NMR structures of membrane proteins in phospholipid bilayers

Abstract

Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins, especially those accomplished with the proteins in phospholipid bilayer environments where they function.

Figure 1

Three views of the structure of the membrane-bound form of fd coat protein in POPC/POPG phospholipid bilayers. The amphipathic in-plane helix is in magenta, the hydrophobic trans-membrane helix is in blue, and the short connecting turn is in yellow. The flexible N- and C- terminal residues are not shown. A. Side view of TM helix, the Trp and Lys sidechains are shown in blue. The dashed gray lines mark the lipid-water boundary. B. Front view and, C. View of in-plane helix looking down from the C-terminus. From Marassi & Opella (2003).

Figure 2

Structures of the Pf1 coat protein. A. and B. The membrane-bound form of the protein. C. and D. The structural form in the intact bacteriophage particle. In the membrane-bound form of the protein, the acidic N-terminus region is exposed to the bacterial periplasmic space (peri) and the basic C-terminal region is exposed to the cytoplasm (cyto). Acidic residues (Asp and Glu) are shown in red, conserved glycine residues in the transmembrane helix are in yellow, basic residues (Arg and Lys) are in blue, and interfacial tyrosines are in pink. Residues R44 and K45 face the cytoplasm in the membrane-bound form and the DNA on the interior of the bacteriophage particles. B. and D. Images obtained by 90^o rotation of A and C around the z axis. From Park et al (2010).

Figure 3

Top and side views of a model of the AChM2 funnel-like pentameric bundle. The channel architecture was calculated using the three-dimensional coordinates of the M2 helix in the lipid bilayer based on restraints from solid-state NMR experiments, and by imposing a symmetric pentameric organization. The top view has the C-terminal synaptic side in front. The wide mouth of the funnel is on the N-terminal, intracellular side of the pore. Middle view shows the side chains from the pore lining residues of Glu1, Ser8, Val15, Leu18, and Gln22. Bottom view contour depicts the pore profile. From Opella et al (1999).

Figure 4

Structural models of the trans-membrane segment of Vpu in a tetramer. The structure was determined by OS solid-state NMR in aligned, stationary phospholipid bilayers. Views of the minimum energy configuration of the tetrameric model; view from above (top) and side view (bottom) with Trp22 protruding out towards lipid head groups. From Park et al, (2003).

Figure 5

Ensembles of NMR structures of the nonphosphorylated form of phospholamban as pentamer with its amphipathic helix in the plane of the membrane bilayer. Top: View along membrane normal, Middle: view of residues 1-17, and Bottom: view perpendicular to membrane normal. From Vostrikov et al, (2013).

Figure 6

NMR structure of rabbit microsomal cytb5. NMR structure of full-length cytb5 obtained from a combined solution and solid-state NMR approach. The soluble heme domain structure (residues 1–104) of full-length cytb5 was solved in DPC micelles by solution NMR. The transmembrane domain structure (residues 106–126) of full-length cytb5 was determined in aligned DMPC/DHPC bicelles using solid-state NMR spectroscopy. From Ahuja et al (2013).

Figure 7

Structure of M2 tetrameter determined by OS solid-state NMR in phospholipid bilayers. Ribbon diagram of the amphipathic and transmembrane helices. From Sharma et al (2010).

Figure 8

Structure of MerF in phospholipid bilayers determined in 14-O-PC lipid bilayers using solid state NMR. From Lu et al (2013).

Figure 9

Structure of p7 in phospholipid bilayers.

Figure 10

Structure of DsbB determined using a combinatorial approach employing x-ray crystallography, solid state NMR, and MD simulations. The UQ cofactor and bond between Tyr153 and Glu26 are shown. From Tang et al (2013).

Figure 11

Solid-state NMR structure of Anabaena sensory rhodospsin. Side view of monomeric protein with retinal shown in orange. From Wang et al (2013a).

Figure 12

NMR structure of the GPCR CXCR1 in phospholipid bilayers determined using RA solid state NMR. From Park et al (2012).