Conserved glycine residues in the cytoplasmic domain of the aspartate receptor play essential roles in kinase coupling and on-off switching

The aspartate receptor of the bacterial chemotaxis pathway serves as a scaffold for the formation of a multiprotein signaling complex containing the receptor and the cytoplasmic pathway components. Within this complex, the receptor regulates the autophosphorylation activity of histidine kinase CheA, thereby controlling the signals sent to the flagellar motor and the receptor adaptation system. The receptor cytoplasmic domain, which controls the on-off switching of CheA, possesses 14 glycine residues that are highly conserved in related receptors. In principle, these conserved glycines could be required for static turns, bends, or close packing in the cytoplasmic domain, or they could be required for conformational dynamics during receptor on-off switching. To determine which glycines are essential and to probe their functional roles, we have substituted each conserved glycine with both alanine and cysteine, and then measured the effects on receptor function in vivo and in vitro. The results reveal a subset of six glycines which are required for receptor function during cellular chemotaxis. Two of these essential glycines (G388 and G391) are located at a hairpin turn at the distal end of the folded cytoplasmic domain, where they are required for the tertiary fold of the signaling subdomain and for CheA kinase activation. Three other essential glycines (G338, G339, and G437) are located at the border between the adaptation and signaling subdomains, where they play key roles in CheA kinase activation and on-off switching. These three glycines form a ring around the four-helix bundle that comprises the receptor cytoplasmic domain, yielding a novel architectural feature termed a bundle hinge. The final essential glycine (G455) is located in the adaptation subdomain where it is required for on-off switching. Overall, the findings confirm that six of the 14 conserved cytoplasmic glycines are essential for receptor function because they enable helix turns and bends required for native receptor structure, and in some cases for switching between the on and off signaling states. An initial working model proposes that the novel bundle hinge enables the four-helix bundle to bend, perhaps during the assembly of the receptor trimer of dimers or during on-off switching. More generally, the findings predict that certain human disease states, including specific cancers, could be triggered by lock-on mutations at essential glycine positions that control the on-off switching of receptors and signaling proteins.     

Signaling domain of the aspartate receptor is a helical hairpin with a localized kinase docking surface: cysteine and disulfide scanning studies.

Cysteine and disulfide scanning has been employed to probe the signaling domain, a highly conserved motif found in the cytoplasmic region of the aspartate receptor of bacterial chemotaxis and related members of the taxis receptor family. Previous work has characterized the N-terminal section of the signaling domain [Bass, R. B., and Falke, J. J. (1998) J. Biol. Chem. 273, 25006-25014], while the present study focuses on the C-terminal section and the interactions between these two regions. Engineered cysteine residues are incorporated at positions Gly388 through Ile419 in the signaling domain, thereby generating a library of receptors each containing a single cysteine per receptor subunit. The solvent exposure of each cysteine is ascertained by chemical reactivity measurements, revealing a periodic pattern of buried hydrophobic and exposed polar residues characteristic of an amphipathic alpha-helix, denoted helix alpha8. The helix begins between positions R392 and Val401, then continues through the last residue scanned, Ile419. Activity assays carried out both in vivo and in vitro indicate that both the buried and exposed faces of this amphipathic helix are critical for proper receptor function and the buried surface is especially important for kinase downregulation. Patterns of disulfide bond formation suggest that helix alpha8, together with the immediately N-terminal helix alpha7, forms a helical hairpin that associates with a symmetric hairpin from the other subunit of the homodimer, generating an antiparallel four helix bundle containing helices alpha7, alpha7', alpha8, and alpha8'. Finally, the protein-interactions-by-cysteine-modification (PICM) method suggests that the loop between helices alpha7 and alpha8 interacts with the kinase CheA and/or the coupling protein CheW, expanding the receptor surface implicated in kinase docking.     

The aspartate receptor cytoplasmic domain: in situ chemical analysis of structure, mechanism and dynamics.

BACKGROUND: Site-directed sulfhydryl chemistry and spectroscopy can be used to probe protein structure, mechanism and dynamics in situ. The aspartate receptor of bacterial chemotaxis is representative of a large family of prokaryotic and eukaryotic receptors that regulate histidine kinases in two-component signaling pathways, and has become one of the best characterized transmembrane receptors. We report here the use of cysteine and disulfide scanning to probe the helix-packing architecture of the cytoplasmic domain of the aspartate receptor. RESULTS: A series of designed cysteine pairs have been used to detect proximities between cytoplasmic helices in the full-length, membrane-bound receptor by measurement of disulfide-bond formation rates. Upon mild oxidation, 25 disulfide bonds from rapidly between three specific pairs of helices, whereas other helix pairs yield no detectable disulfide-bond formation. Further constraints on helix packing are provided by 14 disulfide bonds that retain receptor function in an in vitro kinase regulation assay. Of these functional disulfides, seven lock the receptor in the conformation that constitutively stimulates kinase activity ('lock-on'), whereas the remaining seven retain normal kinase regulation. Finally, disulfide-trapping experiments in the absence of bound kinase reveal large-amplitude relative motions of adjacent helices, including helix translations and rotations of up to 19 A and 180 degrees, respectively. CONCLUSIONS: The 25 rapidly formed and 14 functional disulfide bonds identify helix-helix contacts and their register in the full-length, membrane-bound receptor-kinase complex. The results reveal an extended, rather than compact, domain architecture in which the observed helix-helix interactions are best described by a four-helix bundle arrangement. A cluster of six lock-on disulfide bonds pinpoints a region of the four-helix bundle critical for kinase activation, whereas the signal-retaining disulfides indicate that signal-induced rearrangements of this region are small enough to be accommodated by disulfide-bond flexibility (< or = 1.2 A). In the absence of bound kinase, helix packing within the cytoplasmic domain is highly dynamic.     

Folding topology of the disulfide-bonded dimeric DNA-binding domain of the myogenic determination factor MyoD.

The myogenic determination factor MyoD is a member of the basic-helix-loop-helix (bHLH) protein family. A 68-residue fragment of MyoD encompassing the entire bHLH region (MyoD-bHLH) is sufficient for protein dimerization, sequence-specific DNA binding in vitro, and conversion of fibroblasts into muscle cells. The circular dichroism spectrum of MyoD-bHLH indicates the presence of significant alpha-helical secondary structure; however, the NMR spectrum lacks features of a well-defined tertiary structure. There is a naturally occurring cysteine at residue 135 in mouse MyoD that when oxidized to a disulfide induces MyoD-bHLH to form a symmetric homodimer with a defined tertiary structure as judged by sedimentation equilibrium ultracentrifugation and NMR spectroscopy. Oxidized MyoD-bHLH retains sequence-specific DNA-binding activity, albeit with an apparent 100-1000-fold decrease in affinity. Here, we report the structural characterization of the oxidized MyoD-bHLH homodimer by NMR spectroscopy. Our findings indicate that the basic region is unstructured and flexible, while the HLH region consists of two alpha-helices of unequal length connected by an as yet undetermined loop structure. Qualitative examination of interhelical NOEs suggests several potential arrangements for the two helix 1/helix 2 pairs in the symmetric oxidized dimer. These arrangements were evaluated for whether they could incorporate the disulfide bond, satisfy loop length constraints, and juxtapose the two basic regions. Only a model that aligns helix 1 parallel to helix 1' and antiparallel to helix 2 was consistent with all constraints. Thus, an antiparallel four-helix bundle topology is proposed for the symmetric dimer. This topology is hypothesized to serve as a general model for other bHLH protein domains.     

The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima

The bacterial histidine autokinase CheA contains a histidine phosphotransfer (Hpt) domain that accepts a phosphate from the catalytic domain and donates the phosphate to either target response regulator protein, CheY or CheB. The Hpt domain forms a helix-bundle structure with a conserved four-helix bundle motif and a variable fifth helix. Observation of two nearly equally populated conformations in the crystal structure of a Hpt domain fragment of CheA from Thermotoga maritima containing only the first four helices suggests more mobility in a tightly packed helix bundle structure than previously thought. In order to examine how the structures of Hpt domain homologs may differ from each other particularly in the conformation of the last helix, and whether an alternative conformation exists in the intact Hpt domain in solution, we have solved a high-resolution, solution structure of the CheA Hpt from T. maritima and characterized the backbone dynamics of this protein. The structure contains a four-helix bundle characteristic of histidine phosphotransfer domains. The position and orientation of the fifth helix resembles those in known Hpt domain crystal and solution structures in other histidine kinases. The alternative conformation that was reported in the crystal structure of the CheA Hpt from T. maritima missing the fifth helix is not detected in the solution structure, suggesting a role for the fifth helix in providing stabilizing forces to the overall structure.     

Functional Suppression of HAMP Domain Signaling Defects in the E. coli Serine Chemoreceptor

HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric Escherichia coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: unmodified E residues promote kinase-off output, and methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer 3 of the HAMP bundle and followed in vivo CheA activity with an assay based on Förster resonance energy transfer. We found that an alanine or a serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E and S255A) shifted Tsr output toward the kinase-off state, but two others (S255G and L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations.     

Mutating the four extracellular cysteines in the chemokine receptor CCR6 reveals their differing roles in receptor trafficking,ligand binding,and signaling

CCR6 is the receptor for the chemokine MIP-3 alpha/CCL20. Almost all chemokine receptors contain cysteine residues in the N-terminal domain and in the first, second, and third extracellular loops. In this report, we have studied the importance of all cysteine residues in the CCR6 sequence using site-directed mutagenesis and biochemical techniques. Like all G protein-coupled receptors, mutating disulfide bond-forming cysteines in the first (Cys118) and second (Cys197) extracellular loops in CCR6 led to complete elimination of receptor activity, which for CCR6 was also associated with the accumulation of the receptor intracellularly. Although two additional cysteines in the N-terminal region and the third extracellular loop, which are present in almost all chemokine receptors, are presumed to form a disulfide bond, this has not been demonstrated experimentally for any of these receptors. We found that mutating the cysteines in the N-terminal domain (Cys36) and the third extracellular loop (Cys288) neither significantly affected receptor surface expression nor completely abolished receptor function. Importantly, contrary to several previous reports, we demonstrated directly that instead of forming a disulfide bond, the N-terminal cysteine (Cys36) and the third extracellular loop cysteine (Cys288) contain free SH groups. The cysteine residues (Cys36 and Cys288), rather than forming a disulfide bond, may be important per se. We propose that CCR6 forms only a disulfide bond between the first (Cys118) and second (Cys197) extracellular loops, which confines a helical bundle together with the N-terminus adjacent to the third extracellular loop, creating the structural organization critical for ligand binding and therefore for receptor signaling.     

Site-specific disulfide capture of agonist and antagonist peptides on the C5a receptor

The manner by which peptidic ligands bind and activate their corresponding G-protein-coupled receptors is not well understood. One of the better characterized peptidic ligands is the chemotactic cytokine complement factor 5a (C5a), a 74-amino acid helical bundle. Previous studies showed 6-mer peptide analogs derived from the C terminus of the C5a ligand can bind to C5aR (Kd values approximately 0.1-1 microm) and either agonize or antagonize the receptor (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-3400). Here, we provide direct biochemical data using disulfide trapping to support a model that these peptides bind within a transmembrane helical triad formed by alpha-helices III, VI, and VII. We show that the three amino acids on the C terminus of the peptide analogs bind too weakly to exert a functional effect themselves. However, when a cysteine residue is placed on their N terminus they can be trapped by disulfide interchange to specific cysteines in helix III and VI and not to other cysteines, engineered into the C5aR. The trapped peptides function as agonists or partial antagonists, similar to the non-covalent parents from which they were derived. These data help to further refine the binding mode for C5a to the C5aR and suggest an approach and a binding site that may be applicable to studying other peptide binding receptors.     

A possible degree of motional freedom in bacterial chemoreceptor cytoplasmic domains and its potential role in signal transduction

We describe an array of gaps in an antiparallel four-helix bundle structure, the cytoplasmic domains of bacterial chemoreceptors. For a given helix, the side chain interactions that define a helix’s position are analyzed in terms of residue interfaces, the most important of which are a-a, g-g, d-d, g-d, and a-d. It was found that the interdigitation of the side groups does not entirely fill the space along the long axis of the structure, which results in a rather regular array of gaps. A simulated piston motion of helix CD1 along the helical axis direction by 1.2Å shows that 85% of the side chain interactions still satisfy Van der Waals criteria, while the remaining clashes could be avoided by small rotations of side chains. Therefore, two states could exist in the structure, related by a piston motion. Analysis of the crystal structure of a small four-helix bundle, the P1_short domain of CheA in Thermotoga Maritima, reveals that the two coexisting states related by a 1.3-1.7Å piston motion are defined by the same mechanism. This two-state model is a plausible candidate mechanism for the long distance signal transduction in bacterial chemoreceptors and is qualitatively consistent with literature chemoreceptor mutagenesis results. Such a mechanism could exist in many other structures with interdigitating α-helices.     

Crystal structure of the CheA histidine phosphotransfer domain that mediates response regulator phosphorylation in bacterial chemotaxis

The x-ray crystal structure of the P1 or H domain of the Salmonella CheA protein has been solved at 2.1-A resolution. The structure is composed of an up-down up-down four-helix bundle that is typical of histidine phosphotransfer or HPt domains such as Escherichia coli ArcB(C) and Saccharomyces cerevisiae Ypd1. Loop regions and additional structural features distinguish all three proteins. The CheA domain has an additional C-terminal helix that lies over the surface formed by the C and D helices. The phosphoaccepting His-48 is located at a solvent-exposed position in the middle of the B helix where it is surrounded by several residues that are characteristic of other HPt domains. Mutagenesis studies indicate that conserved glutamate and lysine residues that are part of a hydrogen-bond network with His-48 are essential for the ATP-dependent phosphorylation reaction but not for the phosphotransfer reaction with CheY. These results suggest that the CheA-P1 domain may serve as a good model for understanding the general function of HPt domains in complex two-component phosphorelay systems.     

Replacement of helix 1' enhances the lipid binding activity of apoE3 N-terminal domain

The N-terminal domain of human apolipoprotein E (apoE-NT) harbors residues critical for interaction with members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid free apoE-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational adaptation is required for manifestation of LDLR binding ability. To investigate the structural basis for this conformational change, the short helix connecting helix 1 and 2 in the four-helix bundle was replaced by the sequence NPNG, introducing a beta-turn. Recombinant helix-to-turn (HT) variant apoE3-NT was produced in Escherichia coli, isolated and characterized. Stability studies revealed a denaturation transition midpoint of 1.9 m guanidine hydrochloride for HT apoE3-NT vs. 2.5 M for wild-type apoE3-NT. Wild-type and HT apoE3-NT form dimers in solution via an intermolecular disulfide bond. Native PAGE showed that reconstituted high-density lipoprotein prepared with HT apoE3-NT have a diameter in the range of 9 nm and possess binding activity for the LDLR on cultured human skin fibroblasts. In phospholipid vesicle solubilization assays, HT apoE3-NT was more effective than wild-type apoE3-NT at inducing a time dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. In lipoprotein binding assays, HT apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent that wild-type apoE3-NT. The results indicate that a mutation at one end of the apoE3-NT four-helix bundle markedly enhances the lipid binding activity of this protein. In the context of lipoprotein associated full-length apoE, increased lipid binding affinity of the N-terminal domain may alter the balance between receptor-active and -inactive conformational states.     

Diagnostic cross-linking of paired cysteine pairs demonstrates homologous structures for two chemoreceptor domains with low sequence identity

Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand‐recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence‐divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg_E from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross‐linking between pairs of introduced cysteines. A homology model of the Trg_E domain was created using the homodimeric, four‐helix bundle structure of the Tar_S domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg_E periplasmic domain is very similar to the Tar_S domain. Diagnostic cross‐linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.     

Interaction of Met297 in the seventh transmembrane segment of the tachykinin NK2 receptor with neurokinin A

We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.     

Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.     

A heuristic approach to predicting the tertiary structure of bovine somatotropin

A combination of a heuristic approach and energy minimization was used to predict the three-dimensional structure of bovine somatotropin (bSt), also known as bovine growth hormone, a protein of 191 amino acids. The starting points for energy minimizations were generated from the following two types of inputs: (a) the amino acid sequence and (b) the heuristic inputs, which were derived according to physical, chemical, and biological principles by piecing together all useful information available. The predicted 3-D structure of the bSt molecule has all the features observed in four-helix bundle proteins. The four alpha-helices in bSt are intimately packed to form an assembly with an approximately square cross section. All the adjacent alpha-helices are antiparallel, with a somewhat tilted angle between each of the adjacent pairs so that the assembly of the four helices looks like a left-handed twisted bundle. There are two disulfide bonds in the bSt structure: one "hooking" the middle of a long loop with helix 4 so as to pull the long loop onto the surface of the helix bundle and the other "hooking" the C-terminal segment with the same helix so as to force the C-terminal segment to bend toward the helix bundle. As a consequence, a considerable part of the surface of the four-helix bundle is closely packed or intimately embraced by the loop segments. The predicted bSt structure has a hydrophobic core and a hydrophilic exterior surface. The energetic analysis of the predicted bSt structure indicates that the interaction between helices and loops plays a dominant role in stabilizing the four-helix bundle structure from the viewpoint of both electrostatic and nonbonded interactions. A technique called FOLD was meanwhile developed, by which one can fold a polypeptide chain into any shape as desired. This tool proved to be very useful during the heuristic model-building process.     

A Unified Scheme for Initiation and Conformational Adaptation of Human Apolipoprotein E N-terminal Domain upon Lipoprotein Binding and for Receptor Binding Activity

We report here a high-resolution NMR structure of the complete receptor-binding domain of human apolipoprotein E3 (apoE3-NT). Similar to the crystal structure of apoE-NT, the NMR structure displayed an elongated four-helix bundle. However, additional unique structural features were also observed. The segments in the N and C termini, which were missing in the crystal structure, formed α-helices having extensive tertiary contacts with the bundle, which oriented these short helices at specific positions for receptor binding activity. Several buried hydrophilic residues observed in the bundle were located strategically between helices 1 and 2 and between helices 3 and 4, significantly destabilizing these helix-helix interfaces. In addition, these buried hydrophilic residues formed buried H-bonds, which may play a key role in specific lipid-free helix bundle recovery. A short helix, nHelix C, was fully solvent-exposed and nearly perpendicular to the bundle. This short helix likely plays a critical role in initiating protein-lipid interaction, causing a preferred conformational adaptation of the bundle at the weaker helix-helix interfaces. This produces an open conformation with two lobes of helices, helices 1 and 4 and helices 2 and 3, which may be the competent conformation for receptor binding activity. Thus, the NMR structure suggests a unified scheme for the initiation and helix bundle opening of apoE-NT upon lipoprotein-binding and for receptor binding activity.Human apolipoprotein E (apoE)² is a 299-residue plasma-exchangeable apolipoprotein with the primary function of transporting lipids from one tissue to another. ApoE performs its functions via interactions with the low-density lipoprotein receptor (LDLR) superfamily (1). The high affinity binding of apoE to the receptors allows apoE-associated lipoprotein particles to be targeted for endocytosis and intracellular degradation. As a subclass of high-density lipoprotein, apoE also influences both cholesterol efflux and influx, thus playing an important role in reverse cholesterol transport (2, 3). Three major isoforms of apoE have been identified: ApoE3 has a cysteine at position 112 and an arginine at position 158, whereas apoE2 has cysteines and apoE4 has arginines at both positions. Although these isoforms differ in only two residues, they show profound functional differences. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer disease, cognitive functioning, immunoregulation, cell signaling, and infectious diseases (4).ApoE is a two-domain protein that contains a 22-kDa N-terminal domain (residues 1-191) and a 10-kDa C-terminal domain (residues 216-299) linked by a protease sensitive hinge region. Although the N-terminal domain of apoE (apoE-NT) is primarily responsible for LDL-receptor binding, the C-terminal domain (apoE-CT) binds to lipoprotein with a high affinity (1). The x-ray crystal structure of lipid-free apoE-NT reveals a globular up-and-down four-helix bundle (5). The major receptor-binding region, residues 130-150, is located on the fourth helix. The positively charged residues (Lys and Arg) in this region are critical for interacting with the negatively charged residues in the receptor (1, 6). This structure only contains residues 24-164, whereas the rest of the regions are disordered. However, experimental evidence indicates that regions beyond residues 24-164 are also critical for LDLR binding activity. For example, deletion of residues 167-185 reduces the apoE3 LDLR binding activity to 15%, and a mutation at position Arg-172 reduces the LDLR binding activity to only ∼2% (7). In addition, an E3K mutant of apoE3 enhances the LDLR binding activity by 2-fold (8). Although the x-ray crystal structure of apoE-NT provides a structural explanation of the major receptor-binding domain of apoE, this structure does not explain the above described important experimental data. Thus, our understanding of the structural basis of the receptor binding activity of apoE remains incomplete.Previous studies using truncation mutants have shown that apoE(1-183) displays nearly 100% LDLR binding activity (9), suggesting that residues beyond position 183 are not important in LDLR binding. We report here a high-resolution NMR structure of the complete LDLR-binding domain of apoE3. Interestingly, our NMR structure shows that the N and C termini form α-helical structures that have extensive contacts with the helix bundle, orienting the two termini at specific positions for potential receptor binding. The NMR structure also displays several novel structural features that may provide the structural basis of a unified scheme for initiation and conformational adaptation of apoE-NT upon lipoprotein binding.     

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.     

Different conformations of the kinase-on and kinase-off signaling states in the Aer HAMP domain

HAMP domains are sensory transduction modules that connect input and output domains in diverse signaling proteins from archaea, bacteria, and lower eukaryotes. Here, we employed in vivo disulfide cross-linking to explore the structure of the HAMP domain in the Escherichia coli aerotaxis receptor Aer. Using an Aer HAMP model based on the structure of Archaeoglobus fulgidus Af1503-HAMP, the closest residue pairs at the interface of the HAMP AS-1 and AS-2' helices were determined and then replaced with cysteines and cross-linked in vivo. Except for a unique discontinuity in AS-2, the data suggest that the Aer HAMP domain forms a parallel four-helix bundle that is similar to the structure of Af1503. The HAMP discontinuity was associated with a segment of AS-2 that was recently shown to interact with the Aer-PAS sensing domain. The four-helix HAMP bundle and its discontinuity were maintained in both the kinase-on and kinase-off states of Aer, although differences in the rates of disulfide formation also indicated the existence of different HAMP conformations in the kinase-on and kinase-off states. In particular, the kinase-on state was accompanied by significantly increased disulfide formation rates at the distal end of the HAMP four-helix bundle. This indicates that HAMP signaling may be associated with a tilting of the AS-1 and AS-2' helices, which may be the signal that is transmitted to the kinase control region of Aer.     

The lipid-associated conformation of the low density lipoprotein receptor binding domain of human apolipoprotein E

Apolipoprotein E (apoE) is a 34-kDa exchangeable apolipoprotein that regulates metabolism of plasma lipoproteins by functioning as a ligand for members of the LDL receptor family. The receptor-binding region localizes to the vicinity of residues 130-150 within its independently folded 22-kDa N-terminal domain. In the absence of lipid, this domain exists as a receptor-inactive, globular four-helix bundle. Receptor recognition properties of this domain are manifest upon lipid association, which is accompanied by a conformational change in the protein. Fluorescence resonance energy transfer has been used to monitor helix repositioning, which accompanies lipid association of the apoE N-terminal domain. Site-directed mutagenesis was used to replace naturally occurring Trp residues with phenylalanine, creating a Trp-null apoE3 N-terminal domain (residues 1-183). Subsequently, tyrosine residues in helix 2, helix 3, or helix 4 were converted to Trp, generating single Trp mutant proteins. The lone cysteine at position 112 was covalently modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, which serves as an energy acceptor from excited tryptophan residues. Fluorescence resonance energy transfer analysis of apoE N-terminal domain variants in phospholipid disc complexes suggests that the helix bundle opens to adopt a partially extended conformation. A model is presented that depicts a tandem arrangement of the receptor-binding region of the protein in the disc complex, corresponding to its low density lipoprotein receptor-active conformation.     

Self-association of the histidine kinase CheA as studied by pulsed dipolar ESR spectroscopy

Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells. By measuring the intermolecular dipolar interactions sensed by spin-labels distributed over the protein surface, we show that the soluble CheA kinase aggregates to a small extent through interactions mediated by its regulatory (P5) domain. Direct dipolar distance measurements confirm that a hydrophobic surface at the periphery of P5 subdomain 2 associates CheA dimers in solution. This result is further supported by differential disulfide cross-linking from engineered cysteine reporter sites. We suggest that the periphery of P5 is an interaction site on CheA for other similar hydrophobic surfaces and plays an important role in structuring the signaling particle.