A refinement protocol to determine structure, topology, and depth of insertion of membrane proteins using hybrid solution and solid-state NMR restraints

Abstract

To fully describe the fold space and ultimately the biological function of membrane proteins, it is necessary to determine the specific interactions of the protein with the membrane. This property of membrane proteins that we refer to as structural topology cannot be resolved using X-ray crystallography or solution NMR alone. In this article, we incorporate into XPLOR-NIH a hybrid objective function for membrane protein structure determination that utilizes solution and solid-state NMR restraints, simultaneously defining structure, topology, and depth of insertion. Distance and angular restraints obtained from solution NMR of membrane proteins solubilized in detergent micelles are combined with backbone orientational restraints (chemical shift anisotropy and dipolar couplings) derived from solid-state NMR in aligned lipid bilayers. In addition, a supplementary knowledge-based potential, E (z) (insertion depth potential), is used to ensure the correct positioning of secondary structural elements with respect to a virtual membrane. The hybrid objective function is minimized using a simulated annealing protocol implemented into XPLOR-NIH software for general use.

Fig. 1

Overview of the hybrid refinement protocol for the simultaneous determination of structure and topology of membrane proteins. Step 1: Starting from an extended structure, the simulated annealing protocol minimizes a target function containing only solution NMR data (NOEs, torsion angles, and hydrogen bonds). Step 2: The orientational constraints derived from solid-state NMR are included together with solution NMR restraints to obtain the correct orientation along the Z direction. Step 3: Depth of insertion is determined using rigid body minimization in the presence of the depth of insertion potential, keeping the helical orientation with respect to Z fixed. The resulting structural ensemble is refined using low temperature simulated annealing

Fig. 2

a Definition of (θ, ρ) describing the orientation of helix with respect to membrane normal Z. (θ_{Ib, II}, ρ_Ib,II) are the tilt and rotation angles for the domain Ib,II helix, while (θ_Ia, ρ_Ia) are the tilt and rotation angles for the domain Ia helix. The interhelical angle between the two domains is described by χ. b Helical wheel representation of the reference orientation of domain Ia where ρ_Ia is defined to be zero. The N atom of T8 is aligned to +y axis. ρ_Ia rotates counterclockwise viewing from the top of y–z plane. At ~90 degrees, the hydrophilic residues in blue point into bulk solution. c Reference orientation of transmembrane domain where ρ_Ib,II is defined to be zero

Fig. 3

a Distribution of θ and ρ angles derived from domain Ia and domains Ib and II. b Distribution of interhelical angle χ and ρ_Ia and the comparison with the solution NMR ensemble

Fig. 4

Rigid-body minimization using the knowledge-based E_z potential. a Backbone cartoon representation of a selected PLN conformer before (upper panel) and after rigid-body minimization. b Comparison of the simulated CSA and DC before and after E_z minimization. The only discrepancies are due to residues located in the dynamic loop, which do not have CSA and DC restraints. c Representation of the E_z potential energy function for the domain Ib, II (blue) and domain Ia (red) helices. After minimization, both domains reside in the minima

Fig. 5

a Conformational ensemble (structures and topologies) representing the 20 lowest energy structures in the virtual bilayer. Domain Ia is colored in red, and domains Ib and II are in blue. Hydrophobic side chains of domain Ia are shown in grey. Structure overlay is performed by rotating along Z and translating along X and Y, resulting in no changes in E_z energy and PISEMA data. b Position of the cytoplasmic and transmembrane domains with respect to the depth of insertion E_z potential. c Top view of a. d Distribution of θ and ρ angles in the final structural ensemble

Fig. 6

a Comparison of the experimental and calculated CSA and DC for different values of the CSA tensor components shown in Table 1. The diagonal errors indicate ranges of ±5 ppm and ±0.5 kHz for CSA and DC, respectively. b Effects of the different CSA tensor components on the tilt and rotation angles for the helix defined by domains Ib and II of PLN. Tensors 1 (Wu et al. 1995), 2 (Page et al. 2008), and 3 are shown in red, black, and blue, respectively

Fig. 7

Effects of the resonance misassignment on the calculation of the structural topology of PLN. a Plots of the distribution of the rotation angles for the two ensembles obtained from simulated annealing calculations. The correct assignments give rise to an average rotation angle of 203°, while the incorrect assignment gives rise to an average rotation angle of 189°. b Experimental PISEMA spectra with the two equiprobable assignments for the isoleucine residues of PLN derived from the combinatorial assignment procedures (see text). c Histogram representing the number of violations obtained for residues 44 and 45 in the conformational ensemble generated with the incorrect assignments

Fig. 8

Projection of (θ, ρ) of the hybrid ensemble (20 lowest) for the two helical domains of PLN onto the PISEMA potential surfaces obtained using rigid helix fitting. Three different scoring functions are used to generate the potential surfaces: $\mathbf{a}{\text{score}}_1={\text{RMSD}}_{\text{DC}}+{\text{RMSD}}_{\text{CSA}}\times \frac{{\text{DC}}_{max}}{{\text{CSA}}_{max}},\text{b}{\text{score}}_2={\text{RMSD}}_{\text{DC}}\times \frac{{\text{CSA}}_{max}}{{\text{DC}}_{max}}+{\text{RMSD}}_{\text{CSA}}$ and $\text{c}{\text{score}}_3=\frac{{\text{RMSD}}_{\text{DC}}}{{\text{DC}}_{max}-{\text{DC}}_{min}}+\frac{{\text{RMSD}}_{\text{CSA}}}{{\text{CSA}}_{max}-{\text{CSA}}_{min}}$ These plots demonstrated that the topologies derived from the hybrid method correspond to the lowest energy minima identified in all of the PISEMA potential surfaces