HTH Motif Research Articles

Sequence-selective DNA cleavage by a chimeric metallopeptide.

A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as a folded domain and thus cleaves selected sequences. The de novo artificial nuclease LnP3W represents the first small, underivatized peptide that is both active as a nuclease and sequence selective.

DNA recognition by the KorA proteins of IncP-1 plasmids RK2 and R751

The KorA repressor proteins of IncP-1 plasmids belong to a growing family of plasmid-encoded repressors that regulate partitioning genes, and in the IncP-1 plasmids coordinate these with expression of replication and transfer genes as well. Both KorA RK2 (IncP-1α) and KorA R751 (IncP-1β) recognise the 5′-GTTTAGCTAAAC-3′ palindrome. Reporter gene assays showed that KorA proteins from these two main subgroups of IncP-1 plasmids show specificity for their own promoter/operators and this preference was confirmed with in vitro binding studies using gel mobility shift assays on one representative promoter. Class I (high affinity) operators for KorA RK2 are flanked by an A–A–A/T sequence in the upstream half; the T base was shown to greatly influence strong repression. A C–A–G triplet was present in the same region in the R751 O A sequences and the G base was accordingly found to be important for strong KorA R751 repression. An obvious difference between the two KorA proteins is a histidine to serine change at the C-proximal end of the putative recognition helix of the HTH motif (aa 56). An IncP-1α KorAH56S mutant protein had higher affinity for all operators but had improved more on R751 operators than on RK2 operators. This indicates that KorA of RK2 is not maximised for DNA binding activity and that the aa difference at position 56 may play a role in differentiation between α and β KorA operators.

The Basal Transcription Factors TBP and TFB from the Mesophilic Archaeon Methanosarcina mazeii: Structure and Conformational Changes upon Interaction with Stress-gene Promoters

Transcription of archaeal non-stress genes involves the basal factors TBP and TFB, homologs of the eucaryal TATA-binding protein and transcription factor IIB, respectively. No comparable information exists for the archaeal molecular-chaperone, stress genes hsp70(dnaK), hsp40(dnaJ), and grpE. These do not occur in some archaeal species, but are present in others possibly due to lateral transfer from bacteria, which provides a unique opportunity to study regulation of stress-inducible bacterial genes in organisms with eukaryotic-like transcription machinery. Among the Archaea with the genes, those from the mesophilic methanogen Methanosarcina mazeii are the only ones whose basal (constitutive) and stress-induced transcription patterns have been determined. To continue this work, tbp and tfb were cloned from M. mazeii , sequenced, and the encoded recombinant proteins characterized in solution, separately and in complex with each other and with DNA. M. mazeii TBP ranks among the shortest within Archaea and, contrary to other archaeal TBPs, it lacks tryptophan or an acidic tail at the C terminus and has a basic N-terminal third. M. mazeii TFB is similar in length to archaeal and eucaryal homologs and all have a zinc finger and HTH motifs. Phylogenetically, the archaeal and eucaryal proteins form separate clusters and the M. mazeii molecules are closer to the homologs from Archaeoglobus fulgidus than to any other. Antigenically, M. mazeii TBP and TFB are close to archaeal homologs within each factor family, but the two families are unrelated. The purified recombinant factors were functionally active in a cell-free in vitro transcription system, and were interchangeable with the homologs from Methanococcus thermolithotrophicus. The M. mazeii factors have a similar secondary structure by circular dichroism (CD). The CD spectra changed upon binding to the promoters of the stress genes grpE, dnaK, and dnaJ, with the changes being distinctive for each promoter; in contrast, no effect was produced by the promoter of a non-stress-gene. Factor(s)-DNA modeling predicted that modifications of H bonds are caused by TBP binding, and that these modifications are distinctive for each promoter. It also showed which amino acid residues would contact an extended TATA box with a B recognition element, and evolutionary conservation of the TBP-TFB-DNA complex orientation between two archaeal organisms with widely different optimal temperature for growth (37 and 100 °C).

Altering the DNA-binding specificity of Mu transposase in vitro

We describe the isolation of a variant of Mu transposase (MuA protein) which can recognize altered att sites at the ends of Mu DNA. No prior knowledge of the structure of the DNA binding domain or its mode of interaction with att DNA was necessary to obtain this variant. Protein secondary structure programs initially helped target mutations to predicted helical regions within a subdomain of MuA demonstrated to harbor att DNA binding activity. Of the 54 mutant positions examined, only two showed decreased affinity for att DNA, while eight others affected assembly of the Mu transpososome. A variant impaired in DNA binding [MuA(R146V)], and predicted to be in the recognition helix of an HTH motif, was challenged with altered att sites created from degenerate oligonucleotides to select for novel DNA binding specificity. DNA sequences bound to MuA(R146V) were detected by gel-retardation, and following several steps of PCR amplification/enrichment, were identified by cloning and sequencing. The strategy allowed recovery of an altered att site for which MuA(R146V) showed higher affinity than for the wild-type site, although this site was bound by wild-type MuA as well. The altered association between MuA(R146V) and an altered att site target was competent in transposition. We discuss the strengths and limitations of this methodology, which has applications in dissecting the functional role of specific protein-DNA associations.

Mutational analysis of the global regulator KorA of broad-host-range plasmid RK2

KorA protein encoded in the central control region of IncP plasmid RK2 binds to seven operators on the plasmid genome and acts as a global repressor of genes for replication and stable inheritance functions. At trfAp, the promoter for plasmid replication genes, KorA also causes derepression of trbAp, the promoter for trbA, encoding another global regulator (TrbA), which controls genes required for conjugative transfer. Both KorB, a second global repressor encoded in the central control region, and TrbA also act in the trfAp- trbAp region to down-regulate trfAp, but neither of these extra repressors allows derepression of trbAp. To initiate a functional dissection of KorA, we used random mutagenesis and a positive selection system to identify korA mutants which no longer repressed trfAp. Nine single amino acid changes were obtained, which did not affect polypeptide length or apparent stability. These clustered either in the N-terminal region of the protein (region I) or in the putative HTH motif (region II). No changes were obtained in the C-terminal region (region III). Four truncated KorA proteins, with deletions either from the N-terminal or the C-terminal end, were also screened together with the single mutants. Both the band-shift assay with trfAp DNA and the in vivo promoter-probe assays with either trfAp or trbAp showed that none of the region II mutants could bind to DNA and repress the promoter. The region I mutants with a conservative amino acid substitution retained some DNA binding and repressor activity, as well as the ability to dimerise. However, an in vivo system to detect trans-dominance of the mutants indicated that one region I point mutant together with the two N-terminally truncated mutants had lost their dimerisation ability. Deletions into the basic C terminus of KorA did not abolish dimerisation. The results implicate region I in dimerisation, region II in DNA binding and region III in a yet unspecified role, possibly interaction with other proteins such as KorB.

Solution structure of the IRF-2 DNA-binding domain: a novel subgroup of the winged helix-turn-helix family.

The transcription of interferon (IFN) and IFN-inducible genes is mainly regulated by the interferon regulatory factor (IRF) family of proteins, which recognize a unique AAGTGA hexamer repeat motif in the regulatory region of IFN genes. A DNA-binding domain of approximately 100 amino acids has been commonly found in the IRF family of proteins, but it has no sequence homology to known DNA-binding motifs. Elucidation of the structures of members of the IRF family is therefore useful to the understanding of the regulation and evolution of the immune system at the structural level. The solution structure of the DNA-binding domain of interferon regulatory factor-2 (IRF-2) has been determined by NMR spectroscopy. It is composed of a four-stranded antiparallel beta sheet and three alpha helices, and its global fold is similar to those of the winged helix-turn-helix (wHTH) family of proteins. A long loop (Pro37-Asp51) is found immediately before the HTH motif, which is not found in other wHTH proteins. The NMR signals of residues in this long loop, as well as the second helix of the HTH motif, are strongly affected upon the addition of the hexamer repeat DNA, suggesting that these structural elements participate in DNA recognition and binding. The structural similarity of the DNA-binding domain of IRF-2 with those of proteins in the wHTH family shows that the IRF proteins belong to the wHTH family, even though there is no apparent sequence homology among proteins of the two families. The sequential structure alignment program (SSAP) shows that IRF-2 has a slightly different structure from typical wHTH proteins, mainly in the orientation of helix 2. The IRF family of proteins should therefore be categorized into a subfamily of the wHTH family. The evidence here implies that the evolutional pathway of the IRF family is distinct from that of the other wHTH proteins, in other words, the immune system diverged from an evolutional stem at an early stage.

The Role of the Variable Region in Tet Repressor for Inducibility by Tetracycline

A set of deletions and substitutions to alanine was introduced into the loop separating helices alpha8 and alpha9 of Tn10 Tet repressor (TetR). This region appears as an unstructured loop in the crystal structure of the TetR(D).([Mg-tc]+)2 complex and is the only internal segment of variable length in an alignment of Tet repressors from seven different resistance determinants. In vivo analysis of 10 mutants shows that this loop is important for inducibility by tetracycline (tc), whereas DNA binding is not or only marginally affected. All deletions have an induction-deficient TetRS phenotype, but the corresponding substitutions do not or only slightly affect inducibility. The purified mutant TetR proteins have a reduced affinity for tc in vitro that correlates with their lack of inducibility. The association rate of [Mg-tc]+ to the TetR mutants is enhanced. Since none of the mutated residues contacts tc directly in the crystal structure, we propose that the length of the loop is important for the structural transition between a closed, tc binding and an open, operator binding conformation of TetR. We propose that the deletions in the loop shift the equilibrium between both forms toward the open, operator binding conformation.

Promise: a new database of information on prosthetic centres and metal ions in protein active sites.

The PROMISE (Prosthetic centres and metal ions in protein active sites) database aims to gather together comprehensive sequence, structural, functional and bibliographic information on proteins which possess prosthetic centres, with an emphasis on active site structure and function. The database is available on the World Wide Web at http://bioinf.leeds.ac.uk/promise/ BACKGROUND Of the more than 5000 proteins of known three-dimensional structure, at least half contain metal ions or other non-protein prosthetic groups in their active sites; such prosthetic groups often themselves containing metal ions. Nature has made abundant use of the wide range of chemical properties of the elements, exemplified conspicuously by the complex coordination states of the transition metals. The presence of metal ions and other prosthetic groups confers vitally important properties on the proteins concerned, while the protein environments of the groups modulate the chemistry of the ions in subtle ways. The ‘tuning’ of the chemical reactivity of catalytic centres to the needs of the organism by this interplay of the protein and non-protein components is clearly highly sensitive to the local geometry of the active sites and further modified by the overall molecular structure. The genome sequencing and other structural biology projects are accumulating ever more data, but the need to systematise and analyse the data becomes correspondingly more demanding. In addition to the rapidly growing ‘primary’ databanks of nucleic acid and protein sequences (corresponding already to >150 000 different proteins) and the Protein Databank of three-dimensional (3-D) structures, a number of ‘secondary’ databases have been derived, covering a diverse array of specialised areas. These include classification of proteins in terms of invariant active site groups (3), of ‘fingerprints’ of complex sequence motifs ( 1) or of patterns of chain fold ( 13,19). The PROMISE database is uniquely focused on protein active site structure and on the relationships between protein molecules and non-protein prosthetic centres, combining the relevant sequence, 3-D structural and physico-chemical information (7). The concept of bioinorganic motif (14), a structural feature peculiar for metalloproteins and other complex proteins, may be useful in further discussion. Bioinorganic motifs, some of which are exemplified in Figure 1, endow a protein with function(s) possessed by neither the apoprotein nor prosthetic group alone. These motifs are quite different from sequence motifs (basically strings of text) or protein fold motifs (such as βαβ or HTH motifs). Although there are many families of proteins sharing both fold and bioinorganic motif, there are also evolutionarily unrelated but functionally analogous systems which have similar bioinorganic motif and, vice versa, there are proteins which share the same fold but have distinct active site structures. Bioinorganic motifs often possess unique spectroscopic properties which make it possible to study them relatively independently from the rest of the protein matrix.

Construction of chimeric proteins from the σ;N‐associated transcriptional activators VnfA and AnfA of Azotobacter vinelndii shows that the determinants of promoter specificity lie outside the‘recognition’helix of the HTH motif in the C‐terminal domain

Functional chimeras have been generated from the transcriptional activators VnfA and AnfA, which control expression of the alternative nitrogenases in Azotobacter vinelandii. The activation profiles of the native and chimeric proteins have been determined using lacZ fusions to A. vinelandii anf and vnf promoters in Klebsiella pneumoniae. Replacing the C-terminal domain of AnfA with that of VnfA gives a protein with the promoter specificity of VnfA, confirming that the C-terminal domain contains the determinants of promoter specificity. However, substituting the VnfA sequence from the turn in the helix-turn-helix motif to the C-terminus does not alter the promoter specificity of AnfA. These changes in promoter specificity were reflected in changes in affinity for a VnfA-binding site, as measured by an in vivo repression assay using a lacZ fusion to a synthetic promoter. This supports the assumption that promoter recognition is determined by activator binding to enhancer--like sequences, and shows that the principal determinants of specific DNA-binding lie outside the 'recognition' helix. This may be a general feature of transcriptional activators dependent on sigma N (sigma 54). The chimera with the promoter specificity of VnfA retained the dependence on nitrogenase Fe protein characteristic of AnfA, indicating that this property is not related to particular promoter sequences, but is a function of the central or N-terminal domains of AnfA.