Scientific Report 2006

Molecular Biology

Nuclear Magnetic Resonance Spectroscopy in Protein Structural Biology and Structural Genomics

M. Almeida, W. Augustyniak, L. Columbus, M. Geralt, R. Horst, M. Johnson, B. Pedrini, W.J. Placzek, P. Serrano, K. Wüthrich

Our research program focuses on 2 areas. First, in a collaboration with A. Horwich, Yale University, New Haven, Connecticut, who is a visiting scientist at Scripps Research, we are investigating structural and mechanistic aspects of the function of GroE-type chaperonin systems in Escherichia coli. This research concerns the process of protein folding in healthy and diseased organisms and thus is directly related to the currently extensively discussed protein misfolding diseases. Because of the large size of the GroE-type supramolecular structures, this project depends on continuous improvement of existing solution nuclear magnetic resonance (NMR) techniques and development of new techniques.

Second, we develop and apply NMR methods for use in structural genomics. We participate in the Joint Center for Structural Genomics (JCSG), the JCSG Center for Innovative Membrane Protein Technologies, and the Consortium for Functional and Structural Proteomics of SARS-CoV–Related Proteins (FSPS). On the one hand, we explore the use of automated microscale NMR equipment for the screening of recombinant protein preparations for folded globular domains. On the other hand, we use NMR spectroscopy to determine the structures of selected proteins from the proteomes under study. The following sections highlight our research on proteins from the severe acute respiratory syndrome–associated coronavirus (SARS-CoV) proteome, which is pursued under the auspices of the FSPS (http://sars.scripps.edu) and the JCSG (http://www.jcsg.org).

The SARS-Cov Problem

In 2003, a major global outbreak of SARS was caused by a newly emerged coronavirus. The coronavirus genome is composed of a single plus-strand RNA of about 30 kb and is the largest genome among known RNA viruses. About two thirds of the genome is devoted to encoding the replicase polyprotein, which is cleaved by viral proteases to release the mature nonstructural proteins that perform enzymatic functions of the virus within the host cell. These functions include RNA-processing steps and other functions that are currently unknown. The genome also encodes viral structural proteins, which form part of the mature viral particle along with genomic RNA.

Several of the replicase proteins of coronaviruses have little or no apparent relationship to other known proteins, and little is known about how they function during viral infection. In addition, the reasons for the severe signs and symptoms caused by SARS-CoV in comparison with other human coronaviruses, which usually cause much less severe infections, are currently unknown. We are using NMR spectroscopy for structural and functional investigations of SARS-CoV proteins to gain information about the viral life cycle and to identify possible new antiviral strategies.

Nonstructural Protein 1

Nonstructural protein 1 (nsp1) is the leader protein of the SARS-CoV genome and the first to be translated and cleaved by the viral protease to its mature form. It has little apparent relationship to proteins of other coronaviruses and may perform a function unique to SARS-CoV. We used NMR spectroscopy to investigate the solution structure and dynamics of nsp1. The protein adopts a new 3-dimensional fold, with a distorted, 6-stranded β-barrel covered by an α-helix. This stable, folded globular domain carries a long, flexibly disordered polypeptide “tail” at the C terminus. We used bioinformatics techniques to search for local structural features that might provide insight into the functional properties of this protein. We detected a possible protease active site on one end of the β-barrel, indicating that nsp1 may be a previously unrecognized viral protease. Follow-up studies indicated that formation of a functional active site may require the presence of the long C-terminal tail, or of other protein cofactors.

Nonstructural Protein 3

The viral element nsp3 is a large protein of about 2000 amino acid residues that most likely includes multiple functional domains. We designed smaller constructs of this protein encompassing predicted individual domains, and used 1-dimensional ¹H NMR screening to identify those domains that were independently folded. We then determined the solution structure of the N-terminal domain, nsp3a. Unexpectedly, we found that its structure is similar to that of the α/β roll fold of ubiquitin. This structural motif is most commonly found in proteins of eukaryotes that are involved in cellular signaling pathways. Therefore, the nsp3a domain may be used by the virus to interact with signaling proteins of the host cell and to interfere with cellular pathways in order to increase virulence.

We are also investigating a possible second function of this protein in RNA processing. Using NMR spectroscopy and mass spectrometry, we identified RNA molecules that bind to nsp3a. We are studying these interactions to determine possible enzymatic or scaffolding functions.

Nonstructural Protein 7

The viral component nsp7 is highly conserved between the different coronaviruses and probably performs an essential core function in this virus family. Interestingly, the solution structure of nsp7 also shows a new fold that was not previously observed in any known protein structure. The structure consists of 4 helices, and although many 4-helix bundle proteins are known, nsp7 does not form a bundle. Rather 3 helices are assembled into a flat sheet, with the helices antiparallel, and the fourth helix is stacked across one side of this sheet (Fig. 1). The other surface of the flat sheet contains hydrophobic and negatively charged patches, which most likely are sites for protein-protein interactions. Currently, we are using NMR spectroscopy to identify interactions with other SARS-CoV proteins. Such interactions could be targeted for the design of inhibitory molecules with antiviral activity.

Fig. 1. Ensemble of 20 conformers representing the polypeptide backbone in the solution structure of SARS-CoV nsp7. The 3 helices α2, α3, and α4 assemble into a flat sheet, with the α1 helix stacked diagonally across one surface of this sheet. Reprinted with permission from Peti, W., et al. J. Virol. 79:12905, 2005. Copyright 2005 American Society for Microbiology.

Publications

Almeida, M.S., Herrmann, T., Peti, W., Wilson, I.A., Wüthrich, K. NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima: implications for 216 homologous DUF59 proteins. Protein Sci. 14:2880, 2005.

Columbus, L., Peti, W., Etezady-Esfarjani, T., Herrmann, T., Wüthrich, K. NMR structure determination of the conserved hypothetical protein TM1816 from Thermotoga maritima. Proteins 60:552, 2005.

Horst, R., Bertelsen, E.B., Fiaux, J., Wider, G., Horwich, A.L., Wüthrich, K. Direct NMR observation of a substrate protein bound to the chaperonin GroEL. Proc. Natl. Acad. Sci. U. S. A. 102:12748, 2005.

Peti, W., Herrmann, T., Zagnitko, O., Grzechnik, S.K., Wüthrich, K. NMR structure of the conserved hypothetical protein TM0979 from Thermotoga maritima. Proteins 59:387, 2005.

Peti, W., Johnson, M.A., Herrmann, T., Neuman, B.W., Buchmeier, M.J., Nelson, M., Joseph, J., Page, R., Stevens, R.C., Kuhn, P., Wüthrich, K. Structural genomics of the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance structure of the protein nsp7. J. Virol. 79:12905, 2005.

Peti, W., Page, R., Moy, K., O’Neil-Johnson, M., Wilson, I.A., Stevens, R.C., Wüthrich, K. Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR. J. Struct. Funct. Genomics 6:259, 2005.