Molecular mechanism for functional interaction between DnaA protein and acidic phospholipids: identification of important amino acids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be reactivated from the ADP-bound form to its ATP-bound form through stimulation of ADP release by acidic phospholipids such as cardiolipin. We previously reported that two potential amphipathic helices (Lys-327 to Ile-344 and Asp-357 to Val-374) of DnaA protein are involved in the functional interaction between DnaA and cardiolipin. In relation to one of these helices (Asp-357 to Val-374), we demonstrated that basic amino acids in the helix, especially Lys-372, are vital for this interaction. In this study, we have identified an amino acid in the second potential amphipathic helix (Lys-327 to Ile-344), which would also appear to be involved in the interaction. We constructed three mutant dnaA genes with a single mutation (dnaAR328E, dnaAR334E, and dnaAR342E) and examined the function of the mutant proteins. DnaAR328E, but not DnaAR334E and DnaAR342E, was found to be more resistant to inhibition of its ATP binding activity by cardiolipin than the wild-type protein. The stimulation of ADP release from DnaAR328E by cardiolipin was also weaker than that observed with the other mutants and the wild-type protein. These results suggest that Arg-328 of DnaA protein is involved in the functional interaction of this protein with acidic phospholipids. We propose that acidic phospholipids bind to two basic amino acid residues (Arg-328 and Lys-372) of DnaA protein and change the higher order structure of its ATP-binding pocket, which in turn stimulates the release of ADP from the protein.     

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.     

Identification of amino acids in antiplasmin involved in its noncovalent 'lysine-binding-site'-dependent interaction with plasmin.

The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.     

Identification of amino acids involved in protein structural uniqueness: implication for de novo protein design

Structural uniqueness is characteristic of native proteins and is essential to express their biological functions. The major factors that bring about the uniqueness are specific interactions between hydrophobic residues and their unique packing in the protein core. To find the origin of the uniqueness in their amino acid sequences, we analyzed the distribution of the side chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived deltaS(contact), the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure. The deltaS(contact) values indicate distinct tendencies of the residue pairs to restrict side chain conformation by inter-residue contacts. Of the hydrophobic residues in alpha-helices, aliphatic residues (Leu, Val, Ile) strongly restrict the side chain conformations of each other. In beta-sheets, Met is most strongly restricted by contact with Ile, whereas Leu, Val and Ile are less affected by other residues in contact than those in alpha-helices. In designed and native protein variants, deltaS(contact) was found to correlate with the folding-unfolding cooperativity. Thus, it can be used as a specificity parameter for designing artificial proteins with a unique structure.     

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.     

A conserved acidic amino acid mediates the interaction between modulators and co-chaperones in enterobacteria

Hsp40-like co-chaperones are ubiquitous enzymes that stimulate the protein refolding activity of Hsp70 family chaperones. They are widespread in prokaryotic and eukaryotic systems. In bacteria, the best characterized co-chaperone is the Escherichia coli DnaJ protein. Many γ-proteobacteria encode a functional homologue of DnaJ, known as CbpA, which is expressed in response to starvation and environmental stress. The activity of CbpA is regulated by the “modulator” protein CbpM. Here, we have used a combination of genetics and biochemistry to identify the co-chaperone contact determinant of CbpM. We show that the nature of the interaction is conserved in enterobacteria.     

Identification of amino acids required for the functional up-regulation of human dihydrofolate reductase protein in response to antifolate Treatment

Human dihydrofolate reductase (DHFR) protein levels rapidly increase upon exposure to methotrexate, a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR inhibits its own translation by binding to its cognate mRNA and that methotrexate disrupts the DHFR protein-mRNA complex allowing its translation to resume. In the present study, Chinese hamster ovary cells lacking DHFR were transfected with wild type and mutants of human DHFR to identify amino acids that are essential for increases in DHFR in response to methotrexate. Glu-30, Leu-22, and Ser-118 were involved in the up-regulation of DHFR protein levels by methotrexate and certain other antifolates. Cells transfected with E30A, L22R, and S118A mutants that did not respond to methotrexate up-regulation had higher basal levels of DHFR, consistent with the model, i.e. lack of feedback regulation of these enzymes. Although cells containing the S118A mutant enzyme had higher levels of DHFR and had catalytic activity similar to that of wild type DHFR, they had the same sensitivity to the cytotoxicity of methotrexate, as were cells with wild type DHFR. This finding provides evidence that the adaptive up-regulation of DHFR by methotrexate contributes to the decreased sensitivity to this drug. Based on these observations, a new model is proposed whereby DHFR exists in two conformations, one bound to DHFR mRNA and the other bound to NADPH. The mutants that are not up-regulated by methotrexate are unable to bind their cognate mRNA.     

Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication

DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~ 15 Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication.     

Identification of a putative chromosomal replication origin from Helicobacter pylori and its interaction with the initiator protein DnaA

The key elements of the initiation of Helicobacter pylori chromosome replication, DnaA protein and putative oriC region, have been characterized. The gene arrangement in the H.pylori dnaA region differs from that found in many other eubacterial dnaA regions (rnpA-rmpH-dnaA-dnaN-recF-gyrB). Helicobacter pylori dnaA is flanked by two open reading frames with unknown function, while dnaN-gyrB and rnpA-rmpH loci are separated from the dnaA gene by 600 and 90 kb, respectively. We show that the dnaA gene encoding initiator protein DnaA is expressed in H.pylori cells. The H.pylori DnaA protein, like other DnaA proteins, can be divided into four domains. Here we demonstrate that the C-terminal domain of H.pylori DnaA protein is responsible for DNA binding. Using in silico and in vitro studies, the putative oriC region containing five DnaA boxes has been located upstream of the dnaA gene. DNase I and gel retardation analyses show that the C-terminal domain of H.pylori DnaA protein specifically binds each of five DnaA boxes.     

Mutations in DnaA protein suppress the growth arrest of acidic phospholipid-deficient Escherichia coli cells

Cell growth arrests when the concentrations of anionic phospholipids drop below a critical level in Escherichia coli, with the insufficient amounts of acidic phospholipids adversely affecting the DnaA-dependent initiation of DNA replication at the chromosomal origin (oriC). Mutations have been introduced into the carboxyl region of DnaA, including the portion identified as essential for productive in vitro DnaA-acidic phospholipid interactions. Expression of DnaA proteins possessing certain small deletions or substituted amino acids restored growth to cells deficient in acidic phospholipids, whereas expression of wild-type DnaA did not. The mutations include substitutions and deletions in the phospholipid-interacting domain as well as some small deletions in the DNA-binding domain of DnaA. Marker frequency analysis indicated that initiation of replication occurs at or near oriC in acidic phospholipid- deficient cells rescued by the expression of DnaA having a point mutation in the membrane-binding domain, DnaA(L366K). Flow cytometry revealed that expression in wild-type cells of plasmid-borne DnaA(L366K) and DnaA(Delta363-367) reduced the frequency with which replication was initiated and disturbed the synchrony of initiations.     

Interaction between actin and the effector peptide of MARCKS-related protein. Identification of functional amino acid segments

It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity.     

Diastereoisomeric 4-substituted acidic amino acids in ferns

Diastereoisomeric 4-substituted acidic amino acids occur in characteristic associations in the green parts of some species of the Filicinae. Subspecies of Phyllitis scolopendrium accumulate 2(S),4(R)-4-methylglutamic acid, 2(S)-4-methyleneglutamic acid and the two diastereoisomers of 2(S)-4-hydroxy-4-methylglutamic acid, the last two occurring at relative concentrations of 3: 1. All Asplenium species investigated were distinctive in accumulating 2(S),4(R)-4-methylglutamic acid, the two diastereoisomers of 2(S)-4-hydroxy-4-methylglutamic acid, and the two diastereoisomers of 2(S)-4-hydroxy-2-aminopimelic acid in a characteristic concentration ratio. Some Polystichum species do not accumulate 4-substituted acidic amino acids whereas others accumulate both diastereoisomers of 2(S)-4-hydroxy-4-methylglutamic acid and 'of 2(S)-4-hydroxy-2-aminopimelic acid, and thus resemble Asplenium species. The seasonal variation in the concentration of 4-substituted acidic amino acids in the green parts of Phyllitis, Asplenium and Polystichum species has also been determined.     

Identification of amino acids involved in catalytic process of M. tuberculosis GlmU acetyltransferase

M. tuberculosis GlmU is a bifunctional enzyme with acetyltransferase activity in C-terminus and uridyltransferase activity in N-terminus, and it is involved in the biosynthesis of glycosyl donor UDP-N-acetylglucosamine (UDP-GlcNAc). The crystal structure of M. tuberculosis GlmU clearly determines the active site and catalytic mechanism of GlmU uridyltransferase domain but not succeed in GlmU acetyltransferase domain. Sequence comparison analysis revealed highly conserved amino acid residues in the C-terminus between M. tuberculosis GlmU and GlmU enzymes from other bacteria. To find the essential amino acids related to M. tuberculosis GlmU acetyltransferase activity, we substituted 10 conserved amino acids in the acetyltransferase domain of M. tuberculosis GlmU by site-directed mutagenesis. All the mutant GlmU proteins were largely expressed in soluble and purified by affinity chromatography. Enzyme assays showed that K362A, H374A, Y398A and W460A mutants abolished more than 90?% activity of M. tuberculosis GlmU acetyltransferase and totally lost the affinity with two substrates, suggesting the potential substrate-binding functions. However, K403A, S416A, N456A and E458A mutants exhibited decreased GlmU acetyltransferase activity and lower kinetic parameters, probably responsible for substrate releasing by conformation shifting.     

Determination of taurine and other acidic amino acids in plants

Micromolar concentrations of taurine (about 10 μmol per kg fresh weight) were obtained in the green tissues of higher plants and in the seeds of Phaseolus vulgaris (about 50 μmol/kg). Fruits in general, and also potatoes and fungal sporophores, contained very little taurine. Phosphoserine and phosphoethanolamine showed much higher concentrations in the seeds of Phaseolus vulgaris and in the winter buds of Pinus sylvestris. Their concentrations changed considerably during the growth of the shoot, while that of taurine was more constant.     

Peptide domain involved in the interaction between membrane protein and nucleocapsid protein of SARS-associated coronavirus

Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a novel coronavirus (CoV) that was identified and molecularly characterized in 2003. Previous studies on various coronaviruses indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly and morphogenesis. It is necessary to elucidate the molecular mechanism of SARS-CoV replication and rationalize the anti-SARS therapeutic intervention. In this study, we employed an in vitro GST pull-down assay to investigate the interaction between the membrane (M) and the nucleocapsid (N) proteins. Our results show that the interaction between the M and N proteins does take place in vitro. Moreover, we provide an evidence that 12 amino acids domain (194-205) in the M protein is responsible for binding to N protein. Our work will help shed light on the molecular mechanism of the virus assembly and provide valuable information pertaining to rationalization of future anti-viral strategies.     

Identification of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations

Peptides produced by mild chymotryptic digestion of human erythrocyte protein 4.1 mimic the ability of intact 4.1 to promote the binding of spectrin to F-actin. This complex-promoting activity was found to reside in an 8-kDa peptide which was fully functional when dissociated from other protein 4.1-derived peptides, indicating that noncovalent complexes of multiple peptides were not essential for activity. The 8-kDa peptide was incorporated into a ternary complex with spectrin and F-actin in approximately stoichiometric amounts. Amino acid composition and two-dimensional peptide mapping show that the 8-kDa active peptide is located within the 10-kDa region of protein 4.1 which contains a cAMP-dependent phosphorylated site.