Martin Billeter

Prion (PrPSc)-specific epitope defined by a monoclonal antibody

Prions are infectious particles causing transmissible spongiform encephalopathies (TSEs). They consist, at least in part, of an isoform (PrPSc) of the ubiquitous cellular prion protein (PrPC). Conformational differences between PrPCand PrPScare evident from increased β-sheet content and protease resistance in PrPSc(refs 1,2,3). Here we describe a monoclonal antibody, 15B3, that can discriminate between the normal and disease-specific forms of PrP. Such an antibody has been long sought as it should be invaluable for characterizing the infectious particle as well as for diagnosis of TSEs such as bovine spongiform encephalopathy (BSE) or Creutzfeldt–Jakob disease (CJD) in humans. 15B3 specifically precipitates bovine, murine or human PrPSc, but not PrPC, suggesting that it recognizes an epitope common to prions from different species. Using immobilized synthetic peptides, we mapped three polypeptide segments in PrP as the 15B3 epitope. In the NMR structure of recombinant mouse PrP, segments 2 and 3 of the 15B3 epitope are near neighbours in space, and segment 1 is located in a different part of the molecule. We discuss models forthe PrPSc-specific epitope that ensure close spatial proximity of all three 15B3 segments, either by intermolecular contacts in oligomeric forms of the prion protein or by intramolecular rearrangement.

Protein - DNA contacts in the structure of a homeodomain - DNA complex determined by nuclear magnetic resonance spectroscopy in solution

The 1:1 complex of the mutant Antp(C39––S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three‐dimensional structure were based on the use of the fully 15N‐labelled protein, two‐dimensional homonuclear proton NOESY with 15N(omega 2) half‐filter, and heteronuclear three‐dimensional NMR experiments. Based on nearly complete sequence‐specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H‐1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix‐turn‐helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix‐turn‐helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39––S) homeodomain no defined spatial structure could be found for the apparently flexible N‐terminal segment comprising residues 0‐6.

The structure of the homeodomain and its functional implications

The three-dimensional structure of the homeodomain, as determined by nuclear magnetic resonance spectroscopy, reveals the presence of a helix-turn-helix motif, similar to the one found in prokaryotic gene regulatory proteins. Isolated homeodomains bind with high affinity to specific DNA sequences. Thus, the structure-function relationship is highly conserved in evolution.

Optimizing resolution in multidimensional NMR by three-way decomposition.

Resolution depends on the number of points sampled in a FID; in indirectly detected dimensions it is an important determinant of the total experiment time. Based on the high redundancy present in NMR data, we propose the following timesaving scheme for three-dimensional spectra. An extensive grid of discrete t1- and t2-values is used, which increases resolution while preserving the spectral width. Total experiment time is reduced by avoiding the recording of t3-FIDs for selected pairs of t1 and t2; typically the recording is omitted for about 75% of the (t1,t2) combinations. These data sets are referred to as sparse, and post-experimental processing making optimal use of spectral redundancy provides the missing, non-recorded data. We have previously shown that three-way decomposition (TWD) within the MUNIN approach provides a practical way to process dense NMR data sets. Here, a novel TWD algorithm [Ibraghimov, (2002) Numer. Linear Algebra Appl.9, 551–565] is used to complement a sparselyrecorded time-domain data set by providing the missing FIDs for all (t1,t2) combinations omitted in the experiment. A necessary condition is that for each t1-value at least a few FIDs are recorded, and similar for each t2-value. The method is demonstrated on non-uniformly sampled 15N-NOESY-HSQC data sets recorded for the 14xa0kD protein azurin. The spectra obtained by TWD, reconstruction and ordinary transform to frequency-domain are, in spite of the large number of signals and the high dynamic range typical for NOESYs, highly similar to a corresponding reference spectrum, for which all (t1,t2) combinations were recorded.

Measles virus phosphoprotein retains the nucleocapsid protein in the cytoplasm

Measles virus (MV) proteins were efficiently expressed in COS and Vero cells from vectors based on the strong cytomegalovirus enhancer-promoter and the simian virus 40 origin of replication. When expressed alone, nucleocapsid protein (N) migrates predominantly into the nucleus whereas phosphoprotein (P) is located in the cytoplasm. Coexpression of N and P proteins results in retention of the N protein in the cytoplasm, as seen also in infected cells. The retention of N protein is due to specific interactions with the P protein since coexpression of N with either the matrix or the hemagglutinin protein had no effect. Mapping of the regions of N-P interactions on P protein revealed that the carboxy-terminal 40% of P was sufficient for specific binding to N; however, the carboxy-terminal 60% of P was required for retention of N in the cytoplasm. Thus, the V and C proteins encoded within the first half of the P gene are not involved in the cytoplasmic retention of N protein. N protein might be fortuitously targeted to the nucleus as a result of its many basic amino acids, presumably destined to interact with the MV genome. However, this set of experiments has allowed to analyze in vivo the interactions between the N and P proteins.

MUNIN: A new approach to multi-dimensional NMR spectra interpretation

A new method, MUNIN (Multi-dimensional NMR spectra interpretation), is introduced for the automated interpretation of three-dimensional NMR spectra. It is based on a mathematical concept referred to as three-way decomposition. An NMR spectrum is decomposed into a sum of components, with each component corresponding to one or a group of peaks. Each component is defined as the direct product of three one-dimensional shapes. A consequence is reduction in dimensionality of the spectral data used in further analysis. The decomposition may be applied to frequency-domain or time-domain data, or to a mixture of these. Features of MUNIN include good resolution in crowded regions and the absence of assumptions about line shapes. Uniform sampling of time-domain data, a prerequisite for discrete Fourier transform, is not required. This opens an avenue for the processing of NMR data that do not follow oscillating behaviour, e.g. from relaxation measurements. The application of MUNIN is illustrated for a 1H-15N-NOESY-HSQC, where each component is defined as the set of all NOE peaks formed by a given amide group. As a result, the extraction of structural information simply consists of one-dimensional peak picking of the shape along the NOE-axis obtained for each amide group.

Solution NMR structure determination of proteins revisited

This ‘Perspective’ bears on the present state of protein structure determination by NMR in solution. The focus is on a comparison of the infrastructure available for NMR structure determination when compared to protein crystal structure determination by X-ray diffraction. The main conclusion emerges that the unique potential of NMR to generate high resolution data also on dynamics, interactions and conformational equilibria has contributed to a lack of standard procedures for structure determination which would be readily amenable to improved efficiency by automation. To spark renewed discussion on the topic of NMR structure determination of proteins, procedural steps with high potential for improvement are identified.

Comparison of protein structures determined by NMR in solution and by X-ray diffraction in single crystals.

Following the first determinations of protein structures in the late 1950s and the early 1960s (see for example Kendrew et al. 1960; Perutz, 1964), the three-dimensional structures of several hundred proteins have been elucidated by X-ray diffraction on single crystals. By the end of 1991, approximately 150 entries of proteins with substantially different sequences and a well resolved structure (Hobohm et al. 1992) were deposited in the Protein Data Bank (Bernstein et al. 1977; Abola et al. 1987). In addition, many structures of homologous proteins or of mutants have been described, bringing the total number of entries to about 600. While it was soon accepted that almost all of these structures do indeed give a correct picture of the fold of the active protein in spite of the non-physiological environment of single crystals, it is not clear to what extent structural details are reliably described by these structures. In particular the surface of a protein may be modified due to the dense packing of protein molecules in the crystal lattice. A detailed knowledge of the protein surface is, however, essential for the understanding of the function of the protein.

MUNIN: Application of three-way decomposition to the analysis of heteronuclear NMR relaxation data**

MUNIN (Multidimensional NMR Spectra Interpretation), a recently introduced approach exploiting the mathematical concept of three-way decomposition, is proposed for separation and quantitative relaxation measurements of strongly overlapped resonances in sets of heteronuclear two-dimensional spectra that result from typical relaxation experiments. The approach is general and may also be applied to sets of two-dimensional spectra with arbitrary modulation along the third dimension (e.g., J-coupling, diffusion). Here, the method is applied for the analysis of 15N rotating frame relaxation data.

Fully automated sequence-specific resonance assignments of hetero- nuclear protein spectra

Full automation of the analysis of spectra is a prerequisite for high-throughput NMR studies in structural or functional genomics. Sequence-specific assignments often form the major bottleneck. Here, we present a procedure that yields nearly complete backbone and side chain resonance assignments starting from a set of heteronuclear three-dimensional spectra. Neither manual intervention, e.g., to correct lists obtained from peak picking before feeding these to an assignment program, nor protein-specific information, e.g., structures of homologous proteins, were required. By combining two earlier published procedures, AUTOPSY [Koradi etxa0al. (1998) J. Magn. Reson., 135, 288–297] and GARANT [Bartels etxa0al. (1996) J. Biomol. NMR, 7, 207–213], with a new program, PICS, all necessary steps from spectra analyses to sequence-specific assignments were performed fully automatically. Characteristic features of the present approach are a flexible design allowing as input almost any combination of NMR spectra, applicability to side chains, robustness with respect to parameter choices (such as noise levels) and reproducibility. In this study, automated resonance assignments were obtained for the 14 kD blue copper protein azurin from P.xa0aeruginosa using five spectra: HNCACB, HNHA, HCCH-TOCSY, 15N-NOESY-HSQC and 13C-NOESY-HSQC. Peaks from these three-dimensional spectra were filtered and calibrated with the help of two two-dimensional spectra: 15N-HSQC and 13C-HSQC. The rate of incorrect assignments is less than 1.5% for backbone nuclei and about 3.5% when side chain protons are also considered.