Characterization of a membrane protein folding motif, the Ser zipper, using designed peptides

Polar residues play important roles in the association of transmembrane helices and the stabilities of membrane proteins. Although a single Ser residue in a transmembrane helix is unable to mediate a strong association of the helices, the cooperative interactions of two or more appropriately placed serine hydroxyl groups per helix has been hypothesized to allow formation of a "serine zipper" that can stabilize transmembrane helix association. In particular, a heptad repeat Sera Xxx Xxx Leud Xxx Xxx Xxx (Xxx is a hydrophobic amino acid) appears in both antiparallel helical pairs of polytopic membrane proteins as well as the parallel helical dimerization motif found in the murine erythropoietin receptor. To examine the intrinsic conformational preferences of this motif independent of its context within a larger protein, we synthesized a peptide containing three copies of a SeraLeud heptad motif. Computational results are consistent with the designed peptide adopting either a parallel or antiparallel structure, and conformational search calculations yield the parallel dimer as the lowest energy configuration, which is also significantly more stable than the parallel trimer. Analytical ultracentrifugation indicated that the peptide exists in a monomer-dimer equilibrium in dodecylphosphocholine micelles. Thiol disulfide interchange studies showed a preference for forming parallel dimers in micelles. In phospholipid vesicles, only the parallel dimer was formed. The stability of the SerZip peptide was studied in vesicles prepared from phosphatidylcholine (PC) lipids of different chain length: POPC (C16:0C18:1 PC) and DLPC (C12:0PC). The stability was greater in POPC, which has a good match between the length of the hydrophobic region of the peptide and the bilayer length. Finally, mutation to Ala of the Ser residues in the SerZip motif gave rise to a relatively small decrease in the stability of the dimer, indicating that packing interactions rather than hydrogen-bonding provided the primary driving force for association.     

Thermodynamics of glycophorin A transmembrane helix dimerization in C14 betaine micelles

We have used sedimentation equilibrium analytical ultracentrifugation to measure the free energy change for the glycophorin A transmembrane helix-helix dimerization in C14 betaine micelles. By varying the amount of micellar C14 betaine, we show that the protein association reaction in the micellar C14 phase behaves as an ideal-dilute solution. In this hydrophobic environment, the mole-fraction standard state free energy change for self-association of the SNGpA99 glycophorin A construct is -5.7 (+/-0.3, N=5) kcal mol(-1) at 25 degrees C. Compared with previous results carried out in C(8)E(5) micellar solutions, the free energy of dimerization is 1.3 kcal mol(-1) less favorable in C14 betaine micelles. In contrast, when considered on a per-interface basis, the formation of the glycophorin A transmembrane dimer in C14 betaine micelles may be more favorable than the association of several designed transmembrane peptides.     

Computation and mutagenesis suggest a right-handed structure for the synaptobrevin transmembrane dimer.

Biological membrane fusion involves a highly precise and ordered set of protein-protein interactions. Synaptobrevin is a key player in this process. Mutagenesis studies of its single transmembrane segment suggest that it dimerizes in a sequence specific manner. Using the computational methods developed for the successful structure prediction of the glycophorin A transmembrane dimer, we have calculated a structural model for the synaptobrevin dimer. Our computational search yields a well-populated cluster of right-handed structures consistent with the experimentally determined dimerization motif. The three-dimensional structure contains an interface formed primarily by leucine and isoleucine side-chain atoms and has no interhelical hydrogen bonds. The model is the first three-dimensional picture of the synaptobrevin transmembrane dimer and provides a basis for further focused experimentation on its structure and association thermodynamics.     

A designed protein with packing between left-handed and right-handed helices

A common motif in protein structures is the assembly of alpha-helices. Natural alpha-helical assemblies, such as helical bundles and coiled coils, consist of multiple right-handed alpha-helices. Here we design a protein complex containing both left-handed and right-handed helices, with peptides of D- and L-amino acids, respectively. The two peptides, D-Acid and L-Base, feature hydrophobic heptad repeats and are designed to pack against each other in a "knobs-into-holes" manner. In solution, the peptides form a stable, helical heterotetramer with tight packing in the most solvent-protected core. This motif may be useful for designing protease-resistant, helical D-peptide ligands against biological protein targets.     

Dimerization and folding of LC8, a highly conserved light chain of cytoplasmic dynein

Cytoplasmic dynein is a multisubunit ATPase that transforms chemical energy into motion along microtubules. LC8, a 10 kDa light chain subunit of the dynein complex, is highly conserved with 94% sequence identity between Drosophila and human. The precise function of this protein is unknown, but its ubiquitous expression and conservation suggest a critical role in the function of the dynein motor complex. We have overexpressed LC8 from Drosophila melanogaster and characterized its dimerization and folding using analytical ultracentrifugation, size-exclusion chromatography, circular dichroism, and fluorescence spectroscopy. Sedimentation equilibrium measurements of LC8 at pH 7 reveal a reversible monomer-dimer equilibrium with a dissociation constant of 12 microM at 4 degrees C. At lower pH, LC8 dissociates to a monomer, with a transition midpoint at pH 4.8. Far-UV CD and fluorescence spectra demonstrate that pH-dissociated LC8 retains native secondary and tertiary structures, while the diminished near-UV CD signal shows loss of quaternary structure. The observation that dimeric LC8 dissociates at low pH can be explained by titration of a histidine pair in the dimer interface. Equilibrium denaturation experiments with a protein concentration range spanning almost 2 orders of magnitude indicate that unfolding of LC8 dimer is a two-stage process, in which global unfolding is preceded by dissociation to a folded monomer. The nativelike tertiary structure of the monomer suggests a role for the monomer-dimer equilibrium of LC8 in dynein function.     

Insights into dimerization and four-helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli

The GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long alpha-helices at the NH(2)-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential alpha-helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long alpha-helical tail portion (GrpE1-88) is unable to form a dimer, most likely due to a decrease in alpha-helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short alpha-helices (GrpE1-138 and GrpE88-197) are able to form a dimer and presumably the four-helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer-dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short alpha-helices (GrpE1-112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation.     

Structural and thermodynamic insight into the process of "weak" dimerization of the ErbB4 transmembrane domain by solution NMR

Specific helix-helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure-function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651-678)(2) form a right-handed parallel dimer through the N-terminal double GG4-like motif A(655)GxxGG(660) in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer-dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true ("ideal") solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix-helix interactions, which may be critical for membrane protein activity.     

The membrane environment modulates self-association of the human GpA TM domain—Implications for membrane protein folding and transmembrane signaling

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.     

Detection of alpha-helical coiled-coil dimer formation by spin-labeled synthetic peptides: a model parallel coiled-coil peptide and the antiparallel coiled coil formed by a replica of the ProP C-terminus

Electron paramagnetic resonance spectroscopy was used to determine relative peptide orientation within homodimeric, alpha-helical coiled-coil structures. Introduction of cysteine (Cys) residues into peptides/proteins for spin labeling allows detection of their oligomerization from exchange broadening or dipolar interactions between residues within 25 A of each other. Two synthetic peptides containing Cys substitutions were used: a 35-residue model peptide and the 30-residue ProP peptide. The model peptide is known to form a stable, parallel homodimeric coiled coil, which is partially destabilized by Cys substitutions at heptad a and d positions (peptides C30a and C33d). The ProP peptide, a 30-residue synthetic peptide, corresponds to residues 468-497 of osmoregulatory transporter ProP from Escherichia coli. It forms a relatively unstable, homodimeric coiled coil that is predicted to be antiparallel in orientation. Cys was introduced in heptad g positions of the ProP peptide, near the N-terminus (K473C, creating peptide C473g) or closer to the center of the sequence (E480C, creating peptide C480g). In contrast to the destabilizing effect of Cys substitution at the core heptad a or d positions of model peptides C30a and C33d, circular dichroism spectroscopy showed that Cys substitutions at the heptad g positions of the ProP peptide had little or no effect on coiled-coil stability. Thermal denaturation analysis showed that spin labeling increased the stability of the coiled coil for all peptides. Strong exchange broadening was detected for both C30a and C33d, in agreement with a parallel structure. EPR spectra of C480g had a large hyperfine splitting of about 90 G, indicative of strong dipole-dipole interactions and a distance between spin-labeled residues of less than 9 A. Spin-spin interactions were much weaker for C473g. These results supported the hypothesis that the ProP peptide primarily formed an antiparallel coiled coil, since formation of a parallel dimer should result in similar spin-spin interactions for the spin-labeled Cys at both sites.     

Structural analysis of the chromosome segregation protein Spo0J from Thermus thermophilus

Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The plasmid partitioning loci encode two proteins, ParA and ParB, and a cis-acting centromere-like site denoted parS. The chromosomally encoded homologues of ParA and ParB, Soj and Spo0J, play an active role in chromosome segregation during bacterial cell division and sporulation. Spo0J is a DNA-binding protein that binds to parS sites in vivo. We have solved the X-ray crystal structure of a C-terminally truncated Spo0J (amino acids 1-222) from Thermus thermophilus to 2.3 A resolution by multiwavelength anomalous dispersion. It is a DNA-binding protein with structural similarity to the helix-turn-helix (HTH) motif of the lambda repressor DNA-binding domain. The crystal structure is an antiparallel dimer with the recognition alpha-helices of the HTH motifs of each monomer separated by a distance of 34 A corresponding to the length of the helical repeat of B-DNA. Sedimentation velocity and equilibrium ultracentrifugation studies show that full-length Spo0J exists in a monomer-dimer equilibrium in solution and that Spo0J1-222 is exclusively monomeric. Sedimentation of the C-terminal domain of Spo0J shows it to be exclusively dimeric, confirming that the C-terminus is the primary dimerization domain. We hypothesize that the C-terminus mediates dimerization of Spo0J, thereby effectively increasing the local concentration of the N-termini, which most probably dimerize, as shown by our structure, upon binding to a cognate parS site.     

A conformational rearrangement in gramicidin A: from a double-stranded left-handed to a single-stranded right-handed helix.

A conformational transition is described for the polypeptide, gramicidin A, in which a dimer that forms a left-handed intertwined antiparallel helix is converted to a single-stranded amino terminus to amino terminus right-handed helix. The starting structure is determined here by solution NMR methods while reference is made to the well-established folding motif of gramicidin in a lipid bilayer for the ultimate conformation of this transition. Furthermore, an organic solvent system of benzene and ethanol in which gramicidin has a unique conformation is identified. This conformation is shown to be very similar to that derived from X-ray diffraction of crystals prepared from a similar solvent system.     

Simultaneous formation of right- and left-handed anti-parallel coiled-coil interfaces by a coil2 fragment of human lamin A

The elementary building block of all intermediate filaments (IFs) is a dimer featuring a central α-helical rod domain flanked by the N- and C-terminal end domains. In nuclear IF proteins (lamins), the rod domain consists of two coiled-coil segments, coil1 and coil2, that are connected by a short non-helical linker. Coil1 and the C-terminal part of coil2 contain the two highly conserved IF consensus motifs involved in the longitudinal assembly of dimers. The previously solved crystal structure of a lamin A fragment (residues 305-387) corresponding to the second half of coil2 has yielded a parallel left-handed coiled coil. Here, we present the crystal structure and solution properties of another human lamin A fragment (residues 328-398), which is largely overlapping with fragment 305-387 but harbors a short segment of the tail domain. Unexpectedly, no parallel coiled coil forms within the crystal. Instead, the α-helices are arranged such that two anti-parallel coiled-coil interfaces are formed. The most significant interface has a right-handed geometry, which is accounted for by a characteristic 15-residue repeat pattern that overlays with the canonical heptad repeat pattern. The second interface is a left-handed anti-parallel coiled coil based on the predicted heptad repeat pattern. In solution, the fragment reveals only a weak dimerization propensity. We speculate that the C-terminus of coil2 might unzip, thereby allowing for a right-handed coiled-coil interface to form between two laterally aligned dimers. Such an interface might co-exist with a heterotetrameric left-handed coiled-coil assembly, which is expected to be responsible for the longitudinal A_CN contact.     

Dimerization of the erythropoietin receptor transmembrane domain in micelles

Erythropoietin receptor (EpoR) homodimerization is an initial regulatory step in erythrocyte formation. Receptor dimers form before ligand binding, suggesting that association between receptor proteins is dependent on the receptor itself. EpoR dimerization is an essential step in erythropoiesis, and misregulation of this dimerization has been implicated in several disease states, including multi-lineage leukemias; nevertheless, how EpoR regulates its own dimerization is unclear. In vivo experiments suggest the single-pass transmembrane helix is the strongest candidate for driving ligand-independent association. To address the self-association potential of this transmembrane segment, we studied its interaction energetics in micelles by utilizing a previously successful Staphylococcal nuclease (SN-EpoR TM) fusion protein. This fusion protein strategy allows expression of the EpoR transmembrane domain in Escherichia coli independent of the other EpoR domains. Sedimentation equilibrium analytical ultracentrifugation of the detergent-solubilized SN-EpoR TM demonstrated that the murine EpoR transmembrane domain self-associates to form dimers. Although this interaction is not as stable as the dimerization of the well-studied glycophorin A transmembrane dimer, the murine EpoR transmembrane domain dimer is more stable than the interactions of the colon carcinoma kinase 4 transmembrane domain. The same experiments with the human EpoR transmembrane domain, which differs from the mouse sequence by only three residues, revealed a less favorable interaction than that of the murine sequence and is only slightly more favorable than that expected for non-preferential binding. These results suggest that the mouse and human receptor proteins may differ in the roles they play in signaling.     

The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase

The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. Accurate determination of the dimer dissociation constant and the role of the N-finger (residues 1-7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissociation constant of the wild-type protein was determined to be 14.0microM by analytical ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and analytical ultracentrifugation. A single mutation of M6A was found to be critical to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K(i) of 2.20mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.     

Dimeric structure of transmembrane domain of amyloid precursor protein in micellar environment

Some pathogenic mutations associated with Alzheimer's disease are thought to affect structural-dynamic properties and the lateral dimerization of amyloid precursor protein (APP) in neuron membrane. Dimeric structure of APP transmembrane fragment Gln(686)-Lys(726) was determined in membrane-mimicking dodecylphosphocholine micelles using high-resolution NMR spectroscopy. The APP membrane-spanning α-helix Lys(699)-Lys(724) self-associates in a left-handed parallel dimer through extended heptad repeat motif I(702)X(3)M(706)X(2)G(709)X(3)A(713)X(2)I(716)X(3)I(720)X(2)I(723), whereas the juxtamembrane region Gln(686)-Val(695) constitutes the nascent helix, also sensing the dimerization. The dimerization mechanism of APP transmembrane domain has been described at atomic resolution for the first time and is important for understanding molecular events of APP sequential proteolytical cleavage resulting in amyloid-β peptide.     

The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps

We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.     

Self-assembly of coiled-coil tetramers in the 1.40 A structure of a leucine-zipper mutant

The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel double-stranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.     

Polar residue tagging of transmembrane peptides

Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments. The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes.     

Complex interactions at the helix-helix interface stabilize the glycophorin A transmembrane dimer

To explore the residue interactions in the glycophorin A dimerization motif, an alanine scan double mutant analysis at the helix-helix interface was carried out. These data reveal a combination of additive and coupled effects. The majority of the double mutants are found to be equally or slightly more stable than would be predicted by the sum of the energetic cost of the single-point mutants. The proximity of the mutated sites is not related to the presence of coupling between those sites. Previous studies reveal that a single face of the glycophorin A monomer contains a specific glycine-containing motif (GxxxG) that is thought to be a driving force for the association of transmembrane helices. Double mutant cycles suggest that the relationship of the GxxxG motif to the remainder of the helix-helix interface is complex. Sequences containing mutations that abolish the GxxxG motif retain an ability to dimerize, while a sequence containing a GxxxG motif appears unable to form dimers. The energetic effects of weakly coupled and additive double mutants can be explained by changes in van der Waals interactions at the dimer interface. These results emphasize the fact that the sequence context of the dimer interface modulates the strength of the glycophorin A GxxxG-mediated transmembrane dimerization reaction.