Improved Segmental Isotope Labeling Methods for the NMR Study of Multidomain or Large Proteins: Application to the RRMs of Npl3p and hnRNP L

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Abstract

The study of multidomain or large proteins in solution by NMR spectroscopy has been made possible in recent years by the development of new spectroscopic methods. However, resonance overlap found in large proteins remains a limiting factor, making resonance assignments and structure determination of large proteins very difficult. In this study, we present an expressed protein ligation protocol that can be used for the segmental isotopic labeling of virtually any multidomain or high molecular mass protein, independent of both the folding state and the solubility of the protein fragments, as well as independent of whether the fragments are interacting. The protocol was applied successfully to two different multidomain proteins containing RNA recognition motifs (RRMs), heterogeneous nuclear ribonucleoprotein L and Npl3p. High yields of segmentally labeled proteins could be obtained, allowing characterization of the interdomain interactions with NMR spectroscopy. We found that the RRMs of heterogeneous nuclear ribonucleoprotein L interact, whereas those of Npl3p are independent. Subsequently, the structures of the two RRMs of Npl3p were determined on the basis of samples in which each RRM was expressed individually. The two Npl3p RRMs adopt the expected βαββαβ fold.

Introduction

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for the determination of protein structures in solution as well as for the investigation of protein dynamics, protein–protein and protein–ligand interactions. Currently, however, only proteins with molecular mass of up to ∼30 kDa can be studied structurally by NMR spectroscopy. Large proteins in solution show severe NMR line-broadening owing to slow tumbling and a high signal overlap as the result of the large number of resonances. The introduction of transverse relaxation-optimized spectroscopy1., 2. helped the scope of solution NMR to be extended. Also, problems of overlapping NMR signals can be reduced by isotope labeling by amino acid type,3., 4., 5. or by the recent stereo-array isotope labeling of amino acids.6 However, specific labeling of amino acid residues does not allow protein sequential resonance assignments and stereo-array isotope labeling of amino acids is limited to translation in vitro. An alternative solution for the study of large proteins by NMR would be to isotopically label segments or domains of a large protein. There are two methods that have been used successfully for segmental isotope labeling of proteins: expressed protein ligation (EPL),7., 8., 9., 10. and protein trans-splicing (PTS).11., 12. Segmental isotope labeling of proteins simplifies the structure determination of large proteins, and can provide valuable structural and biological information about multidomain proteins. Segmental isotope labeling allows one to determine whether protein domains interact with each other and helps to define their precise interface, or whether protein domains do not interact and therefore can be structurally (and biologically) characterized independently.

EPL is based on the reaction originally employed in Native Chemical Ligation,13., 14. where a C-terminal α-thioester of one peptide reacts with an N-terminal cysteine residue of the second peptide, resulting in the formation of a native peptide bond.8 However, the synthesis of large peptides has not been reported, as such peptides cannot be synthesized reliably with sufficient yields. EPL is a more convenient approach for generating large protein fragments for ligation.7., 8., 15., 16., 17. EPL relies on bacterial expression of individual fragments in fusion with an intein. Inteins catalyze their own excision, yielding protein fragments with reactive termini optimal for protein ligation. Their self-splicing properties have great value for various applications in protein chemistry, and several inteins are commercially available in systems used for intein-mediated purification of proteins. The IMPACT™-TWIN system introduced by New England Biolabs uses mini-inteins derived from the Synechocystis sp dnaB gene (Ssp DnaB),18 from the Mycobacterium xenopi gyrA gene (Mxe GyrA),19 and from the Methanobacterium thermoautotrophicum rir1 gene (Mth RIR1).20 The Ssp DnaB intein has been engineered to undergo pH or temperature-dependent cleavage at its C terminus, generating protein fragments with the desired N-terminal amino acid residue.21 The Mxe GyrA and Mth RIR1 inteins have been modified to undergo thiol-inducible cleavage at their N terminus, yielding the C-terminal thioester.22 The designed inteins of the IMPACT™-TWIN system thus provide a simple solution for segmental isotope labeling of proteins by EPL. PTS differs from EPL, as it is based on the reconstitution of an intein from two split intein fragments fused to an N and a C extein. After reconstitution, the intein excises itself, resulting in the ligation of the two exteins.8

The successful application of EPL and PTS for segmental isotope labeling of proteins has been reported for several NMR studies. Yamazaki and co-workers segmentally labeled the C-terminal domain of the RNA polymerase α subunit using a trans-splicing system based on the split PI-PfuI intein.12 More recently, the same group reported the segmental labeling of the 52 kDa β subunit of the F1-ATPase, which was used successfully in combination with transverse relaxation-optimized spectroscopy to obtain its backbone resonance assignment, followed by valuable conformational studies.11 Similarly, the potential application of EPL for NMR was demonstrated in subsequent studies. Xu and co-workers chemically ligated two folded recombinant domains (SH2 and SH3) of Abelson protein tyrosine kinase using an intein fusion to generate a C-terminal α-thioester derivate of Abl-SH3 and a factor Xa proteolysis to generate the N-terminal cysteine residue of Abl-SH2 for the ligation reaction.23 Camarero et al used EPL for segmental isotope labeling of two σA factor regions to investigate whether their interaction leads to autoinhibition, i.e. the abolishment of its binding to DNA.10 More recently, our group used EPL to investigate the interactions between the two C-terminal RNA recognition motifs (RRMs) of PTB.24 There, we developed an EPL protocol using an on-column ligation step that allowed us to obtain a very high yield. The segmentally labeled samples were used for a structural investigation of the interactions between the two RRMs, and for the determination of their precise structure.24 Other studies have contributed to the improvement of segmental isotope labeling techniques by finding more convenient ways to perform the ligation reaction in order to increase its yield and to simplify the protocol.24., 25. Significant effort was invested in the development of techniques resulting in the ligation of several protein fragments.9., 26., 27., 28. These works demonstrate the potential of EPL and PTS for the NMR study of large proteins in reducing the signal overlap as well as giving the possibility to investigate the functional properties of the proteins (conformational changes upon the ligand binding) or multidomain proteins by NMR.

Here, we report further improvement of our on-column EPL protocol (Figure 1) and its application to the study of the interactions of two multidomain proteins, hnRNP L and Npl3p. With the three different ligation reactions reported here, we show that EPL can be generally applied to any multidomain proteins, independent of whether the domains interact, are soluble or are folded. These results emphasize the usefulness of this method for the segmental labeling of any given polypeptide chain and their study by NMR. Furthermore, we found that the RRMs of heterogeneous nuclear ribonucleoprotein L (hnRNPL) interact, whereas those of Npl3 are independent. Subsequently, the structures of the two RRMs of Npl3p were determined from samples in which each RRM was expressed individually.

Section snippets

Segmental isotope labeling of two insoluble fragments: hnRNPL RRM3 and RRM4

The hnRNPL is a very abundant RNA-binding protein known to be involved mainly in alternative splicing and degradation of mRNA.29 hnRNP L contains four RRMs (Figure 2(a)). Preliminary studies carried out in our laboratory indicated that RRM3 and RRM4 might interact with each other despite the long linker between the two RRMs (417–461, Figure 2). In order to characterize this interaction structurally by NMR, we decided to segmentally label the RRM34 fragment using our EPL protocol (Figure 1).24

Discussion

Earlier, we described the successful segmental isotopic labeling of two soluble, folded and interacting domains using an on-column EPL protocol.2., 4. To fully exploit the potential of EPL for protein NMR, the EPL protocol must be sufficiently robust to enable ligation of proteins exhibiting less favorable properties. To make EPL as broadly applicable as possible, we have improved our protocol (Figure 1) to allow ligation of insoluble, non-interacting and misfolded domains.24 EPL was applied to

General methods

Ligated Npl3p RRM12 193 and RRM12 211 products were purified by size-exclusion chromatography using a Superdex™ 75 10/300 GL column at a flow-rate of 0.5 ml/min in 50 mM sodium phosphate (pH 7), 200 mM NaCl. SDS-PAGE was used for analysis of the expression and solubility of hnRNPL and Npl3p constructs, as well as estimation of the intein cleavage and ligation efficiencies. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis of all constructs was

Acknowledgements

This investigation was performed within the Structural Biology National Center of Competence in Research (NCCR) ISO-laboratory co-directed by Professor K. Wüthrich and Professor F. Allain. The investigation was supported by grants from the Swiss National Science Foundation and the ETH Zurich through the NCCR Structural Biology and by the Roche Research Fund for Biology at the ETH Zurich to F.H.T.A., who is an EMBO Young Investigator.

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