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.     

ATPase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance for SecA2 function in macrophages

The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.     

Histones bind to contractile proteins and inhibit actomyosin ATPase

Nuclear histones bind to and precipitate the major contractile proteins, actin and myosin. The binding of histone to actin seems to reach saturation at 2:1 ratio, the interaction may serve some regulatory function(s) in intranuclear events. The binding of histone to myosin is not saturable, and, although it inhibits the actin-activated Mg2+-dependent myosin ATPase activity, does therefore not seem of physiological importance.     

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (＞1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.     

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (＞1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.     

N-terminal stathmin-like peptides bind tubulin and impede microtubule assembly

Microtubules are major cytoskeletal components involved in numerous cellular functions such as mitosis, cell motility, or intracellular traffic. These cylindrical polymers of alphabeta-tubulin assemble in a closely regulated dynamic manner. We have shown that the stathmin family proteins sequester tubulin in a nonpolymerizable ternary complex, through their stathmin-like domains (SLD) and thus contribute to the regulation of microtubule dynamics. We demonstrate here that short peptides derived from the N-terminal part of SLDs impede tubulin polymerization with various efficiencies and that phosphorylation of the most potent of these peptides reduces its efficiency as in full-length stathmin. To understand the mechanism of action of these peptides, we undertook a NMR-based structural analysis of the peptide-tubulin interaction with the most efficient peptide (I19L). Our results show that, while disordered when free in solution, I19L folds into a beta-hairpin upon binding to tubulin. We further identified, by means of saturation transfer difference NMR, hydrophobic residues located on the beta2-strand of I19L that are involved in its tubulin binding. These structural data were used together with tubulin atomic coordinates from the tubulin/RB3-SLD crystal structure to model the I19L/tubulin interaction. The model agrees with I19L acting through an autonomous tubulin capping capability to impede tubulin polymerization and provides information to help understand the variation of efficiency against tubulin polymerization among the peptides tested. Altogether these results enlighten the mechanism of tubulin sequestration by SLDs, while they pave the way for the development of protein-based compounds aimed at interfering with tubulin polymerization.     

The variable subdomain of Escherichia coli SecA functions to regulate SecA ATPase activity and ADP release

Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotide-binding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secAΔ519-547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azi(r)) and suppression of signal sequence defects (PrlD). The SecAΔ(519-547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG.     

Signal peptide determinants of SecA binding and stimulation of ATPase activity

A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA. Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional signal sequences but not with transport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.     

SecB modulates the nucleotide-bound state of SecA and stimulates ATPase activity

In Escherichia coli, the formation of SecA-SecB complexes has a direct effect on SecA ATPase activity. The mechanism of this interaction was evaluated and defined using controlled trypsinolysis, equilibrium dialysis at low temperature, and kinetic analyses of the SecA ATPase reaction. The proteolysis data indicate that SecB and the nonhydrolyzable ATP analogue AMP-P-C-P induce similar conformational changes in SecA which result in a more open or extended structure that is suggestive of the ATP-bound form. The effect is synergistic and concentration-dependent, and requires the occupation of both the high- and low-affinity nucleotide binding sites for maximum effect. The equilibrium dialysis experiments and kinetic data support the observation that the SecB-enhanced SecA ATPase activity is the result of an increased rate of ATP hydrolysis rather than an increase in the affinity of ATP for SecA and that the high-affinity nucleotide binding site is conformationally regulated by SecB. It appears that SecB may function as an intermolecular regulator of ATP hydrolysis by promoting the ATP-bound state of SecA. The inhibition of SecA ATPase activity by sodium azide in the presence of IMVs and a functional signal peptide further indicates that SecB promotes the ATP-bound form of SecA.     

Expression and purification of Pseudomonas aeruginosa SecA N-terminal domain: stimulation of ATPase activity of the SecAL43P mutant protein

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium which secretes a wide range of hydrolytic enzymes, toxins, and virulence factors into the extracellular medium. Although P. aeruginosa possesses numerous specific systems for the export of proteins across its double-membrane envelopes, the Sec system is still the major and essential mechanism. However, very little is known about its molecular basis. We constructed, cloned, and expressed the N-terminal 236 amino acids of PaSecA domain (PaSecAN236), and SecAL43P mutants of P. aeruginosa in Escherichia coli BL21.19 (secA(ts)). Here, we describe the purification of PaSecAN236 by using osmotic shock as the first step to efficiently release targeted protein from cells, followed by cation-exchange and size exclusion columns to obtain homogeneous PaSecAN236. The purified PaSecA N-terminal domain was functional in stimulating the ATPase activity of mutant SecAL43P protein of P. aeruginosa.     

Functional role of soluble mitochondrial ATPase subunits

A preparation of soluble mitochondrial ATPase (coupling factor F₁) containing no and minor subunits has been isolated. The minor-subunits-deficient F₁ was found to be competent in ATP hydrolysis. However, it did not demonstrate a coupling effect in EDTA-submitochondrial particles. A portion of the ATPase activity of EDTA particles, stimulated by the minor-subunits-deficient F₁, was insensitive to oligomycin. ATPase activity of Na⁺-particles was changed only slightly by this F₁. It is suggested that and subunits are necessary to form specific contacts between the F₁ molecule and components of the mitochrondrial membrane.Abbreviations SMP submitochondrial particles - F₁ coupling factor (soluble mitochondrial ATPase) - PCB^– phenyl dicarbaundecaborane anions     

Computational differentiation of N-terminal signal peptides and transmembrane helices

Signal peptides and transmembrane helices both contain a stretch of hydrophobic amino acids. This common feature makes it difficult for signal peptide and transmembrane helix predictors to correctly assign identity to stretches of hydrophobic residues near the N-terminal methionine of a protein sequence. The inability to reliably distinguish between N-terminal transmembrane helix and signal peptide is an error with serious consequences for the prediction of protein secretory status or transmembrane topology. In this study, we report a new method for differentiating protein N-terminal signal peptides and transmembrane helices. Based on the sequence features extracted from hydrophobic regions (amino acid frequency, hydrophobicity, and the start position), we set up discriminant functions and examined them on non-redundant datasets with jackknife tests. This method can incorporate other signal peptide prediction methods and achieve higher prediction accuracy. For Gram-negative bacterial proteins, 95.7% of N-terminal signal peptides and transmembrane helices can be correctly predicted (coefficient 0.90). Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 99% (coefficient 0.92). For eukaryotic proteins, 94.2% of N-terminal signal peptides and transmembrane helices can be correctly predicted with coefficient 0.83. Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 87% (coefficient 0.85). The method can be used to complement current transmembrane protein prediction and signal peptide prediction methods to improve their prediction accuracies.     

Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity

Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.     

Functional role of soluble mitochondrial ATPase subunits.

A preparation of soluble mitochondrial ATPase (coupling factor F1) containing no gamma and delta minor subunits has been isolated. The minor-subunits-deficient F1 was found to be competent in ATP hydrolysis. However, it did not demonstrate a "coupling" effect in EDTA-submitochondrial particles. A portion of the ATPase activity of EDTA particles, stimulated by the minor-subunits-deficient F1, was insensitive to oligomycin. ATPase activity of Na+-particles was changed only slightly by this F1. It is suggested that gamma and delta subunits are necessary to form specific contacts between the F1 molecule and components of the mitochrondrial membrane.     

Effect of divalent cations on the ATPase activity of Escherichia coli SecA

It was found that Ca(2+) stimulates the intrinsic SecA ATPase activity in the absence as well as in the presence of liposome. On the other hand, Mg(2+), the general cofactor for ATPase, did not affect the intrinsic SecA ATPase but reduced the portion of ATPase activity enhanced by Ca(2+). The enhancement of SecA ATPase activity correlated well with the increase in 8-anilino-1-naphthalene-sulfonic acid binding of SecA, suggesting that increased exposure of hydrophobic residues stimulates the enzyme activity.     

Effects of signal peptide and adenylate on the oligomerization and membrane binding of soluble SecA

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.     

Functional analysis of expressed peptides that bind yeast STE proteins

Peptides are potentially useful for target validation and other reverse genetic applications. For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules. We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p. A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays. In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast. Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide-protein interactions can be complex and unpredictable. Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes.