A simple motif for protein recognition in DNA secondary structures

DNA in a single-stranded form (ssDNA) exists transiently within the cell and comprises the telomeres of linear chromosomes and the genomes of some DNA viruses. As with RNA, in the single-stranded state, some DNA sequences are able to fold into complex secondary and tertiary structures that may be recognized by proteins and participate in gene regulation. To better understand how such DNA elements might fold and interact with proteins, and to compare recognition features to those of a structured RNA, we used in vitro selection to identify ssDNAs that bind an RNA-binding peptide from the HIV Rev protein with high affinity and specificity. The large majority of selected binders contain a non-Watson-Crick G.T base-pair and an adjacent C:G base-pair and both are essential for binding. This GT motif can be presented in different DNA contexts, including a nearly perfect duplex and a branched three-helix structure, and appears to be recognized in large part by arginine residues separated by one turn of an alpha-helix. Interestingly, a very similar GT motif is necessary also for protein binding and function of a well-characterized model ssDNA regulatory element from the proenkephalin promoter.     

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein–RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein–double-stranded DNA and protein–single-stranded DNA complexes. The interface properties of the protein–RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein–RNA and protein–DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein–RNA complexes, backbone contacts were more dominant in the protein–DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.     

The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold

The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.     

Uncoupling of Nucleotide Hydrolysis and Polymerization in the ParA Protein Superfamily Disrupts DNA Segregation Dynamics

DNA segregation in bacteria is mediated most frequently by proteins of the ParA superfamily that transport DNA molecules attached via the segrosome nucleoprotein complex. Segregation is governed by a cycle of ATP-induced polymerization and subsequent depolymerization of the ParA factor. Here, we establish that hyperactive ATPase variants of the ParA homolog ParF display altered segrosome dynamics that block accurate DNA segregation. An arginine finger-like motif in the ParG centromere-binding factor augments ParF ATPase activity but is ineffective in stimulating nucleotide hydrolysis by the hyperactive proteins. Moreover, whereas polymerization of wild-type ParF is accelerated by ATP and inhibited by ADP, filamentation of the mutated proteins is blocked indiscriminately by nucleotides. The mutations affect a triplet of conserved residues that are situated neither in canonical nucleotide binding and hydrolysis motifs in the ParF tertiary structure nor at interfaces implicated in ParF polymerization. Instead the residues are involved in shaping the contours of the binding pocket so that nucleotide binding locks the mutant proteins into a configuration that is refractory to polymerization. Thus, the architecture of the pocket not only is crucial for optimal ATPase kinetics but also plays a key role in the polymerization dynamics of ParA proteins that drive DNA segregation ubiquitously in procaryotes.     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Chemical and structural probing of the N-terminal residues encoded by FMR1 exon 15 and their effect on downstream arginine methylation

Exon 15 of the fragile X mental retardation protein gene (FMR1) is alternatively spliced into three variants. The amino acids encoded by the 5' end of the exon contain several regulatory determinants including phosphorylation sites and a potential conformational switch. Residues encoded by the 3' end of the exon specify FMRP's RGG box, an RNA binding domain that interacts with G-quartet motifs. Previous studies demonstrated that the exon 15-encoded N-terminal residues influence the extent of arginine methylation, independent of S 500 phosphorylation. In the present study we focus on the role the putative conformational switch plays in arginine methylation. Chemical and structural probing of Ex15 alternatively spliced variant proteins and several mutants leads to the following conclusions: Ex15c resides largely in a conformation that is refractory toward methylation; however, it can be methylated by supplementing extracts with recombinant PRMT1 or PRMT3. Protein modeling studies reveal that the RG-rich region is part of a three to four strand antiparallel beta-sheet, which in other RNA binding proteins functions as a platform for nucleic acid interactions. In the Ex15c variant the first strand of this sheet is truncated, and this significantly perturbs the side-chain conformations of the arginine residues in the RG-rich region. Mutating R 507 in the conformational switch to K also truncates the first strand of the beta-sheet, and corresponding decreases in in vitro methylation were found for this and R 507/R 544 and R 507/R 546 double mutants. These effects are not due to the loss of R 507 methylation as a conformational switch-containing peptide reacted under substrate excess and in methyl donor excess was not significantly methylated. Consistent with this, similar changes in beta-sheet structure and decreases in in vitro methylation were observed with a W 513-K mutant. These data support a novel model for FMRP arginine methylation and a role for conformational switch residues in arginine modification.     

The role of helix stabilizing residues in GCN4 basic region folding and DNA binding

Basic region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a coiled-coil leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The basic region is largely unfolded in the absence of DNA, but adopts a helical conformation upon DNA binding. Although a coil --> helix transition is entropically unfavorable, this conformational change positions the DNA-binding residues appropriately for sequence-specific interactions with DNA. The N-terminal residues of the GCN4 DNA-binding domain, DPAAL, make no DNA contacts and are not part of the conserved basic region, but are nonetheless important for DNA binding. Asp and Pro are often found at the N-termini of alpha-helices, and such N-capping motifs can stabilize alpha-helical structure. In the present study, we investigate whether these two residues serve to stabilize a helical conformation in the GCN4 basic region, lowering the energetic cost for DNA binding. Our results suggest that the presence of these residues contributes significantly to helical structure and to the DNA-binding ability of the basic region in the absence of the leucine zipper. Similar helix-capping motifs are found in approximately half of all bZip domains, and the implications of these findings for in vivo protein function are discussed.     

Identification of sequences important for recognition of vnf genes by the VnfA transcriptional activator in Azotobacter vinelandii

Abstract To analyze regulation of the vanadium-dependent nitrogenase of Azotobacter vinelandii , plasmids carrying vnfE-, vnfH- , or vnfD-lacZ fusions were transferred to Escherichia coli . These genes were expressed only if VnfA was present. Deletions of the vnfE upstream region were constructed and comparison of a region necessary for expression with sequences upstream of other vnf genes indicated a substantially conserved motif, GTAC-N6-GTAC, hypothesized to be the binding site for VnfA. This motif was duplicated with 17 or 18 bases lying between each in the vnfH and vnfD promoters. Deletion analysis of the vnfH promoter indicated that both motifs were necessary for full expression. In footprinting experiments, VnfA significantly protected from methylation the guanine residues within or immediately adjacent to the proposed VnfA recognition motifs. The active form of VnfA is probably interacting dimers, a tetramer, or a higher order oligomer since two regions of dyad symmetry are required for its interaction with the DNA.     

Structures of the potassium-saturated, 2:1, and intermediate, 1:1, forms of a quadruplex DNA

Potassium can stabilize the formation of chair- or edge-type quadruplex DNA structures and appears to be the only naturally occurring cation that can do so. As quadruplex DNAs may be important in the structure of telomere, centromere, triplet repeat and other DNAs, information about the details of the potassium–quadruplex DNA interactions are of interest. The structures of the 1:1 and the fully saturated, 2:1, potassium–DNA complexes of d(GGTTGGTGTGGTTGG) have been determined using the combination of experimental NMR results and restrained molecular dynamics simulations. The refined structures have been used to model the interactions at the potassium binding sites. Comparison of the 1:1 and 2:1 potassium:DNA structures indicates how potassium binding can determine the folding pattern of the DNA. In each binding site potassium interacts with the carbonyl oxygens of both the loop thymine residues and the guanine residues of the adjacent quartet.     

The MDMX Acidic Domain Uses Allovalency to Bind Both p53 and MDMX

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.     

Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs.

Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues. Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.     

Crystal structure and functional analysis of DEAD-box protein Dhh1p

The control of mRNA translation and degradation are critical for proper gene expression. A key regulator of both translation and degradation is Dhh1p, which is a DEAD-box protein, and functions both to repress translation and enhance decapping. We describe the crystal structure of the N- and C-terminal truncated Dhh1p (tDhh1p) determined at 2.1 A resolution. This reveals that, like other DEAD-box proteins, tDhh1p contains two RecA-like domains, although with a unique arrangement. In contrast to eIF4A and mjDEAD, in which no motif interactions exist, in Dhh1p, motif V interacts with motif I and the Q-motif, thereby linking the two domains together. Electrostatic potential mapping combined with mutagenesis reveals that motifs I, V, and VI are involved in RNA binding. In addition, trypsin digestion of tDhh1p suggests that ATP binding enhances an RNA-induced conformational change. Interestingly, some mutations located in the conserved motifs and at the interface between the two Dhh1 domains confer dominant negative phenotypes in vivo and disrupt the conformational switch in vitro. This suggests that this conformational change is required in Dhh1 function and identifies key residues involved in that transition.     

Structure and mechanism of the RuvB Holliday junction branch migration motor

The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks. The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain. The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution. ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces. Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins. Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions.     

On the mechanism of sequence-specific DNA-dependent acetylation of p53: the acetylation motif is exposed upon DNA binding

P53 acetylation requires p300-docking to two contiguous sites in the activation domain that in turn mediates DNA-dependent acetylation of the tetramer. In an attempt to further define the mechanism of DNA-dependent acetylation of p53, an in vitro system has been reconstituted with distinct p53 isoforms and has been used to reveal conformational constraints on p53 acetylation. Two native p53 tetrameric isoforms purified from Sf9 cells differing by the extent of phosphorylation within the C-terminal acetylation site are both acetylated in a sequence-specific DNA-dependent manner. By contrast, p53 purified from an Escherichia coli expression system is in a largely denatured conformation and its acetylation is DNA-independent. Heating native p53 to destroy the folded structure restores DNA-independent acetylation similar to that seen with bacterially expressed p53. There are at least two sites of conformational flexibility in the p53 tetramer: the first in the flexible S10 beta-sheet within the MDM2 ubiquitination sequence and the second in the C-terminal regulatory domain. We analysed therefore whether DNA-dependent acetylation correlated with conformational changes in either of these two regions. DNA-dependent acetylation of p53 is maintained in a dose-dependent manner by low concentrations of consensus site DNA under conditions where flexibility in the S10 beta-sheet region is maintained. Oligonucleotide DNAs that promote acetylation stimulate the binding of monoclonal antibodies PAb421 and ICA-9; two antibodies whose contiguous epitopes overlap the C-terminal acetylation motif. By contrast, bent oligonucleotide DNAs that conceal both the S10 beta-sheet from binding of the monoclonal antibody DO-12 and attenuate binding of the monoclonal antibody PAb421 can preclude acetylation. These data suggest that, in the absence of DNA, the acetylation motif of p53 is in a cryptic state, but after DNA binding, allosteric effects mediate an exposure of the acetylation motif to allow DNA-dependent acetylation of the tetramer.     

The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding

The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys397 and Cys400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity.