Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide‐binding P‐loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker‐A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T‐to‐N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP‐bound conformation and the high‐affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker‐A Thr in sensing the nucleotide's γ‐phosphate and in maintaining an allosteric linkage between the P‐loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate‐remodeling activity.     

A link between sequence conservation and domain motion within the AAA+ family

The AAA+ family of proteins play fundamental roles in all three kingdoms of life. It is thought that they act as molecular chaperones in aiding the assembly or disassembly of proteins or protein complexes. Recent structural studies on a number of AAA+ family proteins have revealed that they share similar structural elements. These structures provide a possible link between nucleotide binding/hydrolysis and the conformational changes which are then amplified to generate mechanical forces for their specific functions. However, from these individual studies it is far from clear whether AAA+ proteins in general share properties in terms of nucleotide induced conformational changes. In this study, we analyze sequence conservation within the AAA+ family and identify two subfamilies, each with a distinct conserved linker sequence that may transfer conformational changes upon ATP binding/release to movements between subdomains and attached domains. To investigate the relation of these linker sequences to conformational changes, molecular dynamics (MD) simulations on X-ray structures of AAA+ proteins from each subfamily have been performed. These simulations show differences in both the N-linker peptide, subdomain motion, and cooperativity between elements of quaternary structure. Extrapolation of subdomain movements from one MD simulation enables us to produce a structure in close agreement with cryo-EM experiments.     

Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana

The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5 and 3 flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.     

Characterization of the mac-1 gene encoding a putative ABC transporter from Myxococcus xanthus

The mac-1 gene of Myxococcus xanthus TA, an antibiotic TA producer, encoded a protein with strong sequence similarity to the antibiotic ATP-binding cassette (ABC) transporter for macrolide antibiotics. The mac-1 gene encoding protein (Mac-1) had two ATP-binding domains containing Walker A and B motifs, and no hydrophobic transmembrane regions. Insertional inactivation of mac-1 caused enhanced sensitivity to oleandomycin, a macrolide antibiotic, while the mac-1 mutant showed normal export of antibiotic TA into the extracellular fluid. The mac-1 mutant could form mounds, but was unable to form fruiting bodies or sporulate under nutrient starvation. A primary role for Mac-1 in M. xanthus may be as a transporter which exports or imports a molecule required for the sporulation process.     

Replicon fusions promoted by the inverted repeats of Tn5. The right repeat is an insertion sequence

Members of three repetitive sequence families were isolated from recombinant λ-genome libraries, and were used to investigate sequence relationships within these families. Studies presented elsewhere show that members of all three repeat sequence families are transcribed tissue-specifically. The thermal stability of intrafamilial heteroduplexes was measured, and the extent of colinearity between related sequences was determined by restriction mapping, heteroduplex visualization, gel blot hybridization, and direct sequencing. One large and very divergent family, named 2108, was shown to consist of an assemblage of many small repeat sequence subfamilies. Each subfamily includes <40 members which are not contiguous in the genome but are very closely related colinear sequence elements several thousand nucleotides in length. The different 2108 subfamilies share only small sequence subelements, which in each subfamily occur in a different linear order and are surrounded by different sequences. A second divergent family consisting of short repetitive sequences, the 2109 family, includes many small internally homologous subfamilies as well. A third family, 2034, displays little internal sequence divergence and no apparent subfamily structure. The repeat sequence subfamilies may be biologically significant units of repetition. Thus specific 2108 subfamilies were shown to be evolutionary conserved to a remarkable degree. Highly homologous 2108 sequences were found shared among sea urchin species which diverged almost 200 million years ago, although only about 10% of the single copy DNA sequences of these species are now homologous enough to crossreact.     

Molecular and evolutionary analysis of two divergent subfamilies of a novel miniature inverted repeat transposable element in the yellow fever mosquito, Aedes aegypti

A novel family of miniature inverted repeat transposable elements (MITEs) named Pony was discovered in the yellow fever mosquito, Aedes aegypti. It has all the characteristics of MITEs, including terminal inverted repeats, no coding potential, A+T richness, small size, and the potential to form stable secondary structures. Past mobility of PONY: was indicated by the identification of two Pony insertions which resulted in the duplication of the TA dinucleotide targets. Two highly divergent subfamilies, A and B, were identified in A. aegypti based on sequence comparison and phylogenetic analysis of 38 elements. These subfamilies showed less than 62% sequence similarity. However, within each subfamily, most elements were highly conserved, and multiple subgroups could be identified, indicating recent amplifications from different source genes. Different scenarios are presented to explain the evolutionary history of these subfamilies. Both subfamilies share conserved terminal inverted repeats similar to those of the Tc2 DNA transposons in Caenorhabditis elegans, indicating that Pony may have been borrowing the transposition machinery from a Tc2-like transposon in mosquitoes. In addition to the terminal inverted repeats, full-length and partial subterminal repeats of a sequence motif TTGATTCAWATTCCGRACA represent the majority of the conservation between the two subfamilies, indicating that they may be important structural and/or functional components of the Pony elements. In contrast to known autonomous DNA transposons, both subfamilies of PONY: are highly reiterated in the A. aegypti genome (8,400 and 9, 900 copies, respectively). Together, they constitute approximately 1. 1% of the entire genome. Pony elements were frequently found near other transposable elements or in the noncoding regions of genes. The relative abundance of MITEs varies in eukaryotic genomes, which may have in part contributed to the different organizations of the genomes and reflect different types of interactions between the hosts and these widespread transposable elements.     

On the definition, nomenclature and classification of water channel proteins (aquaporins and relatives)

A water channel protein (WCP) or a water channel can be defined as a transmembrane protein that has a specific three-dimensional structure with a pore that provides a pathway for water permeation across biological membranes. The pore is formed by two highly conserved regions in the amino acid sequence, called NPA boxes (or motifs) with three amino acid residues (asparagine-proline-alanine, NPA) and several surrounding amino acids. The NPA boxes have been called the "signature" sequence of WCPs. WCPs are a family of proteins belonging to the Membrane Intrinsic Proteins (MIPs) superfamily. In addition, in the MIP superfamily (with more than 1000 members) there are also proteins with no channel activity. The WCP family include three subfamilies: aquaporins, aquaglyceroporins and S-aquaporins. (1) The aquaporins (AQPs) are water selective or specific water channels, also named by various authors as "orthodox", "ordinary", "conventional", "classical", "pure", "normal", or "sensu strictu" aquaporins); (2) The aquaglyceroporins are permeable to water, but also to other small uncharged molecules, in particular glycerol; this family includes the glycerol facilitators, abbreviated as GlpFs, from glycerol permease facilitators. The "signature" sequence for aquaglyceroporins is the aspartic acid residue (D) in the second NPA box. (3) The third subfamily of WCPs have little conserved amino acid sequences around the NPA boxes, unclassifiable to the first two subfamilies. I recommend to use always for this subfamily the name S-aquaporins. They are also named "superaquaporins", "aquaporins with unusual (or deviated) NPA boxes", "subcellular aquaporins", or "sip-like aquaporins". I also recommend to use always the spelling aquaporin (not aquaporine), and, for various AQPs, the abbreviation AQP followed immediately by the number, (e.g. AQP1), with no space or - which might create confusions with "minus".     

A novel gene family NBPF: intricate structure generated by gene duplications during primate evolution

Partial and complete genome duplications occurred during evolution and resulted in the creation of new genes and gene families. We identified a novel and intricate human gene family located primarily in regions of segmental duplications on human chromosome 1. We named it NBPF, for neuroblastoma breakpoint family, because one of its members is disrupted by a chromosomal translocation in a neuroblastoma patient. The NBPF genes have a repetitive structure with high intragenic and intergenic sequence similarity in both coding and noncoding regions. These similarities might expose these genomic regions to illegitimate recombination, resulting in structural variation in the NBPF genes. The encoded proteins contain a highly conserved domain of unknown function, which we have named the NBPF repeat. In silico analysis combined with the isolation of multiple full-length cDNA clones showed that several members of this gene family are abundantly expressed in a large variety of tissues and cell lines. Strikingly, no discernable orthologues could be identified in the completed genomes of fruit fly, nematode, mouse, or rat, but sequences with low homology could be isolated from the draft canine and bovine genomes. Interestingly, this gene family shows primate-specific duplications that result in species-specific arrays of NBPF homologous sequences. Overall, this novel NBPF family reflects the continuous evolution of primate genomes that resulted in large physiological differences, and its potential role in this process is discussed.     

Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein

The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.     

Molecular characterization of the rbcS multi-gene family of Petunia (Mitchell)

Summary The small subunit (RbcS) of ribulose bisphosphate carboxylase (RuBPCase) is encoded by eight genes in Petunia (Mitchell). These genes can be divided into three subfamilies (51, 117 and 71) based upon hybridization to three petunia rbcS cDNA clones. The nucleotide sequence of six of the eight petunia rbcS genes is presented here and the structure of the genes is discussed with respect to their genomic linkage and their expression levels in petunia leaf tissue. The rbcS genes belonging to the same subfamily encode an identical mature RbcS polypeptide, however the different subfamilies encode distinguishable polypeptides. All the genes, except one, contian two introns within the mature subunit coding region; one gene contains one extra intron within the coding region. There are large regions of nucleotide sequence homology within the introns of genes within a subfamily, but significantly less homology between the introns of genes of different subfamilies. A complex pattern of homology within the multiple genes of the 51 subfamily is observed. There are regions within these genes which share high levels of sequence homology; this homology does not extend throughout the whole gene and the regions of homology do not always occur in adjacent genes. Two 3 rbcS gene fragments which we isolated from the petunia genome show high levels of homology to two of the intact rbcS genes.     

The integrase family of tyrosine recombinases: evolution of a conserved active site domain.

The integrases are a diverse family of tyrosine recombinases which rearrange DNA duplexes by means of conservative site-specific recombination reactions. Members of this family, of which the well-studied lambda Int protein is the prototype, were previously found to share four strongly conserved residues, including an active site tyrosine directly involved in transesterification. However, few additional sequence similarities were found in the original group of 27 proteins. We have now identified a total of 81 members of the integrase family deposited in the databases. Alignment and comparisons of these sequences combined with an evolutionary analysis aided in identifying broader sequence similarities and clarifying the possible functions of these conserved residues. This analysis showed that members of the family aggregate into subfamilies which are consistent with their biological roles; these subfamilies have significant levels of sequence similarity beyond the four residues previously identified. It was also possible to map the location of conserved residues onto the available crystal structures; most of the conserved residues cluster in the predicted active site cleft. In addition, these results offer clues into an apparent discrepancy between the mechanisms of different subfamilies of integrases.     

Proteins of theXenopus laevis zinc finger multigene family as targets for CK II phosphorylation

Zn finger proteins (ZFPs) of the C₂/H₂ type inXenopus laevis are encoded by a multigene family comprising several hundred members. Based upon conserved sequence features outside the Zn finger region, ZFPs can be subdivided into distinct subfamilies. Two of such subfamilies are characterized by conserved, N-terminal amino acid sequences termed the FAX and the FAR Domain. Here we present data suggesting that the zinc finger proteins of the FAR-ZFP subfamily are targets for CK II mediated phosphorylation. Expression of these proteins during oogenesis coincides with CK II activity in unfertilized eggs. Additionally, we have found that XIcOF 7.1, a member of the FAX-ZFP subfamily, is also phosphorylated by CK II. The target sites forin vitro phosphorylation are localized within the conserved N-terminal domains but not within the Zn finger regions. However, amino acid sequence comparison revealed that individual phosphoacceptor sites are not generally conserved among all members of the respective ZFP subfamilies. The relevance of a potential CK II phosphorylation for the regulation of ZFP activityin vivo is discussed.     

Comparison of human and mouse T-cell receptor variable gene segment subfamilies

Like the immunoglobulin Igh-V and Igk-V gene families, the human or mouse TCRV gene families may be grouped into subfamilies displaying >75% nucleic acid sequence similarity among their members. Systematic interspecies sequence comparisons reveal that most mouse Tcr-V subfamilies exhibit clear homology to human TCRV subfamilies (>60% amino acid sequence similarity). Homologous pairs of TCRV genes in mice and humans show higher sequence similarity than TCRV genes from different subfamilies within either species, indicating transpecies evolution of TCRV genes. Mouse and human homologues show conservation of their relative map order, particularly in the 3' region and a similar sequential and developmentally programmed expression. When the V regions from both species were analyzed together, local length differences and conserved residues in the loop regions were revealed, characteristic of each of the four TCRV families.The alignment data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the alignment number DS23485. The data are available by the EBI FTP server and file server     

ChvD, a chromosomally encoded ATP-binding cassette transporter-homologous protein involved in regulation of virulence gene expression in Agrobacterium tumefaciens

A yeast two-hybrid screen searching for chromosomally encoded proteins that interact with the Agrobacterium tumefaciens VirB8 protein was carried out. This screen identified an interaction candidate homologous to the partial sequence of a gene that had previously been identified in a transposon screen as a potential regulator of virG expression, chvD. In this report, the cloning of the entire chvD gene is described and the gene is sequenced and characterized. Insertion of a promoterless lacZ gene into the chvD locus greatly attenuated virulence and vir gene expression. Compared to that of the wild-type strain, growth of the chvD mutant was reduced in rich, but not minimal, medium. Expression of chvD, as monitored by expression of beta-galactosidase activity from the chvD-lacZ fusion, occurred in both rich and minimal media as well as under conditions that induce virulence gene expression. The ChvD protein is highly homologous to a family of ATP-binding cassette transporters involved in antibiotic export from bacteria and has two complete Walker box motifs. Molecular genetic analysis demonstrated that disruption of either Walker A box, singly, does not inactivate this protein's effect on virulence but that mutations in both Walker A boxes renders it incapable of complementing a chvD mutant strain. Constitutive expression of virG in the chvD mutant strain restored virulence, supporting the hypothesis that ChvD controls virulence through effects on virG expression.     

The ubiquitin-specific protease family from Arabidopsis. AtUBP1 and 2 are required for the resistance to the amino acid analog canavanine

Ubiquitin-specific proteases (UBPs) are a family of unique hydrolases that specifically remove polypeptides covalently linked via peptide or isopeptide bonds to the C-terminal glycine of ubiquitin. UBPs help regulate the ubiquitin/26S proteolytic pathway by generating free ubiquitin monomers from their initial translational products, recycling ubiquitins during the breakdown of ubiquitin-protein conjugates, and/or by removing ubiquitin from specific targets and thus presumably preventing target degradation. Here, we describe a family of 27 UBP genes from Arabidopsis that contain both the conserved cysteine (Cys) and histidine boxes essential for catalysis. They can be clustered into 14 subfamilies based on sequence similarity, genomic organization, and alignments with their closest relatives from other organisms, with seven subfamilies having two or more members. Recombinant AtUBP2 functions as a bona fide UBP: It can release polypeptides attached to ubiquitins via either alpha- or epsilon-amino linkages by an activity that requires the predicted active-site Cys within the Cys box. From the analysis of T-DNA insertion mutants, we demonstrate that the AtUBP1 and 2 subfamily helps confer resistance to the arginine analog canavanine. This phenotype suggests that the AtUBP1 and 2 enzymes are needed for abnormal protein turnover in Arabidopsis.     

Subfamily structure and evolution of the human L1 family of repetive sequences

Summary Comparative analysis of the available 3′-portions of the human L1 (LINE-1) family of repeated sequences indicates that all the sequences can be classified in two major subfamilies. The division is based on patterns of diagnostic bases shared within L1 subfamilies of sequences but differing between them. The overall ratio of replacement to synonymous positions, occupied by the diagnostic bases in the large open reading frame of the L1 sequence, is 1.15. This indicates that both subfamilies were obtained from genes coding for functional proteins. The L1 subfamilies appear to be of different ages and may represent a “fossil record” of the same active gene at different times in the history of primates. The younger subfamily can be split further into at least two closely related branches of sequences. The above facts combined with the recent data for the Alu subfamily structure show that LINE and SINE families of interspersed repeats share discontinuous patterns in their evolution. These data are consistent with the model that both Alu and L1 families, as well as other pseudogene families, contain active genes producing discrete layers of pseudogenes throughout the history of primates. Models of evolutionary processes that could generate these discontinuities are discussed together with the possible biological role of Alu and L1 genes.