NMR analysis of protein interactions

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Recent technological advances in NMR spectroscopy have alleviated the size limitations for the determination of biomolecular structures in solution. At the same time, novel NMR parameters such as residual dipolar couplings are providing greater accuracy. As this review shows, the structures of protein–protein and protein–nucleic acid complexes up to 50 kDa can now be accurately determined. Although de novo structure determination still requires considerable effort, information on interaction surfaces from chemical shift perturbations is much easier to obtain. Advances in modelling and data-driven docking procedures allow this information to be used for determining approximate structures of biomolecular complexes. As a result, a wealth of information has become available on the way in which proteins interact with other biomolecules. Of particular interest is the fact that these NMR-based methods can be applied to weak and transient protein–protein complexes that are difficult to study by other structural methods.

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.

Section snippets

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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