Nucleotide-dependent interaction of the N-terminal domain of MukB with microtubules

The MukB protein from Escherichia coli has a domain structure that is reminiscent of the eukaryotic motor proteins kinesin and myosin: N-terminal globular domains, a region of coiled-coil, and a specialised C-terminal domain. Sequence alignment of the N-terminal domain of MukB with the kinesin motor domain indicated an approximately 22% sequence identity. These observations raised the possibility that MukB might be a prokaryotic motor protein and, due to the sequence homology shared with kinesin, might bind to microtubules (Mts). We found that a construct encoding the first 342 residues of MukB (Muk342) binds specifically to Mts and shares a number of properties with the motor domain of kinesin. Visualisation of the Muk342 decorated Mt complexes using negative stain electron microscopy indicated that the Muk342 smoothly decorates the outside of Mts. Biochemical data demonstrate that Muk342 decorates Mts with a binding stoichiometry of one Muk342 monomer per tubulin monomer. These findings strongly suggest that MukB has a role in force generation and that it is a prokaryotic homologue of kinesin and myosin.     

First crystallographic structure of a xylanase from glycoside hydrolase family 5: implications for catalysis

The room-temperature structure of xylanase (EC 3.2.1.8) from the bacterial plant pathogen Erwinia chrysanthemi expressed in Escherichia coli, a 45 kDa, 413-amino acid protein belonging to glycoside hydrolase family 5, has been determined by multiple isomorphous replacement and refined to a resolution of 1.42 A. This represents the first structure of a xylanase not belonging to either glycoside hydrolase family 10 or family 11. The enzyme is composed of two domains similar to most family 10 xylanases and the alpha-amylases. The catalytic domain (residues 46-315) has a (beta/alpha)(8)-barrel motif with a binding cleft along the C-terminal side of the beta-barrel. The catalytic residues, Glu165 and Glu253, determined by correspondence to other family 5 and family 10 glycoside hydrolases, lie inside this cleft on the C-terminal ends of beta-strands 4 and 7, respectively, with an O(epsilon)2...O(epsilon)1 distance of 4.22 A. The smaller domain (residues 31-43 and 323-413) has a beta(9)-barrel motif with five of the strands interfacing with alpha-helices 7 and 8 of the catalytic domain. The first 13 N-terminal residues form one beta-strand of this domain. Residues 44, 45, and 316-322 form the linkers between this domain and the catalytic domain.     

Kar3Vik1 Mechanochemistry Is Inhibited by Mutation or Deletion of the C Terminus of the Vik1 Subunit

Force production by kinesins has been linked to structural rearrangements of the N and C termini of their motor domain upon nucleotide binding. In recent crystal structures, the Kar3-associated protein Vik1 shows unexpected homology to these conformational states even though it lacks a nucleotide-binding site. This conservation infers a degree of commonality in the function of the N- and C-terminal regions during the mechanochemical cycle of all kinesins and kinesin-related proteins. We tested this inference by examining the functional effects on Kar3Vik1 of mutating or deleting residues in Vik1 that are involved in stabilizing the C terminus against the core and N terminus of the Vik1 motor homology domain (MHD). Point mutations at two moderately conserved residues near the Vik1 C terminus impaired microtubule gliding and microtubule-stimulated ATP turnover by Kar3Vik1. Deletion of the seven C-terminal residues inhibited Kar3Vik1 motility much more drastically. Interestingly, none of the point mutants seemed to perturb the ability of Kar3Vik1 to bind microtubules, whereas the C-terminal truncation mutant did. Molecular dynamics simulations of these C-terminal mutants showed distinct root mean square fluctuations in the N-terminal region of the Vik1 MHD that connects it to Kar3. Here, the degree of motion in the N-terminal portion of Vik1 highly correlated with that in the C terminus. These observations suggest that the N and C termini of the Vik1 MHD form a discrete folding motif that is part of a communication pathway to the nucleotide-binding site of Kar3.     

Interaction of the N-terminal domain of MukB with the bacterial tubulin homologue FtsZ

The MukB protein is involved in the process of chromosome partition in Escherichia coli and has a domain structure reminiscent of the eukaryotic motor proteins kinesin and myosin. This has led to the suggestion that MukB may function as a motor protein in vivo. In order to test this idea we have recombinantly expressed the N-terminal domain of MukB (residues 1–342) as a poly-His tagged fusion protein for biochemical characterisation. The purified protein (Muk342) is monomeric and has low basal Mg-ATPase (1.23 min⁻¹) and Mg-GTPase (0.17 min⁻¹) activities. Muk342 binds with high affinity to the prokaryotic tubulin homologue FtsZ and we have evidence that FtsZ can stimulate nucleotide turnover by Muk342. These properties are consistent with MukB functioning as a motor protein using FtsZ as a track or anchor for generating force within E. coli.     

The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins

The Escherichia coli MukB, MukE, and MukF proteins form a bacterial condensin (MukBEF) that contributes to chromosome management by compacting DNA. MukB is an ATPase and DNA-binding protein of the SMC superfamily; however, the structure and function of non-SMC components, such as MukF, have been less forthcoming. Here, we report the crystal structure of the N-terminal 287 amino acids of MukF at 2.9 A resolution. This region folds into a winged-helix domain and an extended coiled-coil domain that self-associate to form a stable, doubly domain-swapped dimer. Protein dissection and affinity purification data demonstrate that the region of MukF C-terminal to this fragment binds to MukE and MukB. Our findings, together with sequence analyses, indicate that MukF is a kleisin subunit for E. coli condensin and suggest a means by which it may organize the MukBEF assembly.     

Structure and function of the Escherichia coli RecE protein, a member of the RecB nuclease domain family

The RecB subunit of the Escherichia coli RecBCD enzyme has both helicase and nuclease activities. The helicase function was localized to an N-terminal domain, whereas the nuclease activity was found in a C-terminal domain. Recent analysis has uncovered a group of proteins that have weak amino acid sequence similarity to the RecB nuclease domain and that are proposed to constitute a family of related proteins (Aravind, L., Walker, D. R., and Koonin, E. V. (1999) Nucleic Acids Res. 27, 1223-1242). One is the E. coli RecE protein (exonuclease VIII), an ATP-independent exonuclease that degrades the 5'-terminated strand of double-stranded DNA. We have made mutations in several residues of RecE that align with the critical residues of RecB, and we find that the mutations reduce or abolish the nuclease activity of RecE but do not affect the enzyme binding to linear double-stranded DNA. Proteolysis experiments with subtilisin show that a stable 34-kilodalton C-terminal domain that contains these critical residues has nuclease activity, whereas no stable proteolytic fragments accumulate from the N-terminal portion of RecE. These results show that RecE has a nuclease domain and active site that are similar to RecB, despite the very weak sequence similarity between the two proteins. These similarities support the hypothesis that the nuclease domains of the two proteins are evolutionarily related.     

The elastin-binding protein of Staphylococcus aureus (EbpS) is expressed at the cell surface as an integral membrane protein and not as a cell wall-associated protein.

The elastin-binding proteins EbpS of Staphylococcus aureus strains Cowan and 8325-4 were predicted from sequence analysis to comprise 486 residues. Specific antibodies were raised against an N-terminal domain (residues 1-267) and a C-terminal domain (residues 343-486) expressed as recombinant proteins in Escherichia coli. Western blotting of lysates of wild-type 8325-4 and Newman and the corresponding ebpS mutants showed that EbpS migrated with an apparent molecular mass of 83 kDa. The protein was found exclusively in cytoplasmic membrane fractions purified from protoplasts or lysed cells, in contrast to the clumping factor ClfA, which was cell-wall-associated. EbpS was predicted to have three hydrophobic domains H1-(205-224), H2-(265-280), and H3-(315-342). A series of hybrid proteins was formed between EbpS at the N terminus and either alkaline phosphatase or beta-galactosidase at the C terminus (EbpS-PhoA, EbpS-LacZ). PhoA and LacZ were fused to EbpS between hydrophobic domains H1-H2 and H2-H3, and distal to H3. Expression of enzymatic activity in E. coli showed that EbpS is an integral membrane protein with two membrane-spanning domains H1 and H3. N-terminal residues 1-205 and C-terminal residues 343-486 were predicted to be exposed on the outer face of the cytoplasmic membrane. The ligand-binding domain of EbpS is known from previous studies to be present in the N terminus between residues 14-34 and probing whole cells with anti-EbpS1-267 antibodies indicated that this region is exposed on the surface of intact cells. This was also confirmed by the observation that wild-type S. aureus Newman cells bound labeled tropoelastin whereas the ebpS mutant bound 72% less. In contrast, the C terminus, which carries a putative LysM peptidoglycan-binding domain, is not exposed on the surface of intact cells and presumably remains buried within the peptidoglycan. Finally, expression of EbpS was correlated with the ability of cells to grow to a higher density in liquid culture, suggesting that EbpS may have a role in regulating cell growth.     

Nucleotide-dependent dimerization of the C-terminal domain of the ABC transporter CvaB in colicin V secretion

The cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC constitute the bacteriocin colicin V secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter, and its C-terminal domain (CTD) contains typical motifs for the nucleotide-binding and Walker A and B sites and the ABC signature motif. To study the role of the CvaB CTD in the secretion of colicin V, a truncated construct of this domain was made and overexpressed. Different forms of the CvaB CTD were found during purification and identified as monomer, dimer, and oligomer forms by gel filtration and protein cross-linking. Nucleotide binding was shown to be critical for CvaB CTD dimerization. Oligomers could be converted to dimers by nucleotide triphosphate-Mg, and nucleotide release from dimers resulted in transient formation of monomers, followed by oligomerization and aggregation. Site-directed mutagenesis showed that the ABC signature motif was involved in the nucleotide-dependent dimerization. The spatial proximity of the Walker A site and the signature motif was shown by disulfide cross-linking a mixture of the A530C and L630C mutant proteins, while the A530C or L630C mutant protein did not dimerize on its own. Taken together, these results indicate that the CvaB CTD formed a nucleotide-dependent head-to-tail dimer.     

Structure of the OmpA-like domain of RmpM from Neisseria meningitidis

RmpM is a putative peptidoglycan binding protein from Neisseria meningitidis that has been shown to interact with integral outer membrane proteins such as porins and TonB-dependent transporters. Here we report the 1.9 A crystal structure of the C-terminal domain of RmpM. The 150-residue domain adopts a betaalphabetaalphabetabeta fold, as first identified in Bacillus subtilis chorismate mutase. The C-terminal RmpM domain is homologous to the periplasmic, C-terminal domain of Escherichia coli OmpA; these domains are thought to be responsible for non-covalent interactions with peptidoglycan. From the structure of the OmpA-like domain of RmpM, we suggest a putative peptidoglycan binding site and identify residues that may be essential for binding. Both the crystal structure and solution experiments indicate that RmpM may exist as a dimer. This would promote more efficient peptidoglycan binding, by allowing RmpM to interact simultaneously with two glycan chains through its C-terminal, OmpA-like binding domain, while its (structurally uncharacterized) N-terminal domain could stabilize oligomers of porins and TonB-dependent transporters in the outer membrane.     

A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins

The molecular mechanisms of cold acclimation are still largely unknown; however, it has been established that overwintering plants such as winter wheat increases freeze tolerance during cold treatments. In prokaryotes, cold shock proteins are induced by temperature downshifts and have been proposed to function as RNA chaperones. A wheat cDNA encoding a putative nucleic acid-binding protein, WCSP1, was isolated and found to be homologous to the predominant CspA of Escherichia coli. The putative WCSP1 protein contains a three-domain structure consisting of an N-terminal cold shock domain with two internal conserved consensus RNA binding domains and an internal glycine-rich region, which is interspersed with three C-terminal CX(2)CX(4)HX(4)C (CCHC) zinc fingers. Each domain has been described independently within several nucleotide-binding proteins. Northern and Western blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively. WCSP1 induction was cold-specific because neither abscisic acid treatment, drought, salinity, nor heat stress induced WCSP1 expression. Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers. The capacity to bind dsDNA was nearly eliminated in a mutant protein lacking C-terminal zinc fingers. Structural and expression similarities to E. coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation.     

Crp1p,a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae

A synthetic cruciform DNA (X-DNA) was used for screening cellular extracts of Saccharomyces cerevisiae for X-DNA-binding activity. Three X-DNA-binding proteins with apparent molecular mass of 28kDa, 26kDa and 24kDa, estimated by SDS-PAGE, were partially purified. They were identified as N-terminal fragments originating from the same putative protein, encoded by the open reading frame YHR146W, which we named CRP1 (cruciform DNA-recognising protein 1). Expression of CRP1 in Escherichia coli showed that Crp1p is subject to efficient proteolysis at one specific site. Cleavage leads to an N-terminal subpeptide of approximately 160 amino acid residues that is capable of binding specifically X-DNA with an estimated dissociation constant (K(d)) of 800nM, and a C-terminal subpeptide of approximately 305 residues without intrinsic X-DNA-binding activity. The N-terminal subpeptide is of a size similarly to that of the fragments identified in yeast, suggesting that the same cleavage process occurs in the yeast and the E.coli background. This makes the action of a site-specific protease unlikely and favours the possibility of an autoproteolytic activity of Crp1p. The DNA-binding domain of Crp1p was mapped to positions 120-141. This domain can act autonomously as an X-DNA-binding peptide and provides a new, lysine-rich DNA-binding domain different from those of known cruciform DNA-binding proteins (CBPs). As reported earlier for several other CBPs, Crp1p exerts an enhancing effect on the cleavage of X-DNA by endonuclease VII from bacteriophage T4.     

Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter

The Agrobacterium tumefaciens VirB4 ATPase functions with other VirB proteins to export T-DNA to susceptible plant cells and other DNA substrates to a variety of prokaryotic and eukaryotic cells. Previous studies have demonstrated that VirB4 mutants with defects in the Walker A nucleotide-binding motif are non-functional and exert a dominant negative phenotype when synthesized in wild-type cells. This study characterized the oligomeric structure of VirB4 and examined the effects of Walker A sequence mutations on complex formation and transporter activity. VirB4 directed dimer formation when fused to the amino-terminal portion of cI repressor protein, as shown by immunity of Escherichia coli cells to lambda phage infection. VirB4 also dimerized in Agrobacterium tumefaciens, as demonstrated by the recovery of a detergent-resistant complex of native protein and a functional, histidine-tagged derivative by precipitation with anti-His6 antibodies and by Co2+ affinity chromatography. Walker A sequence mutants directed repressor dimerization in E. coli and interacted with His-VirB4 in A. tumefaciens, indicating that ATP binding is not required for self-association. A dimerization domain was localized to a proposed N-terminal membrane-spanning region of VirB4, as shown by the dominance of an allele coding for the N-terminal 312 residues and phage immunity of host cells expressing cI repressor fusions to alleles for the first 237 or 312 residues. A recent study reported that the synthesis of a subset of VirB proteins, including VirB4, in agrobacterial recipients has a pronounced stimulatory effect on the virB-dependent conjugal transfer of plasmid RSF1010 by agrobacterial donors. VirB4'312 suppressed the stimulatory effect of VirB proteins for DNA uptake when synthesized in recipient cells. In striking contrast, Walker A sequence mutants contributed to the stimulatory effect of VirB proteins to the same extent as native VirB4. These findings indicate that the oligomeric structure of VirB4, but not its capacity to bind ATP, is important for the assembly of VirB proteins as a DNA uptake system. The results of these studies support a model in which VirB4 dimers or homomultimers contribute structural information for the assembly of a transenvelope channel competent for bidirectional DNA transfer, whereas an ATP-dependent activity is required for configuring this channel as a dedicated export machine.     

Subcellular trafficking of the yeast plasma membrane ABC transporter, Pdr5, is impaired by a mutation in the N-terminal nucleotide-binding fold

The plasma membrane ATP-binding cassette (ABC) transporter, Pdr5p, mediates resistance to many different xenobiotic compounds in yeast. We have isolated several mutated forms that fail to confer resistance to cycloheximide and itraconazole. Here, we examined two variants, the expression of which was abnormally low when cells reach the stationary phase of growth. The Pdr5(1157) variant lacked the C-terminal transmembrane domain due to the presence of a nonsense mutation at codon 1158. The second variant, Pdr5(L183P), contained a Leu183Pro substitution close to the Walker A motif in the N-terminal nucleotide-binding domain. This substitution impaired UTPase activity as well as protein stability. The Pdr5(L183P) variant induced the unfolded protein response and was targeted to the proteasome for degradation. Fluorescence microscopy showed that the highly unstable Pdr5(L183P) was mislocalized to endoplasmic reticulum (ER)-associated compartments, whereas the truncated Pdr5(1157) protein was retained in the ER. When threonine 363 (located in the first nucleotide-binding domain, close to the Walker B motif) in Pdr5(L183P) was replaced with isoleucine, this double mutant conferred partial drug resistance. These results suggest that Pdr5p requires a properly folded nucleotide-binding domain for trafficking to the plasma membrane.     

Conserved structural elements in glutathione transferase homologues encoded in the genome of Escherichia coli

Multiple sequence alignments of the eight glutathione (GSH) transferase homologues encoded in the genome of Escherichia coli were used to define a consensus sequence for the proteins. The consensus sequence was analyzed in the context of the three-dimensional structure of the gst gene product (EGST) obtained from two different crystal forms of the enzyme. The enzyme consists of two domains. The N-terminal region (domain I) has a thioredoxin-like alpha/beta-fold, while the C-terminal domain (domain II) is all alpha-helical. The majority of the consensus residues (12/17) reside in the N-terminal domain. Fifteen of the 17 residues are involved in hydrophobic core interactions, turns, or electrostatic interactions between the two domains. The results suggest that all of the homologues retain a well-defined group of structural elements both in and between the N-terminal alpha/beta domain and the C-terminal domain. The conservation of two key residues for the recognition motif for the gamma-glutamyl-portion of GSH indicates that the homologues may interact with GSH or GSH analogues such as glutathionylspermidine or alpha-amino acids. The genome context of two of the homologues forms the basis for a hypothesis that the b2989 and yibF gene products are involved in glutathionylspermidine and selenium biochemistry, respectively.     

Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase.

An oxyanion-translocating ATPase encoded by a bacterial plasmid confers resistance to antiomonials and arsenicals in Escherichia coli by extrusion of the toxic oxyanions from the cytosol. The anion pump is composed of two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein is an oxyanion-stimulated ATPase with two nucleotide-binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA protein can be labeled with [alpha-32P]ATP by a UV-catalyzed reaction. Previously reported mutations in the N-terminal site abolish photoadduct formation. Using site-directed mutagenesis the glycine-rich region of the C-terminal putative nucleotide-binding sequence was altered. Three C-terminal site mutant proteins (GR337, KE340, KN340) were analyzed, as well as one additional N-terminal mutant protein (KE21). Strains bearing the mutated plasmids were arsenite sensitive to varying degrees. The purified ArsA protein from mutant KE340 retained approximately 20% of the wild type oxyanion-stimulated ATPase activity, while the purified proteins from the other mutants were catalytically inactive. The KE21 mutation in the N-terminal nucleotide-binding site eliminated photoadduct formation with [alpha-32P] ATP, while the purified proteins with mutations in the C-terminal site retained the ability to form a photoadduct. Each mutant protein was capable of forming a membrane-bound complex in arsB expressing strains. These results suggest first that both sites are required for resistance and ATPase activity, and second that the conserved lysyl residue in the glycine-rich loop of the C-terminal nucleotide-binding site is not essential for catalytic activity.     

Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis

AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidant enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 A resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escherichia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129-Cys132). A flexible linker (residues 197-209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Modeling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF. Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E. coli thioredoxin. A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol-thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.     

ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry

In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p's ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.     

Domain mapping of Escherichia coli RecQ defines the roles of conserved N- and C-terminal regions in the RecQ family

RecQ DNA helicases function in DNA replication, recombination and repair. Although the precise cellular roles played by this family of enzymes remain elusive, the importance of RecQ proteins is clear; mutations in any of three human RecQ genes lead to genomic instability and cancer. In this report, proteolysis is used to define a two-domain structure for Escherichia coli RecQ, revealing a large (~59 kDa) N-terminal and a small (~9 kDa) C-terminal domain. A short N-terminal segment (7 or 21 residues) is also shown to be sensitive to proteases. The effects of removing these regions of RecQ are tested in vitro. Removing 21 N-terminal residues from RecQ severely diminishes its DNA-dependent ATPase and helicase activities, but does not affect its ability to bind DNA in electrophoretic mobility shift assays. In contrast, removing the ~9 kDa C-terminal domain from RecQ results in a fragment with normal levels of ATPase and helicase activity, but that has lost the ability to stably associate with DNA. These results establish the biochemical roles of an N-terminal sequence motif in RecQ catalytic function and for the C-terminal RecQ domain in stable DNA binding.