research advances

Membrane protein structures enter an exponential growth phase

SBKB [doi:10.1038/fa_sbkb.2010.39]

Adrenergic receptors, ion channels and membrane transport proteins are among the membrane proteins whose structures have recently been determined.

Pentameric structure of the membrane protein FocA (PDB 3KLY), viewed from the periplasm6

The difficulty of determining the structure of membrane proteins means that the rate of submission of such structures to the Protein Data Bank lags far behind that of soluble proteins. But the exponential growth in membrane protein structures over the past few years is now rising to a level at which papers reporting new structures appear almost every month. Recent research by the Protein Structure Initiative (PSI) funded by the National Institute of General Medical Sciences (NIGMS) has contributed to the solution of representative protein structures from several important membrane protein families, particularly structures of human membrane proteins, and to the determination of more structures at resolutions better than 3.0 Å.

G-protein-coupled receptors (GPCRs) are the targets for more than half the current drug therapies and, until recently, the only high-resolution structural model available for this large family was bovine rhodopsin. Structures of GPCRs will aid in drug discovery and in the development of more specific drugs, as well as increase our basic understanding of molecular recognition and signaling. Of particular note are structures of human GPCRs from the adrenergic class, which are targeted by cardiac and asthma medications. Cherezov et al. 1 have determined the crystal structure of the β2-adrenergic receptor, a member of the class A adrenergic family which is targeted by cardiac and asthma medications, in a tour de force that used many strategies pioneered in the PSI, the National Institutes of Health Roadmap, and elsewhere. In a relatively quick follow-up, Jaakola et al. 2 have solved the crystal structure of the A2A adenosine receptor bound to a subtype-selective antagonist. The adenosine receptor class of GPCRs is responsible for mediating the diverse actions of adenosine on cellular activity and is antagonized by caffeine. Other structures from different branches of the GPCR phylogenetic tree are being determined and the community-wide GPCR Dock 2008 and 2010 assessments are helping to evaluate the current state of computational tools and research groups with respect to GPCR modeling and docking. The differences in the determined GPCR structures are extending the analysis of human receptors and providing insights into their pharmacology, ligand recognition and activation.

The efforts of PSI groups and their collaborators have also determin structures from several membrane transporter and protein channel families. CorA family members are the primary Mg2+ ion transporters in bacteria and archea and are ubiquitous. The structure of CorA from Thermotoga maritima determined by Eshagi et al. 3 provides new functional details about this transporter class and reveals a transmembrane pore architecture that differs from that of other cation channels and transporters. The crystal structure of a representative member of the urea transporter (UT) family, one of at least four families of transporters involved in facilitating the selective permeation of urea across cell membranes, was also recently determined by Levin et al. 4 . The structure suggests that UTs are likely to operate by a channel-like mechanism with permeation achieved through the dehydration of urea by a long, narrow selectivity filter.

The reuptake of serotonin, dopamine and noradrenaline (norepinephrine) by neurotransmitter transporters located in the presynaptic membrane terminates signal transmission in the central nervous system. These transporters are the targets of tricyclic antidepressants (TCAs) and selective serotonin-reuptake inhibitors used in the treatment of depression. Zhou et al. 5 have determined the structure of the leucine transporter LeuT, a bacterial homolog of these transporters, bound to leucine and the TCA desipramine, which provides insight into the mechanism of inhibition by this class of drugs. In the not too distant future, structures for their human counterparts will perhaps be forthcoming that could enable a structure-based approach to drug discovery in a field where there are no animal models.

Members of the formate/nitrite transport (FNT) family are important in anerobic bacterial respiration and protection of pathogenic bacteria from the host immune response. The crystal structure of the FNT protein FocA from Vibrio cholerae determined by Waight et al. 6 reveals a fold strikingly similar to that found in aquaporins, indicating a channel-like function, and provides the basis for its formate selectivity.

The Rh family of membrane proteins are known for their roles in red blood cells and in the kidney where they govern waste nitrogen excretion and regulate pH balance. This year, the structure of the first human Rh factor at 2.1 Å resolution, from Gruswitz et al. 7 defined the mechanism and selectivity of the Rh channel and presented a new picture of the human red-cell complex.

Aquaglyceroporin (PfAQP) from the malarial parasite Plasmodium falciparum is important for glycerol uptake by the parasite during the blood stage of its life cycle and may thus be a potential drug target. The crystal structure of PfAQP determined by Newby et al. 8 revealed the basis for its dual selectivity for water and glycerol. And last year, a PSI effort by Ho et al 9 . solved the crystal structure at 1.8 Å resolution of human aquaporin 4, a target for drugs that minimize the deleterious effects of stroke, revealed the exquisite atomic detail of water selectivity in the channel. The structure of aquaporin 4 gives researchers into neuromyelitis optica, a neurodegenerative disease that causes blindness, a continuous supply of the 'well-behaved' membrane protein.

Membrane-associated or membrane-anchored proteins are often difficult to purify and characterize as a result of their dual natures. Embryonic factor 1 (FAC1) is an AMP deaminase (AMPD) essential for the zygote-to-embryo transition in the plant Arabidopsis and is the target of microbial-derived herbicides. Sequence analysis of FAC1 suggests the presence of an amino-terminal transmembrane helix that tethers the cytoplasmic catalytic domain to the intracellular membrane 10 . The crystal structure of this domain 10 provides functional insight into AMPD catalysis. Structural analysis of the membrane-anchored ubiquitin-fold (MUB) proteins, through the use of the solution structure of the human ortholog Bc059385 11 , suggests how the ubiquitin fold may have additional roles in protein trafficking and membrane localization.

Another membrane protein structure solved by the PSI is that of DesA3, a stearoyl CoA desaturase from Mycobacterium tuberculosis. DesA3 is an essential enzyme in fatty-acid biosynthesis and is necessary for the pathogenicity of the bacterium. Chang et al. 12 have investigated how carboxy-terminal degradation of the DesA3 may be used to regulate its enzymatic activity post-translationally. The findings also suggest that other essential genes may be susceptible to this mode of regulation.

Not all the new structures of human membrane proteins were determined by crystallography. Hiller et al. 13 determined the structure of the human channel protein VDAC-1 in detergent micelles using NMR spectroscopy, and thereby increased our understanding of the functional role of cholesterol bound to the channel. Other eukaryotic membrane protein structures also illustrate the importance of cholesterol in binding to, and stabilizing, membrane proteins 14 .

Researchers are also gaining ground in devising methods for expressing integral membrane proteins. For example, Goren et al. 15 describe the successful use of a wheat-germ cell-free translation system to produce membrane proteins in unilamellar liposomes and the benefits of this method in some cases compared with the expression and purification of membrane proteins from in vivo systems.

There is still much to do in the field of membrane protein structure determination. However, great excitement is being generated by the increasing number of structure depositions due to the use of new and improved tools for expression, crystallization, and NMR spectroscopy especially, and our improved understanding of the structures of different membrane protein families. The new PSI:Biology enterprise will contribute to the increasingly significant investment in membrane proteins.

One of the most challenging problems under investigation is to determine the structures of the complexes formed between membrane and soluble proteins, and between membrane proteins and other membrane proteins, which often form the functional entity. Several structures of such complexes have been reported in recent months, usually derived from co-selection and coexpression strategies. New technologies, approaches, methods and improved understanding of the complexes are being developed.

While the first structure of a membrane protein appeared in 1985, 25 years after the first structure of a soluble protein — myoglobin — the stage is now being set for the technology evolution that began ten years ago for soluble proteins with PSI-1 and for membrane proteins five years ago with PSI-II. The potential therapeutic impact of a greater understanding of transmembrane signaling and transport processes is enormous.

Michelle Montoya

  1. V. Cherezov, D. M. Rosenbaum, M. A. Hanson, S. G. Rasmussen, F. S. Thian et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor.

    Science 318, 1258-1265 (2007). doi:10.1126/science.1150577

  2. V. P. Jaakola, M. T. Griffith, M. A. Hanson, V. Cherezov, E. Y. Chien et al. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist.

    Science 322, 1211-1217 (2008). doi:10.1126/science.1164772

  3. S. Eshaghi, D. Niegowski, A. Kohl, D. Martinez Molina, S. A. Lesley et al. Crystal structure of a divalent metal ion transporter CorA at 2.9 angstrom resolution.

    Science 313, 354-357 (2006). doi:10.1126/science.1127121

  4. E. J. Levin, M. Quick & M. Zhou Crystal structure of a bacterial homologue of the kidney urea transporter.

    Nature 462, 757-761 (2009). doi:10.1038/nature08558

  5. Z. Zhou, J. Zhen, N. K. Karpowich, R. M. Goetz, C. J. Law et al. LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake.

    Science 317, 1390-1393 (2007). doi:10.1126/science.1147614

  6. A. B. Waight, J. Love & D. N. Wang Structure and mechanism of a pentameric formate channel.

    Nature Struct. Mol. Biol. 17, 31-37 (2010). doi:10.1038/nsmb.1740

  7. F. Gruswitz, S. Chaudhary, J. D. Ho, A. Schlessinger, B. Pezeshki et al. Function of human Rh based on structure of RhCG at 2.1 Å.

    Proc. Natl Acad. Sci. USA 107, 9638-9643 (2010). doi:10.1073/pnas.1003587107

  8. Z. E. Newby, J. O'Connell, 3rd, Y. Robles-Colmenares, S. Khademi, L. J. Miercke et al. Crystal structure of the aquaglyceroporin PfAQP from the malarial parasite Plasmodium falciparum. Nature Struct.

    Mol. Biol. 15, 619-625 (2008). doi:10.1038/nsmb.1431

  9. J. D. Ho, R. Yeh, A. Sandstrom, I. Chorny, W. E. Harries et al. Crystal structure of human aquaporin 4 at 1.8 Å and its mechanism of conductance.

    Proc. Natl Acad. Sci. USA 106, 7437-7442 (2009). doi:10.1073/pnas.0902725106

  10. B. W. Han, C. A. Bingman, D. K. Mahnke, R. M. Bannen, S. Y. Bednarek et al. Membrane association, mechanism of action, and structure of Arabidopsis embryonic factor 1 (FAC1).

    J. Biol. Chem. 281, 14939-14947 (2006). doi:10.1074/jbc.M513009200

  11. N. B. de la Cruz, F. C. Peterson, B. L. Lytle & B. F. Volkman Solution structure of a membrane-anchored ubiquitin-fold (MUB) protein from Homo sapiens.

    Protein Sci 16, 1479-1484 (2007). doi:10.1110/ps.072834007

  12. Y. Chang, G. E. Wesenberg, C. A. Bingman & B. G. Fox. In vivo inactivation of the mycobacterial integral membrane stearoyl coenzyme A desaturase DesA3 by a C-terminus-specific degradation process.

    J. Bacteriol. 190, 6686-6696 (2008). doi:10.1128/JB.00585-08

  13. S. Hiller, R. G. Garces, T. J. Malia, V. Y. Orekhov, M. Colombini et al. Solution structure of the integral human membrane protein VDAC-1 in detergent micelles.

    Science 321, 1206-1210 (2008). doi:10.1126/science.1161302

  14. M. A. Hanson, V. Cherezov, M. T. Griffith, C. B. Roth, V. P. Jaakola et al. A specific cholesterol binding site is established by the 2.8 Å structure of the human beta2-adrenergic receptor.

    Structure 16, 897-905 (2008). doi:10.1016/j.str.2008.05.001

  15. M. A. Goren, A. Nozawa, S. Makino, R. L. Wrobel & B. G. Fox Cell-free translation of integral membrane proteins into unilamellar liposomes.

    Methods Enzymol. 463, 647-673 (2009). doi:10.1016/S0076-6879(09)63037-8


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