NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates.

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031-4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187-3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.

Building a stable RNA U-turn with a protonated cytidine.

The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5'-UNR-3' (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3' phosphate group of the R residue as well as a hydrogen bond between the 2'-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3' from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.

The First structure of a lantibiotic immunity protein, SpaI from Bacillus subtilis, reveals a novel fold.

Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.

Requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by NMR.

Nuclear magnetic resonance (NMR) structure calculations of the α-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary for obtaining enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables α-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data.

Simultaneous single-structure and bundle representation of protein NMR structures in torsion angle space.

A method is introduced to represent an ensemble of conformers of a protein by a single structure in torsion angle space that lies closest to the averaged Cartesian coordinates while maintaining perfect covalent geometry and on average equal steric quality and an equally good fit to the experimental (e.g. NMR) data as the individual conformers of the ensemble. The single representative 'regmean structure' is obtained by simulated annealing in torsion angle space with the program CYANA using as input data the experimental restraints, restraints for the atom positions relative to the average Cartesian coordinates, and restraints for the torsion angles relative to the corresponding principal cluster average values of the ensemble. The method was applied to 11 proteins for which NMR structure ensembles are available, and compared to alternative, commonly used simple approaches for selecting a single representative structure, e.g. the structure from the ensemble that best fulfills the experimental and steric restraints, or the structure from the ensemble that has the lowest RMSD value to the average Cartesian coordinates. In all cases our method found a structure in torsion angle space that is significantly closer to the mean coordinates than the alternatives while maintaining the same quality as individual conformers. The method is thus suitable to generate representative single structure representations of protein structure ensembles in torsion angle space. Since in the case of NMR structure calculations with CYANA the single structure is calculated in the same way as the individual conformers except that weak positional and torsion angle restraints are added, we propose to represent new NMR structures by a 'regmean bundle' consisting of the single representative structure as the first conformer and all but one original individual conformers (the original conformer with the highest target function value is discarded in order to keep the number of conformers in the bundle constant). In this way, analyses that require a single structure can be carried out in the most meaningful way using the first model, while at the same time the additional information contained in the ensemble remains available.

Characterization of molecular interactions between ACP and halogenase domains in the Curacin A polyketide synthase.

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with potent anti-proliferative activity. During its biosynthesis the growing substrate is bound covalently to an acyl carrier protein (ACP) that is able to access catalytic sites of neighboring domains for chain elongation and modification. While ACP domains usually occur as monomers, the curacin A cluster codes for a triplet ACP (ACP(I)-ACP(II)-ACP(III)) within the CurA PKS module. We have determined the structure of the isolated holo-ACP(I) and show that the ACPs are independent of each other within this tridomain system. In addition, we have determined the structure of the 3-hydroxyl-3-methylglutaryl-loaded holo-ACP(I), which is the substrate for the unique halogenase (Hal) domain embedded within the CurA module. We have identified the interaction surface of both proteins using mutagenesis and MALDI-based identification of product formation. Amino acids affecting product formation are located on helices II and III of ACP(I) and form a contiguous surface. Since the CurA Hal accepts substrate only when presented by one of the ACPs within the ACP(I)-ACP(II)-ACP(III) tridomain, our data provide insight into the specificity of the chlorination reaction.

Structural investigation of the C-terminal catalytic fragment of presenilin 1.

The gamma-secretase complex has a decisive role in the development of Alzheimer's disease, in that it cleaves a precursor to create the amyloid beta peptide whose aggregates form the senile plaques encountered in the brains of patients. Gamma-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. Many of the mutations encountered in early onset familial Alzheimer's disease are linked to presenilin 1, the catalytic component of gamma-secretase, whose active form requires its endoproteolytic cleavage into N-terminal and C-terminal fragments. Although there is general agreement regarding the topology of the N-terminal fragment, studies of the C-terminal fragment have yielded ambiguous and contradictory results that may be difficult to reconcile in the absence of structural information. Here we present the first structure of the C-terminal fragment of human presenilin 1, as obtained from NMR studies in SDS micelles. The structure reveals a topology where the membrane is likely traversed three times in accordance with the more generally accepted nine transmembrane domain model of presenilin 1, but contains unique structural features adapted to accommodate the unusual intramembrane catalysis. These include a putative half-membrane-spanning helix N-terminally harboring the catalytic aspartate, a severely kinked helical structure toward the C terminus as well as a soluble helix in the assumed-to-be unstructured N-terminal loop.