X-ray structure of a water-soluble analog of the membrane protein phospholamban: sequence determinants defining the topology of tetrameric and pentameric coiled coils

Phospholamban (PLB) is a pentameric transmembrane protein that regulates the Ca(2+)-dependent ATPase SERCA2a in sarcoplasmic reticulum membranes. We previously described the computational design of a water-soluble variant of phospholamban, WSPLB, which reproduced many of the structural and functional properties of the native membrane-soluble protein. While the full-length WSPLB forms a pentamer in solution, a truncated variant forms very stable tetramers. To obtain insight into the tetramer-pentamer cytoplasmic switch, we solved the crystal structure of the truncated construct, WSPLB 21-52. This peptide has a heptad sequence repeat with Leu residues at a- and Ile at d-positions from residues 31-52. The crystal structure revealed that WSPLB 21-52 adopted an antiparallel tetrameric coiled coil. This topology contrasts with the parallel topology of an analogue of the coiled-coil of GCN4 with the same Leu(a) Ile(d) repeat. Analysis of these structures revealed how the nature of the partially exposed residues at e- and g-positions influence the topology formed by the bundle. We also constructed a model for the pentameric form of PLB using the coiled-coil parameters derived from a single monomer in the tetrameric structure. This model suggests that both buried and interfacial hydrogen bonds are important for stabilizing the parallel pentamer.     

De novo design of a pentameric coiled-coil: decoding the motif for tetramer versus pentamer formation in water-soluble phospholamban.

Water-soluble phospholamban (WSPLB) is a designed, water-soluble analogue of the pentameric membrane protein phospholamban (PLB), which contains the same core and interhelical residues as PLB, with only the solvent-exposed positions mutated. WSPLB contains the same secondary and quaternary structure as PLB. The hydrophobic cores of PLB and WSPLB contain Leu and Ile at the a- and d-positions of a heptad repeat (abcdefg) from residues 31-52, while residues 21-30 are rich in polar amino acids at these positions. While the full-length WSPLB forms pentamers in solution, truncated peptides lacking residues 21-30 are largely tetrameric. Thus, truncation of residues 1-20 promotes a switch from pentamer to tetramer formation. Here, the motifs for WSPLB pentamerization were elucidated by characterizing a series of peptides, which were progressively truncated in this polar 'switch' region. When fully present, the 'switch' region promotes pentamer formation in WSPLB, by destabilizing a more stable tetrameric species which exists in its absence. We find that the burial of hydrogen bonding residues from 21 to 30 drives WSPLB from a tetramer to a pentamer, with direct implications for coiled-coil design.     

Role of cysteine residues in structural stability and function of a transmembrane helix bundle

To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis to prepare alpha-amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the three Cys residues by Ala (producing Ala-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (from SDS-polyacrylamide gel electrophoresis) and function (inhibition of the Ca-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wild-type PLB and Ala-PLB. Molecular modeling provides structural and energetic insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer formation or inhibitory function; 2) the steric properties of cysteine residues in the PLB transmembrane domain contribute substantially to pentameric stability, whereas the polar or chemical properties of the sulfhydryl group play only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-terminal methionine has neither a functional nor a structural effect in full-length PLB.     

Storage function of cartilage oligomeric matrix protein: the crystal structure of the coiled-coil domain in complex with vitamin D(3)

The five-stranded coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) forms a continuous axial pore with binding capacities for hydrophobic compounds, including prominent cell signalling molecules. Here, we report the X-ray structure of the COMPcc domain in complex with vitamin D(3) at 1.7 A resolution. The COMPcc pentamer harbours two molecules of the steroid hormone precursor in a planar s-trans conformation of the conjugated triene, with the aliphatic tails lying along the molecule axis. A hydrophilic ring of five Gln54 side chains divides the channel into two hydrophobic compartments in which the bound vitamin D(3) pair is fixed in a head-to-head orientation. Vitamin D(3) binding induces a volumetric increase of the cavities of approximately 30% while the main chain distances of the pentamer are retained. This adaptation to the bulky ring systems of the ligands is accomplished by a rotamer re-orientation of beta-branched side chains that form the knobs into holes of the coiled-coil structure. Compared with binding of vitamin D and retinoic acid by their classical receptors, COMP exerts a distinct mechanism of interaction mainly defined by the pattern of hydrophobic core residues.     

Conversion of phospholamban into a soluble pentameric helical bundle

Although membrane proteins and soluble proteins may achieve their final folded states through different pathways, it has been suggested that the packing inside a membrane protein could maintain a similar fold if the lipid-exposed surface were redesigned for solubility in an aqueous environment. To test this idea, the surface of the transmembrane domain of phospholamban (PLB), a protein that forms a stable helical homopentamer within the sarcoplasmic reticulum membrane, has been redesigned by replacing its lipid-exposed hydrophobic residues with charged and polar residues. CD spectra indicate that the full-length soluble PLB is highly alpha-helical. Small-angle X-ray scattering and multiangle laser light scattering experiments reveal that this soluble variant of PLB associates as a pentamer, preserving the oligomeric state of the natural protein. Mutations that destabilize native PLB also disrupt the pentamer. However, NMR experiments suggest that the redesigned protein exhibits molten globule-like properties, possibly because the redesign of the surface of this membrane protein may have altered some native contacts at the core of the protein or possibly because the core is not rigidly packed in wild-type PLB. Nonetheless, our success in converting the membrane protein PLB into a specific soluble helical pentamer indicates that the interior of a membrane protein contains at least some of the determinants necessary to dictate folding in an aqueous environment. The design we successfully used was based on one of the two models in the literature; the alternative design did not give stable, soluble pentamers. This suggests that surface redesign can be employed in gaining insights into the structures of membrane proteins.     

Rational design of peptide inhibitors of the sarcoplasmic reticulum calcium pump

The sequence of phospholamban (PLB) is practically invariant among mammalian species. The hydrophobic transmembrane domain has 10 leucine and 8 isoleucine residues. Two roles have been proposed for the leucines; one subset stabilizes PLB oligomers, while a second subset physically interacts with SERCA. On the basis of the sequence of the PLB transmembrane domain, we chemically synthesized a series of peptides and tested their ability to regulate SERCA in reconstituted membranes. In all, eight peptides were studied: a peptide corresponding to the null-cysteine transmembrane domain of PLB (TM-Ala-PLB), two polyleucine peptides (Leu18 and Leu24), polyalanine peptides containing 4, 7, and 12 leucine residues (Leu4, Leu7, and Leu12, respectively), and a polyalanine peptide containing the 9 leucine residues present in the transmembrane domain of PLB with and without the essential Asn34 residue (Asn1Leu9 and Leu9, respectively). With the exception of Leu18, co-reconstitution of the peptides revealed effects on the apparent calcium affinity of SERCA. The TM-Ala-PLB peptide possessed approximately 70% of the inhibitory function of wild-type PLB. The remaining peptides exhibited significant inhibitory activity decreasing in the following order: Leu12, Leu9, Leu24, Leu7, and Leu4. Replacing Asn34 of PLB in the Leu9 peptide resulted in superinhibition of SERCA. On the basis of these observations, we conclude that a partial requirement for SERCA inhibition is met by a simple hydrophobic surface on a transmembrane alpha-helix. In addition, the superinhibition observed for the Asn34-containing peptide suggests that the model peptides mimic the inhibitory properties of PLB. A model is presented in which surface complementarity around key amino acid positions is enhanced in the interaction with SERCA.     

Synthetic null-cysteine phospholamban analogue and the corresponding transmembrane domain inhibit the Ca-ATPase

Chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) have been used to analyze the structure and function of phospholamban (PLB), a 52-residue integral membrane protein that regulates the calcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-PAGE, EPR, and fluorescence. It has been proposed that inhibition of the pump is due primarily to the monomeric form, with both pentameric stability and inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-PAGE, and compared it with the synthetic null-cysteine TM domain (residues 26-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isolated TM domain is more oligomeric than the full-length parent molecule. These results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic TM domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic domains (residues 1-25). When reconstituted into liposomes containing the Ca-ATPase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule. We conclude that the TM domain of PLB is sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.     

Reexamination of the role of the leucine/isoleucine zipper residues of phospholamban in inhibition of the Ca2+ pump of cardiac sarcoplasmic reticulum

Phospholamban is a small phosphoprotein inhibitor of the Ca(2+)-pump in cardiac sarcoplasmic reticulum, which shows a distinct oligomeric distribution between monomers and homopentamers that are stabilized through Leu/Ile zipper interactions. A two-faced model of phospholamban inhibition of the Ca(2+)-pump was proposed, in which the Leu/Ile zipper residues located on one face of the transmembrane alpha-helix regulate the pentamer to monomer equilibrium, whereas residues on the other face of the helix bind to and inhibit the pump. Here we tested this two-faced model of phospholamban action by analyzing the functional effects of a new series of Leu/Ile zipper mutants. Pentameric stabilities of the mutants were quantified at different SDS concentrations. We show that several phospholamban mutants with hydrophobic amino acid substitutions at the Leu/Ile zipper region retain the ability to form pentamers but at the same time give the same or even stronger (i.e. L37I-PLB) inhibition of the Ca(2+)-pump than do mutants that are more completely monomeric. Steric constraints prevent the Leu/Ile zipper residues sequestered in the interior of the phospholamban pentamer from binding to the Ca(2+)-pump, leading to the conclusion that the zipper residues access the pump from the phospholamban monomer, which is the active inhibitory species. A modified model of phospholamban transmembrane domain action is proposed, in which the membrane span of the phospholamban monomer maintains contacts with the Ca(2+)-pump around most of its circumference, including residues located in the Leu/Ile zipper region.     

Molecular dynamics studies on structure and dynamics of phospholamban monomer and pentamer in membranes

Phospholamban (PLB) is an integral membrane protein of 52 residues that regulates the activity of the sarcoplasmic reticulum calcium pump in cardiac muscle cells through reversible phosphorylation of Ser16. To explore its possible conformations and dynamics in a monomeric state, we have performed comparative molecular dynamics simulations of unphosphorylated and phosphorylated PLB (pPLB) with various orientations in POPC membranes. The simulations indicate that dynamics of the cytoplasmic domain is highly dependent on its interactions with membranes, that is, large conformational changes in the absence of membrane interactions, but very restricted dynamics in their presence. pPLB shows more structural flexibility in its cytoplasmic domain, which is consistent with experimental observations. We have also performed a simulation of a PLB pentameric structure (the so‐called bellflower model), recently determined in micelles, to investigate its behaviors in a POPC membrane. The cytoplasmic domain in each monomer shows uncorrelated dynamics and undergoes large conformational changes toward the membrane surface during the simulation, which supports the so‐called pinwheel model of the PLB pentamer structure. The hydrophobic nature of the pentameric pore excludes water molecules in the pore region, which illustrates that the pore appears to be an energetic barrier for ion and water translocation. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Peptide inhibitors use two related mechanisms to alter the apparent calcium affinity of the sarcoplasmic reticulum calcium pump

The primary sequence of phospholamban (PLB) has provided a template for the rational design of peptide inhibitors of the sarcoplasmic reticulum calcium ATPase (SERCA). In the transmembrane domain of PLB, there are few polar residues and only one is essential (Asn (34)). Using synthetic peptides, we have previously investigated the role of Asn (34) in the context of simple hydrophobic transmembrane peptides. Herein we propose that the role of Asn in SERCA inhibition is position-sensitive and dependent upon the distribution of hydrophobic residues. To test this hypothesis, we synthesized a series of transmembrane peptides based on a 24 amino acid polyalanine sequence having either an alternating Leu-Ala sequence (Leu 12) or Leu residues at the native positions found in PLB (Leu 9). Asn-containing Leu 9 and Leu 12 peptides were synthesized with a single Asn residue located either one amino acid (N+/-1) or one turn of the helix (N+/-4) in either direction from its native position. Co-reconstitution of these peptides with SERCA into proteoliposomes revealed effects on the apparent calcium affinity and cooperativity of SERCA that correlated with the positions of the Asn and Leu residues. The most inhibitory peptides increased the cooperativity of SERCA as indicated by the Hill coefficients, suggesting that calcium-dependent reversibility is an inherent part of the inhibitory mechanism. Kinetic simulations combined with molecular modeling of the interaction between the peptides and SERCA reveal two related mechanisms of inhibition. Peptides that resemble PLB use the same inhibitory mechanism, whereas peptides that are more divergent from PLB alter an additional step in the calcium transport cycle.     

NMR structure of the natural killer cell receptor 2B4 (CD244): implications for ligand recognition

2B4, a transmembrane receptor expressed primarily on natural killer (NK) cells and on a subset of CD8(+) T cells, plays an important role in activating NK-mediated cytotoxicity through its interaction with CD48 on target cells. We report here the atomic-resolution structure of the ligand-binding (D1) domain of 2B4 in solution determined by nuclear magnetic resonance (NMR) spectroscopy. The overall main chain structure resembles an immunoglobulin variable (V) domain fold, very similar to that seen previously for domain 1 of CD2 and CD4. The structure contains nine beta-strands assembled into two beta-sheets conventionally labeled DEB and AGFCC'C' '. The six-stranded sheet (AGFCC'C' ') contains structural features that may have implications for ligand recognition and receptor function. A noncanonical disulfide bridge between Cys2 and Cys99 stabilizes a long and parallel beta-structure between strand A (residues 3-12) and strand G (residues 100-108). A beta-bulge at residues Glu45 and Ile46 places a bend in the middle of strand C' that orients two conserved and adjacent hydrophobic residues (Ile46 and Leu47) inside the beta-sandwich as seen in other V domains. Finally, the FG-loop (implicated in ligand recognition in the CD2-CD58 complex) is dynamically disordered in 2B4 in the absence of a ligand. We propose that ligand binding to 2B4 might stabilize the structure of the FG-loop in the ligand complex.     

Computational design of a water-soluble analog of phospholamban

Membrane proteins and water-soluble proteins share a similar core. This similarity suggests that it should be possible to water-solubilize membrane proteins by mutating only their lipid-exposed residues. We have developed computational tools to design water-soluble variants of helical membrane proteins, using the pentameric phospholamban (PLB) as our test case. To water-solublize PLB, the membrane-exposed positions were changed to polar or charged amino acids, while the putative core was left unaltered. We generated water-soluble phospholamban (WSPLB), and compared its properties to its predecessor PLB. In aqueous solution, WSPLB mimics all of the reported properties of PLB including oligomerization state, helical structure, and stabilization upon phosphorylation. We also characterized the truncated mutant WSPLB (21-52) comprising only the former transmembrane segment of PLB. This peptide shows a decreased specificity for forming a pentameric oligomerization state.     

Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position "d"

We describe the de novo design and biophysical characterization of a model coiled-coil protein in which we have systematically substituted 20 different amino acid residues in the central "d" position. The model protein consists of two identical 38 residue polypeptide chains covalently linked at their N termini via a disulfide bridge. The hydrophobic core contained Val and Ile residues at positions "a" and Leu residues at positions "d". This core allowed for the formation of both two-stranded and three-stranded coiled-coils in benign buffer, depending on the substitution at position "d". The structure of each analog was analyzed by CD spectroscopy and their relative stability determined by chemical denaturation using GdnHCI (all analogs denatured from the two-stranded state). The oligomeric state(s) was determined by high-performance size-exclusion chromatography and sedimentation equilibrium analysis in benign medium. Our results showed a thermodynamic stability order (in order of decreasing stability) of: Leu, Met, Ile, Tyr, Phe, Val, Gln, Ala, Trp, Asn, His, Thr, Lys, Ser, Asp, Glu, Arg, Orn, and Gly. The Pro analog prevented coiled-coil formation. The overall stability range was 7.4 kcal/mol from the lowest to the highest analog, indicating the importance of the hydrophobic core and the dramatic effect a single substitution in the core can have upon the stability of the protein fold. In general, the side-chain contribution to the level of stability correlated with side-chain hydrophobicity. Molecular modelling studies, however, showed that packing effects could explain deviations from a direct correlation. In regards to oligomerization state, eight analogs demonstrated the ability to populate exclusively one oligomerization state in benign buffer (0.1 M KCl, 0.05 M K(2)PO(4)(pH 7)). Ile and Val (the beta-branched residues) induced the three-stranded oligomerization state, whereas Tyr, Lys, Arg, Orn, Glu and Asp induced the two-stranded state. Asn, Gln, Ser, Ala, Gly, Phe, Leu, Met and Trp analogs were indiscriminate and populated two-stranded and three-stranded states. Comparison of these results with similar substitutions in position "a" highlights the positional effects of individual residues in defining the stability and numbers of polypeptide chains occurring in a coiled-coil structure. Overall, these results in conjunction with other work now generate a relative thermodynamic stability scale for 19 naturally occurring amino acid residues in either an "a" or "d" position of a two-stranded coiled-coil. Thus, these results will aid in the de novo design of new coiled-coil structures, a better understanding of their structure/function relationships and the design of algorithms to predict the presence of coiled-coils within native protein sequences.     

Helix capping interactions stabilize the N-terminus of the kinesin neck coiled-coil

In an effort to understand how specific structural features within the kinesin neck, a region of the heavy chain located between the catalytic core and stalk domains, may contribute to motor processivity (an ability to remain attached to the microtubule filament), we have prepared several synthetic peptides corresponding to the neck region of human conventional kinesin and determined their secondary structure content and stability by CD spectroscopy. Our results show that the coiled-coil dimerization domain within the human kinesin neck region corresponds to residues 337 to 369 in solution, and thus is in excellent agreement with the recent X-ray crystallographic structures of rat brain kinesin. Further, we show that the first and last heptads of this region are absolutely critical for creating the high stability and association of the dimeric structure. Interestingly, addition of the 7 N-terminal neck-linker residues (330-336) to the coiled-coil domain significantly increased its stability (Delta GdnHCl midpoint of 1 M or an increase of approximately 1.5 kcal/mol), indicating that a strong structural link exists between the neck-linker and coiled-coil region. Subsequent high-resolution structural analysis of the residues located at the junction of the neck-linker and coiled-coil revealed the presence of the two helix capping motifs, the capping box (a reciprocal interaction of Thr 336 with Gln 339) and the hydrophobic staple (a hydrophobic packing interaction of Leu 335 with Trp 340). Substitution of Leu 335 and Thr 336 (the capping residues) with Gly completely eliminated the increased stability of the coiled-coil region observed in the presence of the neck-linker residues. Correspondingly, substitution of Trp 340, the first hydrophobic core d position residue of the coiled-coil, with an Ala residue resulted in a greater than expected decrease in stability and helicity of the coiled-coil structure. Subsequent analysis of the X-ray structure and substitution analysis of Lys 341 revealed that Trp 340 makes an important interchain hydrophobic interaction with Lys 341 of the opposite chain. Taken together these results reveal that a set of strong intra- and inter-chain interactions made up of the helix "capping box," "hydrophobic staple," and the newly identified "Leu-Trp-Lys sandwich" motifs stabilize the kinesin neck coiled-coil structure, thus preventing it from fraying and unfolding.     

Structural resiliency of an EGF-like subdomain bound to its target protein, thrombin.

The thrombin-bound structures of native peptide fragments from the fifth EGF-like domain of thrombomodulin were determined by use of NMR and transferred NOE spectroscopy. The bound peptides assume an EGF-like structure of an antiparallel beta-sheet, a novel structural motif observed for a bound peptide in protein-peptide complexes. There is a remarkable structural resiliency of this structure motif manifested in its ability to accommodate a different number of residues within the disulfide loop. Docking experiments revealed that the key contacts with thrombin are hydrophobic interactions between the side chains of residues Ile 414 and Ile 424 of thrombomodulin and a hydrophobic pocket on the thrombin surface. Residues Leu 415, Phe 419, and Ile 420, which would have been buried in intact EGF-like domains, are unfavorably exposed in the complex of thrombin with the EGF-like thrombomodulin fragment, thus providing a rationale for the enhancement of binding affinity upon the deletion of Ile 420. The unique beta-sheet structures of the bound peptides are specified by the presence of disulfide bridges in the peptides because a corresponding linear thrombomodulin fragment folds into a sheet structure with a different backbone topology. The different bound conformations for the linear and the cyclized peptides indicate that side-chain interactions within a specific environment may dictate the folding of bound peptides in protein-peptide complexes.     

Structure of the 1-36 amino-terminal fragment of human phospholamban by nuclear magnetic resonance and modeling of the phospholamban pentamer

The structure of a 36-amino-acid-long amino-terminal fragment of phospholamban (phospholamban[1-36]) in aqueous solution containing 30% trifluoroethanol was determined by nuclear magnetic resonance. The peptide, which comprises the cytoplasmic domain and six residues of the transmembrane domain of phospholamban, assumes a conformation characterized by two alpha-helices connected by a turn. The residues of the turn are Ile18, Glu19, Met20, and Pro21, which are adjacent to the two phosphorylation sites Ser16 and Thr17. The proline is in a trans conformation. The helix comprising amino acids 22-36 is well determined (the root mean square deviation for the backbone atoms, calculated for a family of 18 nuclear magnetic resonance structures is 0.57 A). Recently, two molecular models of the transmembrane domain of phospholamban were proposed in which a symmetric homopentamer is composed of a left-handed coiled coil of alpha-helices. The two models differ by the relative orientation of the helices. The model proposed by,Simmerman et al. (H.K. Simmerman, Y.M. Kobayashi, J.M. Autry, and L.R. Jones, 1996, J. Biol. Chem. 271:5941-5946), in which the coiled coil is stabilized by a leucine-isoleucine zipper, is similar to the transmembrane pentamer structure of the cartilage oligomeric membrane protein determined recently by x-ray (V. Malashkevich, R. Kammerer, V Efimov, T. Schulthess, and J. Engel, 1996, Science 274:761-765). In the model proposed by Adams et al. (P.D. Adams, I.T. Arkin, D.M. Engelman, and A.T. Brunger, 1995, Nature Struct. Biol. 2:154-162), the helices in the coiled coil have a different relative orientation, i.e., are rotated clockwise by approximately 50 degrees. It was possible to overlap and connect the structure of phospholamban[1-36] derived in the present study to the two transmembrane pentamer models proposed. In this way two models of the whole phospholamban in its pentameric form were generated. When our structure was connected to the leucine-isoleucine zipper model, the inner side of the cytoplasmic domain of the pentamer (where the helices face one another) was lined by polar residues (Gln23, Gln26, and Asn30), whereas the five Arg25 side chains were on the outer side. On the contrary, when our structure was connected to the other transmembrane model, in the inner side of the cytoplasmic domain of the pentamer, the five Arg25 residues formed a highly charged cluster.     

Stabilizing and destabilizing clusters in the hydrophobic core of long two-stranded alpha-helical coiled-coils

Detailed sequence analyses of the hydrophobic core residues of two long two-stranded alpha-helical coiled-coils that differ dramatically in sequence, function, and length were performed (tropomyosin of 284 residues and the coiled-coil domain of the myosin rod of 1086 residues). Three types of regions were present in the hydrophobic core of both proteins: stabilizing clusters and destabilizing clusters, defined as three or more consecutive core residues of either stabilizing (Leu, Ile, Val, Met, Phe, and Tyr) or destabilizing (Gly, Ala, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Arg, Lys, and Trp) residues, and intervening regions that consist of both stabilizing and destabilizing residues in the hydrophobic core but no clusters. Subsequently, we designed a series of two-stranded coiled-coils to determine what defines a destabilizing cluster and varied the length of the destabilizing cluster from 3 to 7 residues to determine the length effect of the destabilizing cluster on protein stability. The results showed a dramatic destabilization, caused by a single Leu to Ala substitution, on formation of a 3-residue destabilizing cluster (DeltaT(m) of 17-21 degrees C) regardless of the stability of the coiled-coil. Any further substitution of Leu to Ala that increased the size of the destabilizing cluster to 5 or 7 hydrophobic core residues in length had little effect on stability (DeltaT(m) of 1.4-2.8 degrees C). These results suggested that the contribution of Leu to protein stability is context-dependent on whether the hydrophobe is in a stabilizing cluster or its proximity to neighboring destabilizing and stabilizing clusters.     

Solid-state NMR reveals structural changes in phospholamban accompanying the functional regulation of Ca2+-ATPase

Calcium transport across the sarcoplasmic reticulum of cardiac myocytes is regulated by a reversible inhibitory interaction between the Ca2+-ATPase and the small transmembrane protein phospholamban (PLB). A nullcysteine analogue of PLB, containing isotope labels in the transmembrane domain or cytoplasmic domain, was reconstituted into membranes in the absence and presence of the SERCA1 isoform of Ca2+-ATPase for structural investigation by cross-polarization magic-angle spinning (CP-MAS) NMR. PLB lowered the maximal hydrolytic activity of SERCA1 and its affinity for calcium in membrane preparations suitable for structural analysis by NMR. Novel backbone amide proton-deuterium exchange CP-MAS NMR experiments on the two PLB analogues co-reconstituted with SERCA1 indicated that labeled residues Leu42 and Leu44 were situated well within the membrane interior, whereas Pro21 and Ala24 lie exposed outside the membrane. Internuclear distance measurements on PLB using rotational resonance NMR indicated that the sequences Pro21-Ala24 and Leu42-Leu44 adopt an alpha-helical structure in pure lipid bilayers, which is unchanged in the presence of Ca2+-ATPase. By contrast, rotational echo double resonance (REDOR) NMR experiments revealed that the sequence Ala24-Gln26 switches from an alpha-helix in pure lipid membranes to a more extended structure in the presence of SERCA1, which may reflect local structural distortions which change the orientations of the transmembrane and cytoplasmic domains. These results suggest that Ca2+-ATPase has a long-range effect on the structure of PLB around residue 25, which promotes the functional association of the two proteins.     

All-trans retinol, vitamin D and other hydrophobic compounds bind in the axial pore of the five-stranded coiled-coil domain of cartilage oligomeric matrix protein.

The potential storage and delivery function of cartilage oligomeric matrix protein (COMP) for cell signaling molecules was explored by binding hydrophobic compounds to the recombinant five-stranded coiled-coil domain of COMP. Complex formation with benzene, cyclohexane, vitamin D3 and elaidic acid was demonstrated through increases in denaturation temperatures of 2-10 degreesC. For all-trans retinol and all-trans retinoic acid, an equilibrium dissociation constant KD = 0.6 microM was evaluated by fluorescence titration. Binding of benzene and all-trans retinol into the hydrophobic axial pore of the COMP coiled-coil domain was proven by the X-ray crystal structures of the corresponding complexes at 0.25 and 0.27 nm resolution, respectively. Benzene binds with its plane perpendicular to the pore axis. The binding site is between the two internal rings formed by Leu37 and Thr40 pointing into the pore of the COMP coiled-coil domain. The retinol beta-ionone ring is positioned in a hydrophobic environment near Thr40, and the 1.1 nm long isoprene tail follows a completely hydrophobic region of the pore. Its terminal hydroxyl group complexes with a ring of the five side chains of Gln54. A mutant in which Gln54 is replaced by Ile binds all-trans retinol with affinity similar to the wild-type, demonstrating that hydrophobic interactions are predominant.