(1)H/(15)N heteronuclear NMR spectroscopy shows four dynamic domains for phospholamban reconstituted in dodecylphosphocholine micelles

We report the backbone dynamics of monomeric phospholamban in dodecylphosphocholine micelles using (1)H/(15)N heteronuclear NMR spectroscopy. Phospholamban is a 52-amino acid membrane protein that regulates Ca-ATPase in cardiac muscle. Phospholamban comprises three structural domains: a transmembrane domain from residues 22 to 52, a connecting loop from 17 to 21, and a cytoplasmic domain from 1 to 16 that is organized in an "L"-shaped structure where the transmembrane and the cytoplasmic domain form an angle of approximately 80 degrees (Zamoon et al., 2003; Mascioni et al., 2002). T(1), T(2), and (1)H/(15)N nuclear Overhauser effect values measured for the amide backbone resonances were interpreted using the model-free approach of Lipari and Szabo. The results point to the existence of four dynamic domains, revealing the overall plasticity of the cytoplasmic helix, the flexible loop, and part of the transmembrane domain (residues 22-30). In addition, using Carr-Purcell-Meiboom-Gill-based experiments, we have characterized phospholamban dynamics in the micros-ms timescale. We found that the majority of the residues in the cytoplasmic domain, the flexible loop, and the first ten residues of the transmembrane domain undergo dynamics in the micros-ms range, whereas minimal dynamics were detected for the transmembrane domain. Hydrogen/deuterium exchange factors measured at different temperatures support the existence of slow motion in both the loop and the cytoplasmic helix. We propose that these dynamic properties are critical factors in the biomolecular recognition of phospholamban by Ca-ATPase and other interacting proteins such as protein kinase A and protein phosphatase 1.     

NMR analysis of the conformational properties of the trapped on-pathway folding intermediate of the bacterial immunity protein Im7

Previous work shows that the transiently populated, on-pathway intermediate in Im7 folding contains three of the four native alpha-helices docked around a core stabilised by native and non-native interactions. To determine the structure and dynamic properties of this species in more detail, we have used protein engineering to trap the intermediate at equilibrium and analysed the resulting proteins using NMR spectroscopy and small angle X-ray scattering. Four variants were created. In L53AI54A, two hydrophobic residues within helix III are truncated, preventing helix III from docking stably onto the developing hydrophobic core. In two other variants, the six residues encompassing the native helix III were replaced with three (H3G3) or six (H3G6) glycine residues. In the fourth variant, YY, two native tyrosine residues (Tyr55 and Tyr56) were re-introduced into H3G6 to examine their role in determining the properties of the intermediate ensemble. All four variants show variable peak intensities and broad peak widths, consistent with these proteins being conformationally dynamic. Chemical shift analyses demonstrated that L53AI54A and YY contain native-like secondary structure in helices I and IV, while helix II is partly formed and helix III is absent. Lack of NOEs and rapid NH exchange for L53AI54A, combined with detailed analysis of the backbone dynamics, indicated that the hydrophobic core of this variant is not uniquely structured, but fluctuates on the NMR timescale. The results demonstrate that though much of the native-like secondary structure of Im7 is present in the variants, their hydrophobic cores remain relatively fluid. The comparison of H3G3/H3G6 and L53AI54A/YY suggests that Tyr55 and/or Tyr56 interact with the three-helix core, leading other residues in this region of the protein to dock with the core as folding progresses. In this respect, the three-helix bundle acts as a template for formation of helix III and the creation of the native fold.     

Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum

The structure of phospholamban, a 30-kDa oligomeric protein integral to cardiac sarcoplasmic reticulum, was probed using ultraviolet absorbance and circular dichroism spectroscopy. Purified phospholamban was examined in three detergents: octyl glucoside, n-dodecyloctaethylene glycol monoether (C12E8) and sodium dodecyl sulfate (SDS). Ultraviolet absorption spectra of phospholamban reflected its aromatic amino acid content: absorption peaks at 275-277 nm and 253, 259, 265 and 268 nm were attributed to phospholamban's one tyrosine and two phenylalanines, respectively. Phospholamban phosphorylated at serine 16 by the catalytic subunit of cAMP-dependent protein kinase exhibited no absorbance changes when examined in C12E8 or SDS. Circular dichroism spectroscopy at 250-190 nm demonstrated that phospholamban possesses a very high content of alpha-helix in all three detergents and is unusually resistant to denaturation. Dissociation of phospholamban subunits by boiling in SDS increased the helical content, suggesting that the highly ordered structure is not dependent upon oligomeric interactions. The purified COOH-terminal tryptic fragment of phospholamban, containing residues 26-52 and comprising the hydrophobic, putative membrane-spanning domain, also exhibited a circular dichroism spectrum characteristic of alpha-helix. Circular dichroism spectra of phosphorylated and dephosphorylated phospholamban were very similar, indicating that phosphorylation does not alter phospholamban secondary structure significantly. The results are consistent with a two-domain model of phospholamban in which each domain contains a helix and phosphorylation may act to rotate one domain relative to the other.     

Solution Structure of the N-terminal RNP Domain of U1A Protein: The Role of C-terminal Residues in Structure Stability and RNA Binding

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an α-helix that lies across the β-sheet, with residues Ile93, Ile94 and Met97 making contacts with Leu44, Phe56 and Ile58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the β-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135° to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from Ile93 and Ile94 to Ile58, Leu 41, Val62 and His10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.     

Solution structure and backbone dynamics of the DNA-binding domain of mouse Sox-5

The fold of the murine Sox-5 (mSox-5) HMG box in free solution has been determined by multidimensional NMR using (15)N-labeled protein and has been found to adopt the characteristic twisted L-shape made up of two wings: the major wing comprising helix 1 (F10--F25) and helix 2 (N32--A43), the minor wing comprising helix 3 (P51--Y67) in weak antiparallel association with the N-terminal extended segment. (15)N relaxation measurements show considerable mobility (reduced order parameter, S(2)) in the minor wing that increases toward the amino and carboxy termini of the chain. The mobility of residues C-terminal to Q62 is significantly greater than the equivalent residues of non-sequence-specific boxes, and these residues show a weaker association with the extended N-terminal segment than in non-sequence boxes. Comparison with previously determined structures of HMG boxes both in free solution and complexed with DNA shows close similarity in the packing of the hydrophobic cores and the relative disposition of the three helices. Only in hSRY/DNA does the arrangement of aromatic sidechains differ significantly from that of mSox-5, and only in rHMG1 box 1 bound to cisplatinated DNA does helix 1 have no kink. Helix 3 in mSox-5 is terminated by P68, a conserved residue in DNA sequence-specific HMG boxes, which results in the chain turning through approximately 90 degrees.     

NMR observable-based structure refinement of DAP12-NKG2C activating immunoreceptor complex in explicit membranes

NMR observables, such as NOE-based distance measurements, are increasingly being used to characterize membrane protein structures. However, challenges in membrane protein NMR studies often yield a relatively small number of such restraints that can create ambiguities in defining critical side chain-side chain interactions. In the recent solution NMR structure of the DAP12-NKG2C immunoreceptor transmembrane helix complex, five functionally required interfacial residues (two Asps and two Thrs in the DAP12 dimer and one Lys in NKG2C) display a surprising arrangement in which one Asp side chain faces the membrane hydrophobic core. To explore whether these side-chain interactions are energetically optimal, we used the published distance restraints for molecular dynamics simulations in explicit micelles and bilayers. The structures refined by this protocol are globally similar to the published structure, but the side chains of those five residues form a stable network of salt bridges and hydrogen bonds, leaving the Asp side chain shielded from the hydrophobic core, which is also consistent with available experimental observations. Moreover, the simulations show similar short-range interactions between the transmembrane complex and lipid/detergent molecules in micelles and bilayers, respectively. This study illustrates the efficacy of NMR membrane protein structure refinements in explicit membrane systems.     

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide curvacin A

The 3D structure of the membrane-permeabilizing 41-mer pediocin-like antimicrobial peptide curvacin A produced by lactic acid bacteria has been studied by NMR spectroscopy. In DPC micelles, the cationic and hydrophilic N-terminal half of the peptide forms an S-shaped beta-sheet-like domain stabilized by a disulfide bridge and a few hydrogen bonds. This domain is followed by two alpha-helices: a hydrophilic 6-mer helix between residues 19 and 24 and an amphiphilic/hydrophobic 11-mer helix between residues 29 and 39. There are two hinges in the peptide, one at residues 16-18 between the N-terminal S-shaped beta-sheet-like structure and the central 6-mer helix and one at residues 26-28 between the central helix and the 11-mer C-terminal helix. The latter helix is the only amphiphilic/hydrophobic part of the peptide and is thus presumably the part that penetrates into the hydrophobic phase of target-cell membranes. The hinge between the two helices may introduce the flexibility that allows the helix to dip into membranes. The helix-hinge-helix structure in the C-terminal half of curvacin A clearly distinguishes this peptide from the other pediocin-like peptides whose structures have been analyzed and suggests that curvacin A along with the structural homologues enterocin P and carnobacteriocin BM1 belong to a subgroup of the pediocin-like family of antimicrobial peptides.     

Identification of the PXW sequence as a structural gatekeeper of the headpiece C-terminal subdomain fold

The HeadPiece (HP) domain, present in several F-actin-binding multi-domain proteins, features a well-conserved, solvent-exposed PXWK motif in its C-terminal subdomain. The latter is an autonomously folding subunit comprised of three alpha-helices organised around a hydrophobic core, with the sequence motif preceding the last helix. We report the contributions of each conserved residue in the PXWK motif to human villin HP function and structure, as well as the structural implications of the naturally occurring Pro to Ala mutation in dematin HP. NMR shift perturbation mapping reveals that substitution of each residue by Ala induces only minor, local perturbations in the full villin HP structure. CD spectroscopic thermal analysis, however, shows that the Pro and Trp residues in the PXWK motif afford stabilising interactions. This indicates that, in addition to the residues in the hydrophobic core, the Trp-Pro stacking within the motif contributes to HP stability. This is reinforced by our data on isolated C-terminal HP subdomains where the Pro is also essential for structure formation, since the villin, but not the dematin, C-terminal subdomain is structured. Proper folding can be induced in the dematin C-terminal subdomain by exchanging the Ala for Pro. Conversely, the reverse substitution in the villin C-terminal subdomain leads to loss of structure. Thus, we demonstrate a crucial role for this proline residue in structural stability and folding potential of HP (sub)domains consistent with Pro-Trp stacking as a more general determinant of protein stability.     

Role of helix B residues in interfacial activation of a bacterial phosphatidylinositol-specific phospholipase C

The Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), an interfacial enzyme associated with prokaryotic infectivity, is activated by binding to zwitterionic surfaces, particularly phosphatidycholine (PC). Two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure are critical for this interaction. The helix B region (Ile43 to Gly48) in wild-type PI-PLC orients the side chains of Ile43 and Trp47 so that they pack together and form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. In previous studies we reported that in the crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, the helix B region has been reorganized by the mutation into an extended loop. Here we report the construction and characterization (catalytic activity, fluorescence, and NMR studies) of a series of PI-PLC mutants targeting helix B residues and surrounding regions to explore what is needed to stabilize the "membrane-active" conformation of the helix B region. Results strongly suggest that, while hydrophobic groups and presumably an intact helix B are critical for the initial binding of PI-PLC to membranes, disruption of helix B to allow enzyme dimerization is what leads to the activated PI-PLC conformation.     

Defining the intramembrane binding mechanism of sarcolipin to calcium ATPase using solution NMR spectroscopy

Sarcolipin (SLN) is an integral membrane protein that is expressed in both skeletal and cardiac muscle, where it inhibits SERCA (calcium ATPase) by lowering its apparent Ca2+ affinity in a manner similar to that of its homologue phospholamban (PLN). We use solution NMR to map the structural changes occurring within SLN upon interaction with the regulatory target, SERCA, co-reconstituting the two proteins in dodecylphosphocholine (DPC) detergent micelles, a system that preserves the native structure of SLN and the activity of SERCA, with the goal of comparing these interactions with those of the previously studied PLN-SERCA complex. Our analysis of the structural dynamics of SLN in DPC micelles shows this polypeptide to be partitioned into four subdomains: a short unstructured N terminus (residues 1-6), a short dynamic helix (residues 7-14), a more rigid helix (residues 15-26), and an unstructured C terminus (residues 27-31). Upon addition of SERCA, the different domains behave according to their dynamics, molding onto the surface of the enzyme. Remarkably, each domain of SLN behaves in a manner similar to that of the corresponding domains in PLN, supporting the hypothesis that both SLN and PLN bind SERCA in the same groove and with similar mechanisms.     

Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH

Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of approximately 120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix alpha1-alpha2 interface. In PrgI, the major negatively charged surface is not on the "face" but instead is on the "side" of the two-helix bundle, and only residues from helix alpha1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle-packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri.     

Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles

The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers.     

Oxidation of tryptophans in an interhelical hydrophobic cluster of myoglobin alters the thermodynamics of the denaturation transition

Model folding studies of sperm whale myoglobin have illustrated the presence of hydrophobic interfacial regions between elements of secondary structure. The specific oxidation of two tryptophan residues, in the A-H helix contact of sperm whale myoglobin, to the less hydrophobic oxindolylalanine residues is utilized to probe the contribution of hydrophobic packing density in this contact region. The acid denaturation of the modified protein is no longer a simple two-state process exhibiting the presence of stable intermediates. The relative stability of the intermediate is shown to be +5.3 kcal/mol less stable than native myoglobin. This value is consistent with the predicted relative stability, based upon electrostatic model calculations, of the docking of the A helix with a des-A helix myoglobin. The presence of stable intermediate structures in the denaturation pathway of the modified protein is consistent with the proposed role of hydrophobic interactions in damping structural fluctuations and statistical mechanical models of noncooperative protein unfolding. These results demonstrate the relationship between large-scale fluctuations and the frictional forces governing small-scale motions within the protein core.     

Robustness of the long-range structure in denatured staphylococcal nuclease to changes in amino acid sequence

A nativelike low-resolution structure has been shown to persist in the Delta 131 Delta denatured fragment of staphylococcal nuclease, even in the presence of 8 M urea. In this report, the physical-chemical basis of this structure is addressed by monitoring changes in structure reflected in residual dipolar couplings and diffusion coefficients as a function of changes in amino acid sequence. Ten large hydrophobic residues, previously shown to play dominant roles in the stability of the native state, are replaced with polar residues of similar shape. Modest increases in the Stokes radius determined by NMR methods result from replacement of five isoleucine/valine residues with threonine, one leucine with glutamine, and oxidation of four methionines to the sulfoxides. Yet in the presence of all ten hydrophobic to polar substitutions and 8 M urea, the NMR signature of a native-like topology is still largely intact. In addition, removal of 30 residues from either the N-terminus (which deletes a three-strand beta meander) or C-terminus (a long extended segment and the final alpha helix) produces only very small changes in long-range structure. These data indicate that both the general shape of the denatured state and the angular relationships of individual bond angles to the axes describing the spatial distribution of the protein chain are insensitive to large changes in the amino acid sequence, a finding consistent with the conclusion that the long-range structure of denatured proteins is encoded primarily by local steric interactions between side chains and the polypeptide backbone.     

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 15-23), and two extended strands (residues 24-31 and 34-40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.     

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.     

Influence of pH on NMR structure and stability of the human prion protein globular domain

The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121-230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three alpha-helices, comprising residues 144-156, 174-194, and 200-228, and a short anti-parallel beta-sheet, comprising residues 128-131 and 161-164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix alpha1 is elongated at the C-terminal end with residues 153-156 forming a 310-helix, and the population of helical structure in the C-terminal two turns of helix alpha 2 is increased. The protonation of His155 and His187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121-230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154-157 at the C-terminal end of helix alpha 1 and residues 161-164 of beta-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a "starting point" for pH-induced unfolding and thus may be implicated in endosomic PrPC to PrPSc conformational transition resulting in transmissible spongiform encephalopathies.     

Antifreeze protein from shorthorn sculpin: Identification of the ice-binding surface

Shorthorn sculpins, Myoxocephalus scorpius, are protected from freezing in icy seawater by alanine-rich, alpha-helical antifreeze proteins (AFPs). The major serum isoform (SS-8) has been reisolated and analyzed to establish its correct sequence. Over most of its length, this 42 amino acid protein is predicted to be an amphipathic alpha-helix with one face entirely composed of Ala residues. The other side of the helix, which is more heterogeneous and hydrophilic, contains several Lys. Computer simulations had suggested previously that these Lys residues were involved in binding of the peptide to the [11-20] plane of ice in the <-1102> direction. To test this hypothesis, a series of SS-8 variants were generated with single Ala to Lys substitutions at various points around the helix. All of the peptides retained significant alpha-helicity and remained as monomers in solution. Substitutions on the hydrophilic helix face at position 16, 19, or 22 had no obvious effect, but those on the adjacent Ala-rich surface at positions 17, 21, and 25 abolished antifreeze activity. These results, with support from our own modeling and docking studies, show that the helix interacts with the ice surface via the conserved alanine face, and lend support to the emerging idea that the interaction of fish AFPs with ice involves appreciable hydrophobic interactions. Furthermore, our modeling suggests a new N terminus cap structure, which helps to stabilize the helix, whereas the role of the lysines on the hydrophilic face may be to enhance solubility of the protein.