Signal Peptide Binding Research Articles

Isolation and characterisation of two wheat β-expansin genes expressed during male gametophyte development

Two novel beta-expansin genes, TaEXPB1 and TaEXPB2, were isolated from wheat microspores by suppression subtractive hybridisation. Northern blot and reverse transcription PCR analyses showed that the expression of both genes was restricted to early stages of male gametophyte development (from microspores to immature pollen). A homology search showed high similarity of the newly discovered genes to generative beta-expansins in grass pollen (group 1 pollen allergens). Southern hybridisation revealed that the isolated genes belong to a distinct group within the subfamily of beta-expansin genes in the wheat genome. A comparison of full-length cDNAs with the corresponding genomic sequences showed that there are two introns in the TaEXPB1 gene, whereas TaEXPB2 has three introns. Both genes were predicted to encode highly similar basic proteins (pI 9.0) with molecular masses of approximately 29 kDa consisting of a signal peptide, catalytic, and polysaccharide binding domains, which include conserved cysteines and tryptophans and motifs characteristic for beta-expansins.

Identification of the Preprotein Binding Domain of SecA

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.

A structural step into the SRP cycle.

Co-translational targeting of secretory and membrane proteins to the translocation machinery is mediated by the signal recognition particle (SRP) and its membrane-bound receptor (SR) in all three domains of life. Although the overall composition of the SRP system differs, the central ribonucleoprotein core and the general mechanism of GTP-dependent targeting are highly conserved. Recently, structural studies have contributed significantly to our understanding of the molecular organization of SRP. SRP appears as a structurally flexible particle modulated and regulated by its interactions with the ribosome-nascent chain complex, the translocon and the SR. The SRP core (SRP54 with its cognate RNA binding site) plays a central role in these interactions and communicates the different binding states by long-range interdomain communication. Based on recent structures of SRP54, a model for signal peptide binding stimulating the GTP affinity during the first step of the SRP cycle is presented. The model is placed in the context of the recent structures of mammalian SRP bound to a ribosome-nascent chain complex and of a subcomplex of SRP-SR.

Crystal structure of the complete core of archaeal signal recognition particle and implications for interdomain communication.

Targeting of secretory and membrane proteins by the signal recognition particle (SRP) is evolutionarily conserved, and the multidomain protein SRP54 acts as the key player in SRP-mediated protein transport. Binding of a signal peptide to SRP54 at the ribosome is coordinated with GTP binding and subsequent complex formation with the SRP receptor. Because these functions are localized to distinct domains of SRP54, communication between them is essential. We report the crystal structures of SRP54 from the Archaeon Sulfolobus solfataricus with and without its cognate SRP RNA binding site (helix 8) at 4-A resolution. The two structures show the flexibility of the SRP core and the position of SRP54 relative to the RNA. A long linker helix connects the GTPase (G domain) with the signal peptide binding (M) domain, and a hydrophobic contact between the N and M domains relates the signal peptide binding site to the G domain. Hinge regions are identified in the linker between the G and M domains (292-LGMGD) and in the N-terminal part of the M domain, which allow for structural rearrangements within SRP54 upon signal peptide binding at the ribosome.

High-level expression of human glycosyltransferases in insect cells as biochemically active form

cDNAs, encoding human β1,4-galactosyltransferase (hGalT I, EC 2.4.1.22), human Galβ1,3(4)-GlcNAc α2,3-sialyltransferase (hST3GalIII, EC 2.4.99), and human Galβ1,4-GlcNAc α2,6-sialyltransferase (hST6Gal I, EC 2.4.99.1), were cloned from human cell lines. In order to express these glycosyltransferases as secreted form in insect cells, cDNAs were inserted into a novel baculovirus transfer vector equipped with the mouse IgM signal peptide and IgG binding domain of the Staphylococcus aureus protein A as an N-terminal fusion partner. About 14 mg hGalT I, 8 mg hST3GalIII, and 6.4 mg hST6Gal I were purified from 1 liter of recombinant baculovirus infected insect cell culture media. The specific activities of recombinant hGalT I and hST6Gal I were determined as 0.65 and 1.6 U/mg protein, respectively. These results indicated that the recombinant hGalT I and hST6Gal I retained enzyme activities at similar level to those of the authentic one although they were fused with the IgG binding domain at the N-terminus. Taken together, the mouse IgM signal peptide and IgG binding domain of the protein A could be efficiently used as an N-terminus fusion partner for the over-expression of heterologous proteins in insect cells.

Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes.

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.

Systematic site-directed mutagenesis of human protein SRP54: interactions with signal recognition particle RNA and modes of signal peptide recognition.

The amino acid residues of human protein SRP54 which are required for binding to SRP RNA were identified by generating 40 nonoverlapping tri-alanine alterations within its methionine-rich M-domain (SRP54M). The mutant polypeptides were expressed in Escherichia coli, and their ability to bind to human and Methanococcus jannaschii SRP RNA were determined in vitro. Residues at positions 379-387, 394-396, 400-405, and 409-411 of human SRP54 were within the predicted RNA binding site, and their alteration abolished the binding activities of the mutant polypeptides as expected. Changes at positions 418-423 had intermediate effects. Polypeptides containing mutations of 328-TLR-330 were inactive although these residues were far away from the presumed RNA binding site in the crystal structure of the free protein. Using the structures of the E. coli Ffh/4.5S core and of the human SRP54m dimer as templates, a molecular model of the complex between human SRP RNA helix 8 and a single SRP54M molecule was constructed in which Leucine 329 was positioned in closer proximity to the RNA binding domain. This representation was supported by studies of the SRP54m monomer/dimer ratio using gel filtration. The results were consistent with a change in the shape of the signal peptide binding groove upon binding of SRP54 to SRP RNA. We propose that the SRP RNA and a small region centered at a bulky nonpolar amino acid residue at position 329 of protein SRP54 play a critical role in the SRP-dependent binding and release of signal peptides.

Allosteric Communication between Signal Peptides and the SecA Protein DEAD Motor ATPase Domain

SecA, the preprotein translocase ATPase is built of an amino-terminal DEAD helicase motor domain bound to a regulatory C-domain. SecA recognizes mature and signal peptide preprotein regions. We now demonstrate that the amino-terminal 263 residues of the ATPase subdomain of the DEAD motor are necessary and sufficient for high affinity signal peptide binding. Binding is abrogated by deletion of residues 219-244 that lie within SSD, a novel substrate specificity element of the ATPase subdomain. SSD is essential for protein translocation, is unique to SecA, and is absent from other DEAD proteins. Signal peptide binding to the DEAD motor is controlled in trans by the C-terminal intramolecular regulator of ATPase (IRA1) switch. IRA1 mutations that activate the DEAD motor ATPase also enhance signal peptide affinity. This mechanism coordinates signal peptide binding with ATPase activation. Signal peptide binding causes widespread conformational changes to the ATPase subdomain and inhibits the DEAD motor ATPase. This involves an allosteric mechanism, since binding occurs at sites that are distinct from the catalytic ATPase determinants. Our data reveal the physical determinants and sophisticated intramolecular regulation that allow signal peptides to act as allosteric effectors of the SecA motor.

Crystal Structure of a Bacterial Signal Peptidase Apoenzyme: IMPLICATIONS FOR SIGNAL PEPTIDE BINDING AND THE SER-LYS DYAD MECHANISM

We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 A resolution in a different space group (P4(1)2(1)2) from that of the previously published acyl-enzyme inhibitor-bound structure (P2(1)2(1)2) (Paetzel, M., Dalbey, R.E., and Strynadka, N.C.J. (1998) Nature 396, 186-190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 OgammaH) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. A similar mechanism has been proposed for the LexA family of proteases. A structural comparison of SPase and members of the LexA family of proteases reveals a difference in the side-chain orientation for the general base lysine, both of which are stabilized by an adjacent hydroxyl group. To gain insight into how signal peptidase recognizes its substrates, we have modeled a signal peptide into the binding site of SPase. The model is built based on the recently solved crystal structure of the analogous enzyme LexA (Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W., and Strynadka, N. C. J. (2001) Cell 106, 1-10) with its bound cleavage site region.

Glowing zebrafish: Integration, transmission, and expression of a single luciferase transgene promoted by noncovalent DNA–nuclear transport peptide complexes

The development of vehicles driving foreign DNA into the cell nucleus is essential for effective cellular gene transfer applications. We report that noncovalent binding of nuclear localization signal (NLS) peptides to plasmid DNA enhances nuclear uptake of the DNA and promotes germline integration, inheritance, and expression of a single copy of a luciferase reporter gene in zebrafish. As few as 10 DNA–NLS complexes (0.06 fg plasmid DNA) cytoplasmically injected are sufficient to produce germline-transgenic zebrafish bearing a single copy of the transgene. This corresponds to a 105-fold reduction in DNA concentration compared to commonly used procedures. Use of 103 or 104 DNA–NLS complexes augments the number of transgene integrations, which occur mostly within 1–4 distinct insertion sites in the genome. In situ hybridization analyses and transmission studies show that transgene integration into the germline and somatic tissues is mosaic, and that the extent of mosaicism is negatively correlated with the amount of DNA–NLS injected. In addition, a larger proportion of zebrafish harboring a single copy of the transgene expresses luciferase, albeit at a 10-fold lower level than those containing numerous transgene insertions. The data demonstrate the potential use of nuclear targeting peptides noncovalently bound to vector DNA to enhance the efficiency of biotechnological nonviral gene transfer applications. Mol. Reprod. Dev. 55:8–13, 2000. © 2000 Wiley-Liss, Inc.

Glowing zebrafish: integration, transmission, and expression of a single luciferase transgene promoted by noncovalent DNA-nuclear transport peptide complexes.

The development of vehicles driving foreign DNA into the cell nucleus is essential for effective cellular gene transfer applications. We report that noncovalent binding of nuclear localization signal (NLS) peptides to plasmid DNA enhances nuclear uptake of the DNA and promotes germline integration, inheritance, and expression of a single copy of a luciferase reporter gene in zebrafish. As few as 10 DNA-NLS complexes (0.06 fg plasmid DNA) cytoplasmically injected are sufficient to produce germline-transgenic zebrafish bearing a single copy of the transgene. This corresponds to a 10(5)-fold reduction in DNA concentration compared to commonly used procedures. Use of 10(3) or 10(4) DNA-NLS complexes augments the number of transgene integrations, which occur mostly within 1-4 distinct insertion sites in the genome. In situ hybridization analyses and transmission studies show that transgene integration into the germline and somatic tissues is mosaic, and that the extent of mosaicism is negatively correlated with the amount of DNA-NLS injected. In addition, a larger proportion of zebrafish harboring a single copy of the transgene expresses luciferase, albeit at a 10-fold lower level than those containing numerous transgene insertions. The data demonstrate the potential use of nuclear targeting peptides noncovalently bound to vector DNA to enhance the efficiency of biotechnological nonviral gene transfer applications.

Crystal Structure of the Conserved Subdomain of Human Protein SRP54M at 2.1 Å Resolution: Evidence for the Mechanism of Signal Peptide Binding

Protein SRP54 is an integral part of the mammalian signal recognition particle (SRP), a cytosolic ribonucleoprotein complex which associates with ribosomes and serves to recognize, bind, and transport proteins destined for the membrane or secretion. The methionine-rich M-domain of protein SRP54 (SRP54M) binds the SRP RNA and the signal peptide as the nascent protein emerges from the ribosome. A focal point of this critical cellular function is the detailed understanding of how different hydrophobic signal peptides are recognized efficiently and transported specifically, despite considerable variation in sequence. We have solved the crystal structure of a conserved functional subdomain of the human SRP54 protein (hSRP54m) at 2.1Å resolution showing a predominantly alpha helical protein with a large fraction of the structure available for binding. RNA binding is predicted to occur in the vicinity of helices 4 to 6. The N-terminal helix extends significantly from the core of the structure into a large but constricted hydrophobic groove of an adjacent molecule, thus revealing molecular details of possible interactions between alpha helical signal peptides and human SRP54.

Modulation of the binding of signal peptides to lipid bilayers by dipoles near the hydrocarbon-water interface.

Interactions between signal (leader) sequences and membranes are critical to protein insertion and translocation across membranes. In this paper, circular dichroism, tryptophan fluorescence, electrophoretic mobility, dipole potential, and binding measurements were used to study the interaction of the signal sequence of the Escherichia coli LamB protein with various lipid bilayers. By modifying specific chemicophysical properties of both the signal sequence and bilayer, we analyzed some of the key factors underlying peptide-lipid interactions. We synthesized three analogues of the LamB signal peptide differing in their net charge (-2 to +4) and studied their binding to bilayers containing combinations of neutral lipids [egg phosphatidylcholine (EPC), sphingomyelin, cholesterol, ketocholesterol, and nitroxide-containing phospholipid] and a charged lipid (phosphatidylserine). All three peptides bound to EPC bilayers and underwent a random coil to alpha-helix transition upon binding. Microelectrophoresis experiments revealed that both the N and C termini were near the outer surface of the bilayer, suggesting that the peptides adopted a "hammock" configuration with both termini exposed to the aqueous phase and the core of the alpha-helix located near the hydrocarbon-water interface. The binding of these LamB peptides was not markedly dependent on the bilayer area per molecule, compressibility modulus, or dipole potential, but did depend on the charge of the peptide and bilayer interfacial region. Moreover, the binding of LamB peptides was essentially eliminated in bilayers composed of phospholipids with a nitroxide moiety at the 7 position in one of their acyl chains or in EPC bilayers containing equimolar ketocholestanol. We propose that the incorporation of nitroxide or ketone groups into the hydrocarbon region near the lipid headgroup increases the effective width of the hydrophilic interfacial region and prevents some of the hydrophobic amino acids in the alpha-helix from reaching the nonpolar hydrocarbon core, thereby diminishing the free energy of partitioning and inhibiting peptide binding. These results point to an important role for interfacial dipoles in peptide-lipid interactions.

Domain Interactions in E. coli SRP: Stabilization of M Domain by RNA Is Required for Effective Signal Sequence Modulation of NG Domain

The E. coli protein, Ffh, binds to 4.5S RNA through its M domain to form the signal recognition particle (SRP). The other domain of Ffh (NG) is a GTPase, which binds and is coordinately regulated by its receptor, FtsY. We find that the helical M domain is inherently flexible. Binding of 4.5S RNA to Ffh stabilizes the M domain yet has little apparent effect on the binding of signal peptides. However, in the absence of the RNA, signal peptide binding results in a global destabilization of Ffh, which is prevented by binding of 4.5S RNA. Signal peptide binding to isolated NG domain also causes a pronounced destabilization, implicating the NG domain in direct recognition of signal peptide.

Time resolution of binding and membrane insertion of a mitochondrial signal peptide: correlation with structural changes and evidence for cooperativity.

Utilizing a recently developed novel fluorescence technique [Wall et al. (1995) Mol. Membr. Biol. 12, 183-192], it is shown that the interactions of p25, the leader peptide of subunit IV of cytochrome c oxidase, with phospholipid membranes can be identified in real time. p25 is observed to bind following stopped-flow mixing of the peptide with phospholipid membranes with rate constants up to about 700 s-1 and then insert into the membrane with rate constants on the order of 0.4 s-1. Comparison of these processes with similarly time-resolved experiments performed with a stopped-flow CD spectrometer revealed that p25 does not become alpha-helical upon binding to the membrane. Following membrane insertion, however, p25 was observed to adopt an alpha-helical configuration. The temperature dependency of these processes was then found to yield activation energies for the respective components of the p25-membrane interaction.

Nucleotide Sequence of a Gene Encoding a 58.5-Kilodalton Barley Dehydrin That Lacks a Serine Tract

Dehydrins, also known as the late embryogenesis abundant (LEA) D-11 family of proteins (Close et al., 1993, and refs. therein), generally accumulate in plants in response to water deficit, embryo desiccation, cold temperature, reduction in externa1 osmotic potential, or application of ABA. Over 40 examples of deduced amino acid sequences of higher plant dehydrins have been published to date, and there is immunological evidence of related proteins in organisms as distant from higher plants as cyanobacteria (Close and Lammers, 1993). Known members of this family of proteins are characterized by the presence of highly conserved repeating motifs including the Lys-rich 15amino acid consensus EKKGIMDKIKEKLPG, which is always located near the carboxy terminus (the lone exception being cotton LEA D-11). A related but slightly less conserved consensus sequence is usually repeated at least once and up to 10 additional times in published examples of plant dehydrins. Dehydrins can be divided into two main classes, those containing approximately nine consecutive Ser residues and those without this Ser tract. The Ser residues in this tract can be phosphorylated, and it has been proposed that phosphorylation of Ser's is related to the binding of nuclear localization signal peptides (Goday et al., 1994). Dehydrins have a nucleocytoplasmic location (Asghar et al., 1994; Goday et al., 1994). The majority of dehydrins identified to date fall into the class that contains the Ser tract, and the minority fall into the latter class, without the Ser tract. Those in the former class usually contain two or three Lys-rich consensus blocks. Dehydrins in the latter class usually contain six or more consensus blocks. There are exceptions to these generalizations, and each class can be subdivided further based on intervening amino acid sequences located between the consensus blocks. Examples of dehydrins that lack a Ser tract include spinach CAP85 (61.5 kD with 11 repeats) (Neven et al., 1993), wheat WCS120 (39.0 kD with 6 repeats) (Houde et al., 1992)' and wheat COR39 (39.1 kD

Effect of charged residue substitutions on the thermodynamics of signal peptide-lipid interactions for the Escherichia coli LamB signal sequence

We have used tryptophan fluorescence spectroscopy to characterize the binding affinities of an Escherichia coli LamB signal peptide family for lipid vesicles. These peptides harbor charged residue substitutions in the hydrophobic core region. Titrations of peptides with vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-3-phosphoglycerol (65:35 mol%), in conjunction with evaluation of peptide dissociation rates from these vesicles, were used to determine binding parameters quantitatively. We find that under low ionic strength conditions, point mutations introducing negatively charged aspartate residues substantially reduce peptide affinity relative to the wild-type peptide. However, the difference between wild-type and mutant peptide affinities was much lower under approximately physiological ionic strength. In addition, the lipid affinities of model surface-binding and transmembrane peptides were determined. These comparative studies with signal and model peptides permitted semi-quantitative deconvolution of signal peptide binding into electrostatic and hydrophobic components. We find that both interactions contribute significantly to binding, although the theoretically available hydrophobic free energy is largely offset by unfavorable polar-group effects. The implications of these results for understanding the potential roles of the signal sequence in protein translocation are discussed.

Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane.

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the 'cold' synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.

Studies on the binding of a synthetic signal peptide to pancreatic rough microsomal membranes

Studies on the binding of a synthetic signal peptide to pancreatic rough microsomal membranes