Increasing the accuracy of solution NMR structures of membrane proteins by application of residual dipolar couplings. High-resolution structure of outer membrane protein A

Abstract

The structure determination of membrane proteins is one of the most challenging applications of solution NMR spectroscopy. The paucity of distance information available from the highly deuterated proteins employed requires new approaches in structure determination. Here we demonstrate that significant improvement in the structure accuracy of the membrane protein OmpA can be achieved by refinement with residual dipolar couplings (RDCs). The application of charged polyacrylamide gels allowed us to obtain two alignments and accurately measure numerous heteronuclear dipolar couplings. Furthermore, we have demonstrated that using a large set of RDCs in the refinement can yield a structure with 1 A rms deviation to the backbone of the high-resolution crystal structure. Our simulations with various data sets indicate that dipolar couplings will be critical for obtaining accurate structures of membrane proteins.

Figure 1

Stereo-figure showing comparison of ten lowest energy conformers of OmpA (green and gray) and the high-resolution crystal structure (red). The structured fragment of OmpA including residues 5-15, 35-55, 77-100, 122-142, 162-170 is shown in green.

Figure 2

Comparison of structures of the three periplasmic turns in the refined structure of OmpA and the high-resolution crystal structure (red). The two conformations observed for turns 1 and 3 (A and C, respectively) are shown in blue and green.

Figure 3

Conformational heterogeneity revealed by comparison of anisotropic couplings measured for OmpA in a 50+M gel. A) spectral region of ¹H-¹⁵N TROSY-HSQC showing major and two minor peaks for Tyr129; B and C) comparison of anisotropic ¹J_HN couplings for three peaks of Tyr129; D and E) comparison of ¹J_C′Cα couplings for major (D) and minor peak (E) for Met53; F and G) comparison of ¹J_NC′ couplings for Met53.

Figure 4

Effect of the number of restraints on precision and accuracy of OmpA structure. Comparison of high-resolution crystal structure of OmpA (green) and 10 lowest energy structures calculated with different data sets. Structured part of the protein is shown in blue and periplasmic turns are in red. Structures have been calculated using A) distance restraints and dihedral angles; precision 1.82 ± 0.22 Å, accuracy 2.94 ± 0.18 Å; B) distances, dihedral angles and RDCs; precision 0.90 ± 0.20 Å, accuracy 1.92 ± 0.15 Å; C) distance restraints, hydrogen bonds and dihedral angles; precision 1.10 ± 0.15 Å, accuracy 1.66 ± 0.11 Å; D) distance restraints, hydrogen bonds, dihedral angles and RDCs; precision 0.48 ± 0.09 Å, accuracy 1.02 ± 0.02 Å. Precision is calculated as backbone r.m.s. deviations for backbone atoms between 10 lowest energy structures and accuracy is calculated as r.m.s. deviations between 10 lowest energy structures and the crystal structure.