Featured Technology Archive
Leveraging the Power of Molecular Fragment Replacement to Solve Protein Structures by NMR
The protein structures deposited in the PDB during the Protein Structure Initiative (PSI) have been determined by x-ray crystallography and NMR spectroscopy. While
the majority of these structures were solved using x-ray crystallography, nearly 500 were solved using NMR. NMR spectroscopists determine protein structure by
assigning chemical shifts to the backbone atoms and deriving distance and global structural information from nuclear Overhauser enhancements (NOEs). In some cases,
additional information is needed to accurately solve the three-dimensional protein structure such as experimentally determined distance or orientation restraints.
To enhance this information, the experimental data can be used to search the PDB for compatible regions of structures and build a 3D structure model in a method
called molecular fragment replacement (MFR). MFR was first employed in x-ray crystallography where molecular fragments were built to fit experimentally determined
electron density (Jones and Thirup, 1986). The first application to NMR was demonstrated by in the laboratory of Ad Bax (Delaglio F, et al., 2000). Within the PSI, it has been applied
to solving NMR structures of water-soluble proteins in conjunction with CS (chemical shift)-Rosetta
by a collaborative
effort of the PSI:Biology High-Throughput Center Northeast Structural Genomics
consortium (NESG; Guy Montelione, PI) in 2008
(Shen, et al., 2008).
This application was originally proposed as a way to automate structure determination, and therefore to
decrease the time from target selection to structure determination. Building on their previous work, NESG incorporated orientation restraints - residual dipolar
couplings (RDCs) - into the CS-Rosetta structure prediction method and used it to successfully solve structures with backbone-only NMR data
(Raman, et al., 2010).
More recently, one of the PSI:Biology Membrane Centers, Membrane Protein Structures by Solution NMR
(MPSbyNMR), under the direction of James Chou, PhD, at Harvard Medical School, combined NMR and MFR to solve the structure of the 300-residue transmembrane receptor murine mitochondrial uncoupling protein 2 (UCP2; Berardi MJ et al., 2011).
In this study
, the UCP2 sample was reconstituted in micelles for solution NMR experiments. Typically, NOEs are
assigned, but in this case, it was difficult to gather enough unambiguous NOEs due to backbone resonance overlap. Further orientation information was needed, so RDCs
were measured. Using this experimental data, researchers employed the MFR method, searching the Protein Data Bank to construct a database containing over 300,000
seven-residue fragments and selecting the ones with the best fit. These fragments were then used to determine backbone structure using a four-step procedure to
assign corresponding protein segments without overlapping, filling gaps, and extending the fragments. Fifteen structured segments were identified through this
molecular fragment searching method and used in the subsequent structure calculations. These data, in addition to experimentally determined distance restraints,
allowed the researchers to solve the solution structure of this protein and gain information about the region of substrate binding to this integral membrane protein.
The successful application of this technique to the study of this large transport protein will enable its use to discover the mechanisms of other membrane-embedded
transporters and translocators. In the future, researchers hope to capitalize on the power of NMR and MFR to gain insight into the biological function of these
molecules by probing the conformational changes coupled to substrate transport.
Berardi MJ, et al. “Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching.” Nature 476:109-114 (2011).
Delaglio F, Kontaxis G, Bax A. "Protein structure determination using molecular fragment replacement and NMR dipolar couplings." J. Biomol. NMR 38:289-302 (2000).
Jones TA and Thirup S. “Using known substructures in protein model building and crystallography.” EMBO J. 5:819-22 (1986).
Raman S, et al. “NMR structure determination for larger proteins using backbone-only data.” Science 327:1014-1018 (2010).
Shen Y, et al., “Consistent blind protein structure generation from NMR chemical shift data.” PNAS 105:4685-4690 (2008).
NMR solution structure of ALG13: The sugar donor subunit of a yeast N-acetylglucosamine transferase. Northeast Structural Genomics
Consortium target YG1.
Structure of the mitochondrial uncoupling protein 2 determined by NMR molecular fragment replacement. 2LCK DOI:10.2210/pdb2lck/pdb