Technical Highlight - June 2011
Short description: A new method from the Rosetta suite allows for the determination of symmetric oligomeric protein structures in solution using primarily RDCs and chemical shifts.
Most proteins function in the cell as part of large symmetric oligomeric structures. However, structural characterization of these complexes using solution NMR methods has been limited by a number of factors. High-molecular weight complexes have slower tumbling times, limiting the resolution of NMR spectra. Their large size also complicates the use of NOE methods to derive interface restraints, and their structural symmetry gives rise to poor spectral quality and makes it difficult to distinguish whether resulting crosspeaks are due to intra- or intermolecular interactions.
Previous methods for examining dimeric or higher-order symmetric complexes relied upon distance restraints provided by interface NOEs or chemical shift mapping data (which can be ambiguous), as well as small-angle X-ray scattering (SAXS) experiments or residual dipolar couplings (RDCs) data. When distance restraints have been unavailable, docking studies using experimental RDC data and structural models of the protein monomer can be performed, but they are challenging and error-prone. In addition, such studies need a previously determined crystal or solution structure.
Rosetta, the de novo structural prediction algorithm, can symmetrically dock oligomers of varying size and topology provided the structure of a monomeric subunit is known. Its fold-and-dock protocol can also model interleaved topologies of large symmetric complexes. RosettaOligomers, a new, automated method presented by Sgourakis and colleagues (PSI NESG), is an extension of Rosetta that can determine the structure of symmetric protein systems, intertwined or not, without the need for a structural model. RosettaOligomers uses the CS-Rosetta protocol to first produce a model ensemble for a monomeric subunit based upon backbone chemical shift assignments. RDC restraints are then applied to dock the subunits in the complex. If no model convergence is achieved, suggesting an interleaved topology, the fold-and-dock protocol is used, guided by the chemical shift and RDC data.
The authors evaluated RosettaOligomers against several symmetric dimer systems whose structures were previously determined, as well as against the p53 tetramer, finding their structural models to agree with the experimentally determined ones. For larger symmetric systems, they expect that additional data provided by sparse NOEs or SAXS would help determine the correct native oligomeric structure. This methodology should be a useful tool in high-throughput structural genomics initiatives.
N.G. Sgourakis et al. Determination of the structures of symmetric protein oligomers from NMR chemical shifts and residual dipolar couplings.
J. Am. Chem. Soc. 133, 6288-6298 (2011). doi:10.1021/ja111318m