequence positions relative to the intact nsp3 (Fig. 1) are indicated by numbers. Helical secondary structures are red, β-strands 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 RNA-binding site.

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.

On 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. Novel
β -barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus. J. Virol. 81:3151, 2007.

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. 367:788, 2007.

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.


Kurt Wüthrich, Ph.D.
Cecil H. and Ida M. Green Professor of Structural Biology

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