NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Franzin, Carla M.; Gong, Xiaomin; Thai, Khang; Yu, Jinghua; Marassi, Francesca M.

doi:10.1016/j.ymeth.2006.08.011

Cited by 36 publications

(36 citation statements)

References 77 publications

(80 reference statements)

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“…The Arg hydrocarbon side chains are sufficiently long to reach the membrane surface, while allowing H4 to penetrate the membrane below the water-lipid interface. Solution I is also consistent with the solid-state NMR spectra of FXYD1 in lipid bilayers, which indicate the presence of a helical segment associated with the membrane surface (56). Solution II, however, has an opposite amphiphilic polarity to the membrane, with apolar side chains facing out and charged side chains facing in.…”

Section: Resultssupporting

confidence: 68%

Structure of the Na,K-ATPase Regulatory Protein FXYD1 in Micelles

et al. 2007

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FXYD1 is a major regulatory subunit of the Na,K-ATPase and the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinases A and C in heart and skeletal muscle sarcolemma. It is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here, we present the three-dimensional structure of FXYD1 determined in micelles by NMR spectroscopy. Structure determination was made possible by measuring residual dipolar couplings in weakly oriented micelle samples of the protein. This allowed us to obtain the relative orientations of the helical segments and information about the protein dynamics. The structural analysis was further facilitated by the inclusion of distance restraints, obtained from paramagnetic spin label relaxation enhancements, and by refinement with a micelle depth restraint, derived from paramagnetic Mn line broadening effects. The structure of FXYD1 provides the foundation for understanding its intra-membrane association with the Na,K-ATPase alpha subunit and suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein to modulate its interaction with the alpha subunit.

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Section: Resultssupporting

confidence: 68%

Structure of the Na,K-ATPase Regulatory Protein FXYD1 in Micelles

et al. 2007

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“…Both H/D exchange experiments and paramagnetic relaxation enhancement experiments are general methods for characterizing the structural fold of membrane proteins in detergent micelles (32). Our data have shown that although TM1 was well shielded from a soluble paramagnetic probe, Mn 2ϩ , it was highly accessible to D 2 O implying a selective pore-like structure (Fig.…”

Section: Discussionmentioning

confidence: 69%

Ligand Binding in the Conserved Interhelical Loop of CorA, a Magnesium Transporter from Mycobacterium tuberculosis

Sharma

Qin

et al. 2009

Journal of Biological Chemistry

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CorA is a constitutively expressed magnesium transporter in many bacteria. The crystal structures of Thermotoga maritima CorA provide an excellent structural framework for continuing studies. Here, the ligand binding properties of the conserved interhelical loop, the only portion of the protein exposed to the periplasmic space, are characterized by solution nuclear magnetic resonance spectroscopy. Through titration experiments performed on the isolated transmembrane domain of Mycobacterium tuberculosis CorA, it was found that two CorA substrates (Mg 2؉ and Co 2؉ ) and the CorA-specific inhibitor (Co(III) hexamine chloride) bind in the loop at the same binding site. This site includes the glutamic acid residue from the conserved "MPEL" motif. The relatively large dissociation constants indicate that such interactions are weak but not atypical for channels. The present data support the hypothesis that the negatively charged loop could act as an electrostatic ring, increasing local substrate concentrations before transport across the membrane.The heterogeneous membrane environment is very challenging to mimic for structural, dynamic, and functional studies of membrane proteins. It is not surprising, therefore, that different aspects of the structure can be brought to light under different conditions. Recently, an excellent set of crystal structures of the CorA Mg 2ϩ transporter have been published (1-3), and whereas many CorA mysteries were solved, the highly conserved periplasmic interhelical loops in the pentameric structure were not well resolved. Here, solution nuclear magnetic resonance (NMR) spectroscopy of the transmembrane domain resolves these loops, and the secondary structure and ion binding in this domain are characterized.The 2TM-GxN family of transporters is a large group of integral membrane proteins responsible for metal ion transport (especially for magnesium) across membranes (4, 5). CorA is a prototypical member in this family responsible for magnesium influx as well as efflux in some cases (5, 6). In the extensive phylogenetic analysis, it was shown that CorA is characterized by a universally conserved "GMN" motif in an interhelical loop connecting two conserved transmembrane helices at the C terminus of the full-length protein (4). As the only constitutively expressed magnesium transporter, CorA can play an important role in the viability of pathogens, such as Helicobacter pylori (7).Pentameric CorA from Thermotoga maritima forms two distinguishable domains; that is, a large cytoplasmic domain and a small transmembrane domain (1-3). In this latter domain there are two transmembrane helices connected by an interhelical periplasmic loop. The first transmembrane helix (TM1) 2 lines the pore, whereas the second transmembrane helix (TM2) forms an outer ring of helices, which appears to have only weak interactions with the TM1 helices.Different mechanisms of substrate transport for CorA have been proposed (1-3, 6, 8), whereas the structure and function of the conserved loop is still an open issue. Ba...

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“…7. There was no abnormal broadening or clustering of the resonances, which would be a warning sign that the polypeptide was aggregated or improperly folded [29,30]. The majority of the 1 H-15 N cross-peaks lie between 7.5 and 9.0 ppm.…”

Section: Structural Analysismentioning

confidence: 98%