The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous GPCRs such as rhodopsin and the β-adrenergic receptor exhibits unique structural and functional characteristics. Computational models also predict the existence of such a structural motif within the CB1 cannabinoid receptor, another member of the G-protein coupled receptor superfamily. To gain insights into the conformational properties of this GPCR component, a peptide corresponding to helix 8 of the CB1 receptor with a small contiguous segment from transmembrane helix 7 (TM7) was chemically synthesized and its secondary structure determined by circular dichroism (CD) and solution NMR spectroscopy. Our studies in DPC and SDS micelles revealed significant α-helical structure while in an aqueous medium, the peptide exhibited a random coil configuration. The relative orientation of helix 8 within the CB1 receptor was obtained from intermolecular ³¹P-¹H and ¹H-¹H NOE measurements. Our results suggest that in the presence of an amphipathic membrane environment, helix 8 assumes an alpha helical structure with an orientation parallel to the phospholipid membrane surface and perpendicular to TM7. In this model, positively charged side chains interact with the lipid headgroups while the other polar side chains face the aqueous region. The above observations may be relevant to the activation/deactivation of the CB1 receptor.     

Solution structure of the fifth and sixth transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the fifth and sixth transmembrane segments of the bovine mitochondrial oxoglutarate carrier (OGC) and of the hydrophilic loop that connects them were studied by CD and NMR spectroscopies. Peptides F215-R246, W279-K305 and P257-L278 were synthesized and structurally characterized. CD data showed that at high concentrations of TFE and SDS all peptides assume α-helical structures. ¹H-NMR spectra of the three peptides in TFE/water were fully assigned and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides were generated by distance geometry calculations. A well-defined α–helix was found in the region L220-V243 of peptide F215-R246 (TMS-V), in the region P284-M303 of peptide W279-K305 (TMS-VI) and in the region N261-F275 of peptide P257-L278 (hydrophilic loop). The helix L220-V243 exhibited a sharp kink at P239, while a little bend around P291 was observed in the helical region P284-M303. Fluorescence studies performed on peptide W279-K305, alone and together with other transmembrane segments of OGC, showed that the W279 fluorescence was quenched upon addition of peptide F215-R246, but not of peptides K21-K46, R78-R108 and P117-A149 suggesting a specific interaction between TMS-V and TMS-VI of OGC.     

Solution Structure and Phospholipid Interactions of the Isolated Voltage-Sensor Domain from KvAP

Voltage-sensor domains (VSDs) are specialized transmembrane segments that confer voltage sensitivity to many proteins such as ion channels and enzymes. The activities of these domains are highly dependent on both the chemical properties and the physical properties of the surrounding membrane environment. To learn about VSD-lipid interactions, we used nuclear magnetic resonance spectroscopy to determine the structure and phospholipid interface of the VSD from the voltage-dependent K⁺ channel KvAP (prokaryotic Kv from Aeropyrum pernix). The solution structure of the KvAP VSD solubilized within phospholipid micelles is similar to a previously determined crystal structure solubilized by a nonionic detergent and complexed with an antibody fragment. The differences observed include a previously unidentified short amphipathic α-helix that precedes the first transmembrane helix and a subtle rigid-body repositioning of the S3-S4 voltage-sensor paddle. Using ¹⁵N relaxation experiments, we show that much of the VSD, including the pronounced kink in S3 and the S3-S4 paddle, is relatively rigid on the picosecond-to-nanosecond timescale. In contrast, the kink in S3 is mobile on the microsecond-to-millisecond timescale and may act as a hinge in the movement of the paddle during channel gating. We characterized the VSD-phospholipid micelle interactions using nuclear Overhauser effect spectroscopy and showed that the micelle uniformly coats the KvAP VSD and approximates the chemical environment of a phospholipid bilayer. Using paramagnetically labeled phospholipids, we show that bilayer-forming lipids interact with the S3 and S4 helices more strongly than with S1 and S2.     

Solution structure of the first and second transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the first and the second transmembrane segment of the bovine mitochondrial oxoglutarate carrier (OGC) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. Peptides 21–46 and 78–108 of its primary sequence were synthesized and structurally characterized in membrane-mimetic environments. CD data showed that at high concentrations of TFE (>50%) and SDS (>2%) both peptides assume α-helical structures, whereas in more hydrophilic environments only peptide 78–108 has a helical structure. ¹H-NMR spectra of the two peptides in TFE/water and SDS were fully assigned, and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides in TFE/water were generated by distance geometry calculations. A well-defined α-helix was found in the region K24-V39 of peptide 21–46 and in the region A86–F106 of peptide 78–108. We cannot exclude that in intact OGC the extension of these helices is longer. The helix of peptide 21–46 is essentially hydrophobic, whereas that of peptide 78–108 is predominantly hydrophilic.     

Truncated hemoglobins: trimming the classical ‘three-over-three’ globin fold to a minimal size

Truncated hemoglobins (trHbs) host the heme in a “two-over-two’ α-helical sandwich which results from extensive editing of the classical ‘three-over-three’ globin fold. The three-dimensional structure of trHbs is based on four main α-helices, arranged in a sort of α-helical bundle composed of two antiparallel helix pairs (B/E and G/H). Most notably, trHbs deviate from the conventional globin fold in that they display an extended loop substituting for the heme proximal F-helix observed in globins. Moreover, since efficient adaptation of a 110–130 amino acid trHb chain to host the porphyrin ring firstly requires specific chain flexibility, trHbs contain three invariant Gly-based motifs. Inspection of the trHb three-dimensional trHb structures shows that an apparent protein cavity or tunnel would connect the protein surface to an inner region very close to the heme distal site. Such a structural feature, never observed before in (non) vertebrate globins, may have substantial implications for ligand diffusion and binding properties in trHbs.     

The structural properties of the transmembrane segment of the integral membrane protein phospholamban utilizing C CPMAS, H, and REDOR solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca²⁺ regulator. ¹³C cross-polarization magic angle spinning spectra of ¹³C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding ¹³C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an α-helical conformation as monitored by an enhancement of the α-helical carbonyl ¹³C resonance in the corresponding NMR spectrum. ¹³C-¹⁵N REDOR solid-state NMR spectroscopic experiments revealed the distance between the ¹³C carbonyl carbon of Leu39 and the ¹⁵N amide nitrogen of Leu42 to be 4.2 ± 0.2Å indicating an α-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three ²H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11° ± 5° tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer.     

Crystal structure of a core domain of stomatin from Pyrococcus horikoshii Illustrates a novel trimeric and coiled-coil fold

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. However, the function of stomatin is not fully understood. An open reading frame, PH1511, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes p-stomatin, a prokaryotic stomatin. Here, we report the first crystal structure of a stomatin ortholog, the core domain of the p-stomatin PH1511p (residues 56-234 of PH1511p, designated as PhSto^CD). PhSto^CD forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 Å on each side, and three α-helical segments of about 60 Å in length extend from the apexes of the triangle. The α/β domain of PhSto^CD is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. According to chemical cross-linking experiments, PhSto^CD would be able to assemble into an oligomeric form. The coiled-coil fold observed in the crystal probably contributes to self-association.     

Structural repeats and evolution of tobacco mosaic virus coat protein and RNA

The three-dimensional structure of the tobacco mosaic virus (TMV) coat protein disk suggests a possible pathway for the early evolution of the virus self-assembly mechanism.The coat protein contains a 2-fold repeated structural pattern in the folding of both its four alpha helices (A,B,C,D), which run alternately forward and back along the radius of the disk, and the four-stranded antiparallel pleated sheet which links these helices to the hydrophobic girdle at the outer rim of the disk. Helices A and B can be approximately superposed on C and D by a screw rotation about a molecular pseudo-dyad axis which lies nearly parallel to the plane of the protein disk. This operation relates 29 pairs of α-carbon positions with a root-mean-square deviation of 1.77 Å. A second pseudo-dyad in the pleated-sheet region relates 14 more atom pairs with a deviation of 2.32 Å and forms a distorted continuation of the relationship between the helices. The helix dyad also relates repeated pairs of functionally important amino acids which take part in intersubunit contacts.We have analysed these structural repeats and tested their significance by comparing them with repeats in other “helix quartet” proteins, cytochrome b⁵ and the hemerythrins, as well as with an irregular helix cluster in thermolysin. TMV is noticeably more repetitive than the others, including hemerythrin which is thought to have evolved by gene duplication.We propose that the primitive TMV coat protein was a dimeric structure of two smaller units paired about a 2-fold axis. Each unit was a pair of helices, linked at the inner radius of the virus rod by a short bend, where the RNA binding site formed, and connected at the outer radius by two short strands of beta sheet. A tandem gene duplication joined the two units and formed the present helix quartet. The flexible loop which now runs into the centre of the virus and connects helix C to helix D developed later. The assembly origin RNA may have evolved from part of the coat protein RNA which codes for this loop.     

Horse Prion Protein NMR Structure and Comparisons with Related Variants of the Mouse Prion Protein

The NMR structure of the horse (Equus caballus) cellular prion protein at 25 °C exhibits the typical PrP^C [cellular form of prion protein (PrP)] global architecture, but in contrast to most other mammalian PrP^Cs, it contains a well-structured loop connecting the β2 strand with the α2 helix. Comparison with designed variants of the mouse prion protein resulted in the identification of a single amino acid exchange within the loop, D167S, which correlates with the high structural order of this loop in the solution structure at 25 °C and is unique to the PrP sequences of equine species. The β2-α2 loop and the α3 helix form a protein surface epitope that has been proposed to be the recognition area for a hypothetical chaperone, “protein X,” which would promote conversion of PrP^C into the disease-related scrapie form and thus mediate intermolecular interactions related to the transmission barrier for transmissible spongiform encephalopathies (TSEs) between different species. The present results are evaluated in light of recent indications from in vivo experiments that the local β2-α2 loop structure affects the susceptibility of transgenic mice to TSEs and the fact that there are no reports on TSE in horses.     

The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Amino Acid Insertion Reveals a Necessary Three-Helical Intermediate in the Folding Pathway of the Colicin E7 Immunity Protein Im7

The small (87-residue) α-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an α-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and ?-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.     

Structural stability of short subsequences of the tropomyosin chain

The native tropomyosin molecule is a parallel, registered, α-helical coiled coil made from two 284-residiic chains. Long excised subsequences (≥ 95 residues) form the same structure with comparable thermal stability. Here, we investigate local stability using shorter subsequences (20-50 residues) that are chemically synthesized or excised from various regions along the protein chain. Thermal unfolding studies of such shorter peptides by CD in the same solvent medium used in extant studies of the parent protein indicate very low helix content, almost no coiled-coil formation, and high thermal lability of such secondary structure as does form. This behavior is in stark contrast to extant data on leucine-zipper peptides and short “designed” synthetic peptides, many of which have high α-helix content and form highly stable coiled coils. The existence of short coiled coils calls into question the older idea that short subsequences of a protein have little structure. The present study supports the older view, at least in its application to tropomyosin. The intrinsic local α-helical propensity and helix–helix interaction in this prototypical α-helical protein is sufficiently weak as to require not only dimerization, but macro-molecular amplification in order to attain its native conformation in common benign media near neutral pH. © 1995 John Wiley & Sons, Inc.     

The transmembrane domain of caveolin-1 exhibits a helix–break–helix structure

Caveolin is an integral membrane protein that is found in high abundance in caveolae. Both the N- and C- termini lie on the same side of the membrane, and the transmembrane domain has been postulated to form an unusual intra-membrane horseshoe configuration. To probe the structure of the transmembrane domain, we have prepared a construct of caveolin-1 that encompasses residues 96–136 (the entire intact transmembrane domain). Caveolin-1(96–136) was over-expressed and isotopically labeled in E. coli, purified to homogeneity, and incorporated into lyso-myristoylphosphatidylglycerol micelles. Circular dichroism and NMR spectroscopy reveal that the transmembrane domain of caveolin-1 is primarily α-helical (57–65%). Furthermore, chemical shift indexing reveals that the transmembrane domain has a helix–break–helix structure which could be critical for the formation of the intra-membrane horseshoe conformation predicted for caveolin-1. The break in the helix spans residues 108 to 110, and alanine scanning mutagenesis was carried out to probe the structural significance of these residues. Our results indicate that mutation of glycine 108 to alanine does not disrupt the structure, but mutation of isoleucine 109 and proline 110 to alanine dramatically alters the helix–break–helix structure. To explore the structural determinants further, additional mutagenesis was performed. Glycine 108 can be substituted with other small side chain amino acids (i.e. alanine), leucine 109 can be substituted with other β-branched amino acids (i.e. valine), and proline 110 cannot be substituted without disrupting the helix–break–helix structure.     

The First N-terminal Amino Acids of α-Synuclein Are Essential for α-Helical Structure Formation In Vitro and Membrane Binding in Yeast

α-Synuclein (α-syn), a protein implicated in Parkinson's disease, is structurally diverse. In addition to its random-coil state, α-syn can adopt an α-helical structure upon lipid membrane binding or a β-sheet structure upon aggregation. We used yeast biology and in vitro biochemistry to detect how sequence changes alter the structural propensity of α-syn. The N-terminus of the protein, which adopts an α-helical conformation upon lipid binding, is essential for membrane binding in yeast, and variants that are more prone to forming an α-helical structure in vitro are generally more toxic to yeast. β-Sheet structure and inclusion formation, on the other hand, appear to be protective, possibly by sequestering the protein from the membrane. Surprisingly, sequential deletion of residues 2 through 11 caused a dramatic drop in α-helical propensity, vesicle binding in vitro, and membrane binding and toxicity in yeast, part of which could be mimicked by mutating aspartic acid at position 2 to alanine. Variants with distinct structural preferences, identified here by a reductionist approach, provide valuable tools for elucidating the nature of toxic forms of α-syn in neurons.     

An Empirical Correlation between Secondary Structure Content and Averaged Chemical Shifts in Proteins

It is shown that the averaged chemical shift (ACS) of a particular nucleus in the protein backbone empirically correlates well to its secondary structure content (SSC). Chemical shift values of more than 200 proteins obtained from the Biological Magnetic Resonance Bank are used to calculate ACS values, and the SSC is estimated from the corresponding three-dimensional coordinates obtained from the Protein Data Bank. ACS values of ¹H_α show the highest correlation to helical and sheet structure content (correlation coefficient of 0.80 and 0.75, respectively); ¹H_N exhibits less reliability (0.65 for both sheet and helix), whereas such correlations are poor for the heteronuclei. SSC estimated using this correlation shows a good agreement with the conventional chemical shift index-based approach for a set of proteins that only have chemical shift information but no NMR or x-ray determined three-dimensional structure. These results suggest that even chemical shifts averaged over the entire protein retain significant information about the secondary structure. Thus, the correlation between ACS and SSC can be used to estimate secondary structure content and to monitor large-scale secondary structural changes in protein, as in folding studies.     

Structural biology of human cannabinoid receptor-2 helix 6 in membrane-mimetic environments

We detail the structure and dynamics of a synthetic peptide corresponding to transmembrane helix 6 (TMH6) of human cannabinoid receptor-2 (hCB2) in biomembrane-mimetic environments. The peptide’s NMR structural biology is characterized by two α-helical domains bridged by a flexible, nonhelical hinge region containing a highly-conserved CWFP motif with an environmentally sensitive, Pro-based conformational switch. Buried within the peptide’s flexible region, W²⁵⁸ may hydrogen-bond with L²⁵⁵ to help stabilize the Pro-kinked hCB2 TMH6 structure and position C²⁵⁷ advantageously for interaction with agonist ligands. These characteristics of hCB2 TMH6 are potential structural features of ligand-induced hCB2 activation in vivo.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly ¹⁵N and ¹³C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the ¹H, ¹³C, and ¹⁵N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

Solution Conformation and Dynamics of the HIV-1 Integrase Core Domain

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a critical enzyme involved in infection. It catalyzes two reactions to integrate the viral cDNA into the host genome, 3′ processing and strand transfer, but the dynamic behavior of the active site during catalysis of these two processes remains poorly characterized. NMR spectroscopy can reveal important structural details about enzyme mechanisms, but to date the IN catalytic core domain has proven resistant to such an analysis. Here, we present the first NMR studies of a soluble variant of the catalytic core domain. The NMR chemical shifts are found to corroborate structures observed in crystals, and confirm prior studies suggesting that the α4 helix extends toward the active site. We also observe a dramatic improvement in NMR spectra with increasing MgCl₂ concentration. This improvement suggests a structural transition not only near the active site residues but also throughout the entire molecule as IN binds Mg²⁺. In particular, the stability of the core domain is linked to the conformation of its C-terminal helix, which has implications for relative domain orientation in the full-length enzyme. ¹⁵N relaxation experiments further show that, although conformationally flexible, the catalytic loop of IN is not fully disordered in the absence of DNA. Indeed, automated chemical shift-based modeling of the active site loop reveals several stable clusters that show striking similarity to a recent crystal structure of prototype foamy virus IN bound to DNA.