Methodology for rigorous modeling of protein conformational changes by Rosetta using DEER distance restraints

Abstract

We describe an approach for integrating distance restraints from Double Electron-Electron Resonance (DEER) spectroscopy into Rosetta with the purpose of modeling alternative protein conformations from an initial experimental structure. Fundamental to this approach is a multilateration algorithm that harnesses sets of interconnected spin label pairs to identify optimal rotamer ensembles at each residue that fit the DEER decay in the time domain. Benchmarked relative to data analysis packages, the algorithm yields comparable distance distributions with the advantage that fitting the DEER decay and rotamer ensemble optimization are coupled. We demonstrate this approach by modeling the protonation-dependent transition of the multidrug transporter PfMATE to an inward facing conformation with a deviation to the experimental structure of less than 2Å Cα RMSD. By decreasing spin label rotamer entropy, this approach engenders more accurate Rosetta models that are also more closely clustered, thus setting the stage for more robust modeling of protein conformational changes.

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…The corresponding measurements are usually executed (i) during ∼12 h, (ii) at cryogenic temperatures (from 10 to 60 K, a recent trityl spin-label permitted DEER acquisition at 150 K), (iii) using Q-band (34 GHz) or even the less common W-band (94 GHz) spectrometers, (iv) on cellular samples of ∼50 μL (10–20 million mammalian cells are enough) containing down to submicromolar concentrations of doubly spin-labeled proteins/nucleic acids, preferably in deuterated buffers. − ,,− Hence, the measured distance distribution reports the ensemble of frozen conformations, independently of the size and location of the studied objects. The distance extraction resolves an ill-posed problem from noisy data, which can bias the distribution shape. ,,− The global accuracy depends also on the size and rigidity of the two paramagnetic tags. ,− …”

Integration of Nanometer-Range Label-to-Label Distances and Their Distributions into Modelling Approaches

“…A very small further improvement was achieved by shifting the domains by 1–3 Å. A similar approach implemented in RosettaEPR resulted in improved modeling of a conformational change of the transition from the outward to the inward state of the multidrug transporter PfMATE [ 76 ].…”

“…Therefore, a direct fit to the primary data can reduce the uncertainty. This strategy has been used for high-resolution docking of protomers in the dimer of the sodium/proton antiporter NhaA [ 78 ] and in high-resolution approaches involving reweighting of rotamer populations [ 75 , 76 ]. With such approaches, a good estimate for the distance distribution is available during background fitting.…”

Integration of Nanometer-range Label-to-label Distances and their Distributions into Modelling Approaches

“…A very small further improvement was achieved by shifting the domains by 1 -3 Å. A similar approach implemented in RosettaEPR resulted in improved modeling of a conformational change of the transition from the outward to the inward state of the multidrug transporter PfMATE [73].…”

“…Therefore, a direct fit to the primary ⃗ V data can reduce the uncertainty. This strategy has been used for high-resolution docking of protomers in the dimer of the sodium/proton antiporter NhaA [75] and in high-resolution approaches involving reweighting of rotamer populations [72,73]. With such approaches, a good estimate for the distance distribution is available during background fitting.…”