Do proteins facilitate the formation of cholesterol-rich domains?

Both biological and model membranes can exhibit the formation of domains. A brief review of some of the diverse methodologies used to identify the presence of domains in membranes is given. Some of these domains are enriched in cholesterol. The segregation of lipids into cholesterol-rich domains can occur in both pure lipid systems as well as membranes containing peptides and proteins. Peptides and proteins can promote the formation of cholesterol-rich domains not only by preferentially interacting with cholesterol and being sequestered into these regions of the membrane, but also indirectly as a consequence of being excluded from cholesterol-rich domains. The redistribution of components is dictated by the thermodynamics of the system. The formation of domains in a biological membrane is a consequence of all of the intermolecular interactions including those among lipid molecules as well as between lipids and proteins.     

NMR structures of the transmembrane domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green? fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.     

Mesoscale organization of domains in the plasma membrane – beyond the lipid raft

The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid–protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the “lipid raft” concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms “membrane raft” or “membrane nanodomain” are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.     

Molecular dynamics investigation of the ω-current in the Kv1.2 voltage sensor domains

Voltage sensor domains (VSD) are transmembrane proteins that respond to changes in membrane voltage and modulate the activity of ion channels, enzymes, or in the case of proton channels allow permeation of protons across the cell membrane. VSDs consist of four transmembrane segments, S1-S4, forming an antiparallel helical bundle. The S4 segment contains several positively charged residues, mainly arginines, located at every third position along the helix. In the voltage-gated Shaker K(+) channel, the mutation of the first arginine of S4 to a smaller uncharged amino acid allows permeation of cations through the VSD. These currents, known as ω-currents, pass through the VSD and are distinct from K(+) currents passing through the main ion conduction pore. Here we report molecular dynamics simulations of the ω-current in the resting-state conformation for Kv1.2 and for four of its mutants. The four tested mutants exhibit various degrees of conductivity for K(+) and Cl(-) ions, with a slight selectivity for K(+) over Cl(-). Analysis of the ion permeation pathway, in the case of a highly conductive mutant, reveals a negatively charged constriction region near the center of the membrane that might act as a selectivity filter to prevent permeation of anions through the pore. The residues R1 in S4 and E1 in S2 are located at the narrowest region of the ω-pore for the resting state conformation of the VSD, in agreement with experiments showing that the largest increase in current is produced by the double mutation E1D and R1S.     

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.Abbreviations X:Y(Z) fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxyl terminus     

Starch-binding domains in the CBM45 family--low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism

Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosumα-glucan, water dikinase and the Arabidopsis thaliana plastidial α-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T(m) of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.     

Bacterial FHA domains: neglected players in the phospho-threonine signalling game?

Forkhead-associated (FHA) domains bind phospho-threonine peptides and are known to mediate phosphorylation-dependent protein–protein interactions in a variety of eukaryotic settings. However, their role in bacterial physiology and signalling has been largely neglected. We have surveyed bacterial FHA domains and discovered that they are implicated in many bacterial processes, including regulation of cell shape, type III secretion, sporulation, pathogenic and symbiotic host–bacterium interactions, carbohydrate storage and transport, signal transduction and ethambutol resistance. The way is now open to identify the targets of each FHA domain, and their roles in cellular physiology, and perhaps even to develop novel FHA-blocking antibacterial agents.     

Do life’s three domains mirror the origins of sex?

Conclusions The sexual theory of cell evolution has clear corollaries. All higher life would be initially sexual, and of two types (A- or Eu-karyotes). Relations would be dynamic with alternative unions and dissolution of the union of the two constituent moneric species, even as their viability as free-living species tended to decline in subsequent evolution. Superior bioenergetics meant eukaryotes were retained in multicellular species. However, some a-karyotes would be phylogenetically older than eukaryotes, a point which if verified, would render such biota of fundamental significance.     

Calcium negatively regulates an intramolecular interaction between the N-terminal recoverin homology and EF-hand motif domains and the C-terminal C1 and catalytic domains of diacylglycerol kinase α

The type I diacylglycerol kinase (DGK) isozymes (α, β and γ) contain a shared recoverin homology (RVH) domain, a tandem repeat of Ca2+-binding EF-hand motifs, two cysteine-rich C1 domains, and the catalytic domain. We previously reported that a DGKα mutant lacking the RVH domain and EF-hands was constitutively active, implying that the N-terminal region (NTR) of DGKα, consisting of the RVH domain and EF-hand motifs, intramolecularly interacts with and masks the activity of the C-terminal region (CTR), containing the C1 and catalytic domains. In this study, we demonstrate that a glutathione S-transferase (GST)-fused DGKα-NTR construct physically binds to a green fluorescent protein (GFP)-fused DGKα-CTR construct. Moreover, co-precipitation of GFP-DGKα-CTR with GST-DGKα-NTR was clearly attenuated by the addition of 1 μM Ca2+. This result indicates that Ca2+ induces dissociation of the physical interaction between DGKα-NTR and DGKα-CTR. In addition to previously reported calcium-dependent changes in the hydrophobicity and net surface charge, Ca2+ also appeared to induce a decrease in the α-helical content of DGKα-NTR. These results suggest that Ca2+-induced conformational changes in the NTR release the intramolecular association between the NTR and the CTR of DGKα.     

Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin

Proteolytic digestion of bovine β-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15–20), AASDISLLDAQSAPLR (residues 25–40), IPAVFK (residues 78–83) and VLVLDTDYK (residues 92–100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55–64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of β-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of β-lactoglobulin could be useful to increase its antimicrobial function.     

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp. Received: 21 March 1996 / Received revision: 11 July 1996 / Accepted: 19 July 1996     

Understanding the role of domain–domain linkers in the spatial orientation of domains in multi-domain proteins

Inter-domain linkers (IDLs)’ bridge flanking domains and support inter-domain communication in multi-domain proteins. Their sequence and conformational preferences enable them to carry out varied functions. They also provide sufficient flexibility to facilitate domain motions and, in conjunction with the interacting interfaces, they also regulate the inter-domain geometry (IDG). In spite of the basic intuitive understanding of the inter-domain orientations with respect to linker conformations and interfaces, we still do not entirely understand the precise relationship among the three. We show that IDG is evolutionarily well conserved and is constrained by the domain–domain interface interactions. The IDLs modulate the interactions by varying their lengths, conformations and local structure, thereby affecting the overall IDG. Results of our analysis provide guidelines in modelling of multi-domain proteins from the tertiary structures of constituent domain components.     

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.     

Organization of the variant domains of α satellite DNA on human chromosome 21

The de novo creation of long, homogeneous, satellite DNA domains was postulated previously to occur by saltatory amplification. In this paper, pulsed field gel electrophoresis analysis of the α satellite DNA block organization of the human chromosome 21 supports this hypothesis. Double-dimension electrophoresis indicated that the variant copies of the basic α satellite repeat of chromosome 21 are organized in a single 3,150 Kblong domain. It was also established that the other satellite DNAs found in man (β, II, and III) are organized independently of the α satellite DNA block of the same chromosome. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Elastase inhibition by the C-terminal domains of α-crystallin and small heat-shock protein

α-Crystallin, an abundant eye-lens protein and a stress protein in other tissues, shows structural and functional similarities with the small heat-shock proteins. One of the properties in common is the inhibition of elastase. We now report that the separated subunits of α-crystallin, αA and αB,also exhibit elastase inhibition, whereas phosphorylation of these subunits apparently has no influence on the inhibitory capacity. Furthermore, for both αA-crystallin and mouse HSP25 the putative C-terminal structural domain, comprising the major region of homology between these proteins, is sufficient to give elastase inhibition. With database search no homology could be found between the three proteins under investigation and any of the known consensus sequences of proteinase inhibitor families.     

Enriching the viral–host interactomes with interactions mediated by SH3 domains

Protein–protein interactions play an essential role in the regulation of most cellular processes. The process of viral infection is no exception and many viral pathogenic strategies involve targeting and perturbing host–protein interactions. The characterization of the host protein subnetworks disturbed by invading viruses is a major goal of viral research and may contribute to reveal fundamental biological mechanisms and to identify new therapeutic strategies. To assist in this approach, we have developed a database, VirusMINT, which stores in a structured format most of the published interactions between viral and host proteome. Although SH3 are the most ubiquitous and abundant class of protein binding modules, VirusMINT contains only a few interactions mediated by this domain class. To overcome this limitation, we have applied the whole interactome scanning experiment approach to identify interactions between 15 human SH3 domains and viral proline-rich peptides of two oncogenic viruses, human papillomavirus type 16 and human adenovirus A type 12. This approach identifies 114 new potential interactions between the human SH3 domains and proline-rich regions of the two viral proteomes.     

Effects of the association between the α-subunit thigh and the β-Subunit EGF2 domains on integrin activation and signaling

Integrin bidirectional signaling is mediated by conformational change. It has been shown that the separation of the α- and β-subunit transmembrane/cytoplasmic tails and the lower legs is required for transmitting integrin bidirectional signals across the plasma membrane. In this study, we address whether the separation of the αβ knee is critical for integrin activation and outside-in signaling. By introducing three disulfide bonds to restrict dissociation of the α-subunit thigh domain and β-subunit I-EGF2 domain, we found that two of them could completely abolish integrin inside-out activation, whereas the other could not. This disulfide-bonded mutant, in the context of the activation mutation of the cytoplasmic domain, had intermediate affinity for ligands and was able to mediate cell adhesion. Our data suggest that there exists rearrangement at the interface between the thigh domain and the I-EGF2 domain during integrin inside-out activation. None of the disulfide-bonded mutants could mediate cell spreading upon adhering to immobilized ligands, suggesting that dissociation of the integrin two knees is required for integrin outside-in signaling. Disrupting the interface by introducing a glycan chain into either subunit is sufficient for high affinity ligand binding and cell spreading.     

How the projection domains of NF-L and α-internexin determine the conformations of NF-M and NF-H in neurofilaments

Making use of a numerical self-consistent field method and polymer brush concepts, we model the solvated corona of neurofilaments (NF) composed of projection domains (unstructured tails) of constituent proteins. Projections are modeled with amino acid resolution. We focus on the importance of the two shortest ones (α-internexin and NF-L) in regulating the conformations of the two longer ones (NF-M and NF-H) in an isolated NF. We take the wild-type NF with no α-internexin as the reference, for which the phosphorylation-induced translocation of M- and H-tails has been examined previously. We demonstrate that a subbrush of L-tails creates an electrostatic potential profile with an approximately parabolic shape. An experimentally relevant (2:1) ratio of L- to α-projections reduces the charge density of the L subbrush and shifts the translocation transition of the H-tails to slightly higher degrees of phosphorylation. Replacing all L-tails by α-projections destroys the substructure of the NF corona and this alters the NF response to the phosphorylation of long tails.