Transmembrane Aspartate Research Articles

Custom Design of a Humidifier Chamber for InMeso Crystallization.

Membrane proteins are indispensable for every living organism, yet their structural organization remains underexplored. Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography, which have significantly increased the structural coverage of membrane proteins across various kingdoms, certain scientific methods, such as time-resolved crystallography, still mostly rely on crystallization techniques, such as lipidic cubic phase (LCP) or in meso crystallization. In this study, we present an open-access blueprint for a humidity control chamber designed for LCP/in meso crystallization experiments using a Gryphon crystallization robot. Using this chamber, we have obtained crystals of a transmembrane aspartate transporter GltTk from Thermococcus kodakarensis in a lipidic environment using in meso crystallization. The data collected from these crystals allowed us to perform an analysis of lipids bound to the transporter. With this publication of our open-access design of a humidity chamber, we aim to improve the accessibility of in meso protein crystallization for the scientific community.

A negatively charged transmembrane aspartate residue controls activation of the relaxin-3 receptor RXFP3

Relaxin-3 is an insulin/relaxin superfamily neuropeptide involved in the regulation of food intake and stress response via activation of its cognate receptor RXFP3, an A-class G protein-coupled receptor (GPCR). In recent studies, a highly conserved ExxxD motif essential for binding of relaxin-3 has been identified at extracellular end of the second transmembrane domain (TMD2) of RXFP3. For most of the A-class GPCRs, a highly conserved negatively charged Asp residue (Asp2.50 using Ballesteros–Weinstein numbering and Asp128 in human RXFP3) is present at the middle of TMD2. To elucidate function of the conserved transmembrane Asp128, in the present work we replaced it with other residues and the resultant RXFP3 mutants all retained quite high ligand-binding potency, but their activation and agonist-induced internalization were abolished or drastically decreased. Thus, the negatively charged transmembrane Asp128 controlled transduction of agonist-binding information from the extracellular region to the intracellular region through maintaining RXFP3 in a metastable state for efficient conformational change induced by binding of an agonist.

Intramembrane-cleaving Proteases

In the past decade or so, membrane-embedded proteases that carry out hydrolysis on the transmembrane region of their substrates have been discovered. These I-CLiPs2 (1) somehow create an environment for water and the hydrophilic residues needed for catalysis and bend or unwind their helical substrates to make the amide bonds susceptible to hydrolysis. Despite the distinction of being membrane-embedded and cleaving TMDs, the residues essential for catalysis by these I-CLiPs are virtually the same as those found in aqueous proteases, clear examples of convergent evolution toward a common mechanism. Described herein are the different types of I-CLiPs and an update on their structural and mechanistic features and biological roles.

Evidence that both ligand binding and covalent adaptation drive a two-state equilibrium in the aspartate receptor signaling complex.

The transmembrane aspartate receptor of bacterial chemotaxis regulates an associated kinase protein in response to both attractant binding to the receptor periplasmic domain and covalent modification of four adaptation sites on the receptor cytoplasmic domain. The existence of at least 16 covalent modification states raises the question of how many stable signaling conformations exist. In the simplest case, the receptor could have just two stable conformations ("on" and "off") yielding the two-state behavior of a toggle-switch. Alternatively, covalent modification could incrementally shift the receptor between many more than two stable conformations, thereby allowing the receptor to function as a rheostatic switch. An important distinction between these models is that the observed functional parameters of a toggle-switch receptor could strongly covary as covalent modification shifts the equilibrium between the on- and off-states, due to population-weighted averaging of the intrinsic on- and off-state parameters. By contrast, covalent modification of a rheostatic receptor would create new conformational states with completely independent parameters. To resolve the toggle-switch and rheostat models, the present study has generated all 16 homogeneous covalent modification states of the receptor adaptation sites, and has compared their effects on the attractant affinity and kinase activity of the reconstituted receptor-kinase signaling complex. This approach reveals that receptor covalent modification modulates both attractant affinity and kinase activity up to 100-fold, respectively. The regulatory effects of individual adaptation sites are not perfectly additive, indicating synergistic interactions between sites. The three adaptation sites at positions 295, 302, and 309 are more important than the site at position 491 in regulating attractant affinity and kinase activity, thereby explaining the previously observed dominance of the former three sites in in vivo studies. The most notable finding is that covalent modification of the adaptation sites alters the receptor attractant affinity and the receptor-regulated kinase activity in a highly correlated fashion, strongly supporting the toggle-switch model. Similarly, certain mutations that drive the receptor into the kinase activating state are found to have correlated effects on attractant affinity. Together these results provide strong evidence that chemotaxis receptors possess just two stable signaling conformations and that the equilibrium between these pure on- and off-states is modulated by both attractant binding and covalent adaptation. It follows that the attractant and adaptation signals drive the same conformational change between the two settings of a toggle. An approach that quantifies the fractional occupancy of the on- and off-states is illustrated.

Multiple effects of aspartate mutant presenilin 1 on the processing and trafficking of amyloid precursor protein.

PS1 deficiency and expression of PS1 with substitutions of two conserved transmembrane aspartate residues ("PS1 aspartate variants") leads to the reduction of Abeta peptide secretion and the accumulation of amyloid precursor protein (APP) C-terminal fragments. To define the nature of the "dominant negative" effect of the PS1 aspartate variants, we stably expressed PS1 harboring aspartate to alanine substitutions at codons 257 (D257A) or 385 (D385A), singly or in combination (D257A/D385A), in mouse neuroblastoma, N2a cells. Expression of the PS1 aspartate variants resulted in marked accumulation of intracellular and cell surface APP C-terminal fragments. While expression of the D385A PS1 variant reduced the levels of secreted Abeta peptides, we now show that neither the PS1 D257A nor D257A/D385A variants impair Abeta production. Surprisingly, the stability of both immature and mature forms of APP is dramatically elevated in cells expressing PS1 aspartate variants, commensurate with an increase in the cell surface levels of APP. These findings lead us to conclude that the stability and trafficking of APP can be profoundly modulated by coexpression of PS1 with mutations at aspartate 257 and aspartate 385.

Presenilins, processing of beta-amyloid precursor protein, and notch signaling.

Given the evidence that presenilin is required for processing of Notch and APP at transmembrane sites, there are a number of possibilities for the function of presenilin. One is that presenilin is required for the proper trafficking of Notch and APP to their protease(s), which may reside in an intracellular compartment. Another is that presenilin is required for the proper biogenesis or trafficking of the γ-secretase. Another is that presenilin is an essential cofactor for γ-secretase, and yet another is that presenilin itself is γ-secretase. The current data cannot distinguish among these possible functions.Wolfe et al. 1999xWolfe, M.S, Xia, W, Ostaszewski, B.L, Diehl, T.S, Kimberly, W.T, and Selkoe, D.J. Nature. 1999; 398: 513–517Crossref | PubMed | Scopus (1410)See all ReferencesWolfe et al. 1999 favor the idea that presenilin is itself γ-secretase. They focus on two transmembrane aspartate residues conserved in all known presenilins. Expression in cultured cells of presenilin constructs in which either of these two residues is mutated results in loss of presenilin activity as assayed by production of Aβ, even though these cells express wild-type presenilin-1 endogenously. The authors therefore conclude that these constructs act as dominant-negative presenilins. While wild-type presenilins are cleaved at a site in the cytosolic loop, the authors find that the aspartate mutants are not cleaved in this manner; they take this result as circumstantial evidence that this cleavage is mediated by presenilin-1 itself. They also point out that γ-secretase has some characteristics of aspartyl proteases and speculate that the conserved aspartate residues may contribute to an active protease site in presenilin.Wolfe et al. further find that Aβ can be produced by in vitro translation of an APP-derived construct in the presence of microsomes derived from wild-type cells but not when the microsomes are prepared from presenilin-1 mutant cells. The authors argue that presenilin-1 is not required for trafficking of APP, as little or no vesicular trafficking is expected to occur in their in vitro microsomal preparations, and they suggest that presenilin is required in the same subcellular compartment in which γ-secretase resides. An alternative possibility is that presenilin is required for the proper production, processing, or localization of γ-secretase or an essential cofactor. Presenilin could also have multiple functions, including trafficking of APP, only one of which is required in microsomes. As Wolfe et al. point out, conclusive evidence that presenilin is γ-secretase will require a purified, reconstituted system, which may be technically difficult to accomplish. A somewhat more tractable, but less conclusive, approach would be to determine whether the newly identified γ-secretase inhibitors can bind directly to presenilin with the appropriate kinetics.Even should the presenilins not have a role in trafficking, endocytosis appears to have an important role in the processing of APP and in the function of Notch. Inhibitors of endocytosis partially block the processing of APP by γ-secretase in cultured cells. shibire, which encodes the Drosophila dynamin small GTPase and is required for endocytosis, appears to be required for activation of Notch upon ligand binding (Seugnet et al. 1997xSeugnet, L, Simpson, P, and Haenlin, M. Dev. Biol. 1997; 192: 585–598Crossref | PubMed | Scopus (199)See all ReferencesSeugnet et al. 1997). Trafficking of APP and Notch could regulate accessibility to their protease(s). Hence, determining the subcellular compartments for various processing events may contribute importantly to understanding the events in Notch signaling and Aβ production as well as their regulation.The normal function of the presenilins is still elusive, and many other questions remain to be answered. How do the mutations associated with AD affect presenilin function? What is the extent of overlap between the Notch and APP processing machineries? Other genes implicated in the Notch pathway might also have a role in APP processing. Are Notch and APP the only molecules that require presenilins for proteolysis? A careful analysis of the phenotypes of presenilin mutants may reveal defects not found in Notch mutants. By unraveling the connection between Notch and the presenilins, researchers may find answers to long-standing questions regarding both Notch signaling and the pathogenesis of Alzheimer’s disease.*To whom correspondence should be addressed (e-mail: ynjan@itsa.ucsf.edu).

Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity.

Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp --> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 --> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 --> Ala mutation still inhibited gamma-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease.

Detection of a Conserved α-Helix in the Kinase-docking Region of the Aspartate Receptor by Cysteine and Disulfide Scanning

The transmembrane aspartate receptor of Escherichia coli and Salmonella typhimurium propagates extracellular signals to the cytoplasm, where its cytoplasmic domain regulates the histidine kinase, CheA. Different signaling states of the cytoplasmic domain modulate the kinase autophosphorylation rate over at least a 100-fold range. Biochemical and genetic studies have implicated a specific region of the cytoplasmic domain, termed the signaling subdomain, as the region that transmits regulation from the receptor to the kinase. Here cysteine and disulfide scanning are applied to the N-terminal half of the signaling subdomain to probe its secondary structure, solvent exposure, and protein-protein interactions. The chemical reactivities of the scanned cysteines exhibit the characteristic periodicity of an alpha-helix with distinct solvent-exposed and buried faces. This helix, termed alpha7, ranges approximately from residue 355 through 386. Activity measurements probing the effects of cysteine substitutions in vivo and in vitro reveal that both faces of helix alpha7 are critical for kinase activation, while the buried face is especially critical for kinase down-regulation. Disulfide scanning of the region suggests that helix alpha7 is not in direct contact with its symmetric partner (alpha7') from the other subunit; presently, the structural element that packs against the buried face of the helix remains unidentified. Finally, a novel approach termed "protein interactions by cysteine modification" indicates that the exposed C-terminal face of helix alpha7 provides an essential docking site for the kinase CheA or for the coupling protein CheW.

Cysteine and disulfide scanning reveals two amphiphilic helices in the linker region of the aspartate chemoreceptor.

The transmembrane aspartate receptor of E. coli and S. typhimurium mediates cellular chemotaxis toward aspartate by regulating the activity of the cytoplasmic histidine kinase, CheA. Ligand binding results in transduction of a conformational signal through the membrane to the cytoplasmic domain where both kinase regulation and adaptation occur. Of particular interest is the linker region, E213 to Q258, which connects and transduces the conformational signal between the cytoplasmic end of the transmembrane signaling helix (alpha 4/TM2) and the major methylation helix of the cytoplasmic domain (alpha 6). This linker is crucial for stable folding and function of the homodimeric receptor. The present study uses cysteine and disulfide scanning mutagenesis to investigate the secondary structure and packing surfaces within the linker region. Chemical reactivity assays reveal that the linker consists of three distinct subdomains: two alpha-helices termed alpha 4 and alpha 5 and, between them, an ordered region of undetermined secondary structure. When cysteine is scanned through the helices, characteristic repeating patterns of solvent exposure and burial are observed. Activity assays, both in vivo and in vitro, indicate that each helix possesses a buried packing face that is crucial for proper receptor function. The interhelical subdomain is at least partially buried and is also crucial for proper receptor function. Disulfide scanning places helix alpha 4 distal to the central axis of the homodimer, while helix alpha 5 is found to lie at the subunit interface. Finally, sequence alignments suggest that all three linker subdomains are highly conserved among the large subfamily of histidine kinase-coupled sensory receptors that possess methylation sites for use in covalent adaptation.

Use of 19F NMR to probe protein structure and conformational changes.

19F NMR has proven to be a powerful technique in the study of protein structure and dynamics because the 19F nucleus is easily incorporated at specific labeling sites, where it provides a relatively nonperturbing yet sensitive probe with no background signals. Recent applications of 19F NMR in mapping out structural and functional features of proteins, including the galactose-binding protein, the transmembrane aspartate receptor, the CheY protein, dihydrofolate reductase, elongation factor-Tu, and D-lactose dehydrogenase, illustrate the utility of 19F NMR in the analysis of protein conformational states even in molecules too large or unstable for full NMR structure determination. These studies rely on the fact that the chemical shift of 19F is extremely sensitive to changes in the local conformational environment, including van der Waals packing interactions and local electrostatic fields. Additional information is provided by solvent-induced isotope shifts or line broadening of the 19F resonance by aqueous and membrane-bound paramagnetic probes, which may reveal the proximity of a 19F label to bulk solvent or a biological membrane. Finally, the effect of exchanging conformations on the 19F resonance can directly determine the kinetic parameters of the conformational transition.

Transmembrane signaling by the aspartate receptor: engineered disulfides reveal static regions of the subunit interface.

Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18-75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro methylation and phosphorylation assays.

19F nuclear magnetic resonance studies of aqueous and transmembrane receptors. Examples from the Escherichia coli chemosensory pathway

In general, protein conformational changes are beststudied in the appropriate native environment, whetherit be aqueous solution or a biological membrane. 19Fnuclear magnetic resonance (NMR) provides a valuabletool with which to carry out such studies, even for avarietyofproteinstoolargeorinsoluble for multidimen­sional NMRtechniques. Recombinant DNAtechniquessignificantly extend the usefulness of19F NMR, both byfacilitating resonance assignments, and by enablingseparation ofmultidomain structures into smaller, moreaccessible units. Thepresentstudyillustrates the type ofinformation provided by this combined approach usingtwo receptorsfrom theE. coli chemosensory pathway asexamples: the periplasmic o-galactose and o-glucosereceptorand the transmembrane aspartate transducer.

Structure and dynamics of Escherichia coli chemosensory receptors. Engineered sulfhydryl studies

Cysteine residues introduced by site-directed mutagenesis have been used to probe the conformation and dynamics of two receptors in the E. coli chemotaxis pathway. (a) Thermal motions of the polypeptide backbone were investigated in the periplasmic D-galactose and D-glucose receptor, a globular protein of known structure. Disulfide bond formation between pairs of engineered sulfhydryls were used to trap collisions during the relative motions of surface alpha-helices I and X. Motions with amplitudes ranging from 4.5 to 15.2 A were detected on timescales ranging from 10(-4) to 10(-1) s, respectively. These results suggest that thermal backbone motions may have larger amplitudes than previously thought. (b) Conformational features of the transmembrane aspartate transducer have been investigated. Engineered sulfhydryls were used to ascertain the location and orientations of two putative transmembrane alpha-helices in the primary structure, to investigate the packing of these helices, to determine the oligomer and surface structures, and to detect thermal and ligand-induced dynamics of the polypeptide backbone. A model for the folded conformation of the transducer oligomer is reviewed.