NMR analysis of protein interactions
Introduction
In recent years, we have seen great improvements in NMR spectroscopy as a tool for the study of biomolecular interactions. The sensitivity of the technique has been enhanced by the advent of high-field spectrometers (900 MHz) and cryogenic probes. Novel methodologies (transverse relaxation-optimized spectroscopy (TROSY) [1]; residual dipolar couplings [2, 3]) have enabled the structural analysis of larger molecules and complexes. Also, better use can now be made of spectral information such as chemical shift perturbations (CSPs) through improved modelling and docking procedures. Consequently, it is possible to characterize larger and biologically more relevant biomolecular complexes with higher accuracy. In this review, we discuss recent advances in NMR structural studies of the interactions of proteins with other proteins, RNA and DNA. Interactions with small molecules and peptides is not included, as these have been adequately covered in other reviews [4, 5].
Biomolecular complexes reported since 2003 range from full de novo determined structures calculated from NMR-derived restraints (such as nuclear Overhauser effects (NOEs) and residual dipolar couplings (RDCs)) to models obtained by docking individual components based on knowledge of the interface from chemical shift perturbations. Although in the latter case the coordinate accuracy is necessarily lower, the information on the interaction gained is often quite important in guiding subsequent research.
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Methodological developments
Next to the ‘classical’ approach based on the use of intermolecular NOEs, in combination with RDCs when available [6], the characterization of protein interactions has greatly benefited from the incorporation of interface mapping information in the computational modelling of complexes. NMR is particularly powerful in mapping interfaces, allowing the study of weak and transient complexes that can be very difficult to study by other experimental techniques.
The use of CSP data obtained from NMR
Protein–protein interactions
NMR studies of protein–protein complexes have varied from full structure determination to NMR-filtered docking and modeling using interface information. We limit our discussion mainly to complexes for which the atomic coordinates have been deposited into the Protein Data Bank (PDB; http://www.rcsb.org) (see Table 1).
Since 2003, the structures of 14 protein–protein complexes have been solved using intermolecular NOEs detected from 13C/15N-filtered NOESY experiments in combination with RDCs when
Protein–RNA interactions
One of the most abundant RNA binding motifs with an occurrence of 1.5–2% in the human genome is the RNA recognition motif (RRM) recently reviewed in [43, 44]. RRMs are often found as tandem repeats within a protein together with other domains. Specificity mainly comes from direct interactions between the RNA bases and the protein side chains and main chains. Recently, the complex of the two N-terminal domains RRM1 and RRM2 of nucleolin with the nucleolin responsive element (b2NRE) was solved by
Protein–DNA interactions
A structure has been reported for the ternary complex of the POU domain of Oct1, Sox2 and the Hoxb1-DNA regulatory element [56••]. A modular approach was used to build the structure based on the binary DNA complexes of POU and Sox2 (or rather the homologous SRY). The structure (shown in Figure 3) was refined by extensive use of RDCs. Both transcription factors co-interact while bound to adjacent sites at the DNA. Comparison with various other regulatory sites sheds light on the mechanism of
Conclusions
It is clear that NMR has become a powerful and versatile method for the study of biomolecular interactions. Several spectacular protein–protein and protein–nucleic acid complexes in the molecular weight rang 40–50 kDa have been solved, and larger assemblies are within reach. The identification of interaction surfaces by relatively simple NMR experiments such as 15N-HSQC is becoming very popular. The resulting chemical shift perturbations (or any information related to the interaction) can now be
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Dr Peter Wright (Scripps Institute) and Dr Marius Clore (NIH) for providing the material for Figure 2, Figure 3, respectively.
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2018, Methods in EnzymologyCitation Excerpt :This chapter has been written for the non-NMR expert and focuses on the use of NMR spectroscopy to study IDP complexes where disorder is at least partly retained. Thus, we will not go into details in cases where complete folding upon binding is achieved, as relevant and well-written reviews and textbooks are available (see Bonvin, Boelens, & Kaptein, 2005; Gronenborn & Clore, 1995; Hass & Ubbink, 2014; Schieborr et al., 2005; Thompson, Beck, & Campbell, 2015). Likewise, NMR methods for the study of free IDPs have been well described (Brutscher et al., 2015; Jensen et al., 2009; Jensen, Zweckstetter, Huang, & Blackledge, 2014; Konrat, 2014).
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