Two high-mobility group box domains act together to underwind and kink DNA.

High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A is thought to be responsible for the majority of HMGB1-DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA.

PKCα phosphorylation of RhoGDI2 at Ser31 disrupts interactions with Rac1 and decreases GDI activity.

Rho family GTPases control a diverse range of cellular processes, and their deregulation has been implicated in human cancer. Guanine nucleotide dissociation inhibitors (GDIs) bind and sequester GTPases in the cytosol, restricting their actions. RhoGDI2 is a member of the GDI family that acts as a metastasis suppressor in a variety of cancer types; however, very little is known about the regulation of this protein. Here, we present a mechanism for inactivation of RhoGDI2 via protein kinase C (PKC) phosphorylation of Ser31 in a region that contacts GTPases. In cells, RhoGDI2 becomes rapidly phosphorylated at Ser31 in response to phorbol 12-myristate 13-acetate stimulation. Based on the effects of pharmacological inhibitors and knockdown by siRNA, we determine that conventional type PKCα is responsible for this phosphorylation. Phospho-mimetic S31E-RhoGDI2 exhibits reduced binding to Rac1 relative to wild type, with a concomitant failure to reduce levels of activated endogenous Rac1 or remove Rac1 from membranes. These results reveal a mechanism of downregulation of RhoGDI2 activity through PKC-mediated phosphorylation of Ser31. We hypothesize that this mechanism may serve to neutralize RhoGDI2 function in tumors that express RhoGDI2 and active PKCα.

Crystallization and rhenium MAD phasing of the acyl-homoserinelactone synthase EsaI.

Acyl-homoserine-L-lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria. AHLs are produced by AHL synthases from two substrates, S-adenosyl-L-methionine and acyl-acyl carrier protein. The AHL synthase EsaI, which is homologous to the AHL synthases from other pathogenic bacterial species, has been crystallized in the primitive tetragonal space group P4(3), with unit-cell parameters a = b = 66.40, c = 47.33 A. The structure was solved by multiple-wavelength anomalous diffraction with a novel use of the rhenium anomalous signal. The rhenium-containing structure has been refined to a resolution of 2.5 A and the perrhenate ion binding sites and liganding residues have been identified.

Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases.

DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.

Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA.

HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single "prebent" site on the DNA, K(d) = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. We conclude that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail.

Nonsequence-specific DNA recognition: a structural perspective.

Chromosomal proteins that form essential architectural components of chromatin bind and bend DNA with an intrinsic low degree of sequence preference. Comparisons made between two recently determined structures of high mobility group (HMG) protein-DNA complexes and other nonsequence-specific protein-DNA complexes reveal the structural basis of this important mode of DNA binding.

The structure of a chromosomal high mobility group protein-DNA complex reveals sequence-neutral mechanisms important for non-sequence-specific DNA recognition.

The high mobility group (HMG) chromosomal proteins, which are common to all eukaryotes, bind DNA in a non-sequence-specific fashion to promote chromatin function and gene regulation. They interact directly with nucleosomes and are believed to be modulators of chromatin structure. They are also important in V(D)J recombination and in activating a number of regulators of gene expression, including p53, Hox transcription factors and steroid hormone receptors, by increasing their affinity for DNA. The X-ray crystal structure, at 2.2 A resolution, of the HMG domain of the Drosophila melanogaster protein, HMG-D, bound to DNA provides the first detailed view of a chromosomal HMG domain interacting with linear DNA and reveals the molecular basis of non-sequence-specific DNA recognition. Ser10 forms water-mediated hydrogen bonds to DNA bases, and Val32 with Thr33 partially intercalates the DNA. These two 'sequence-neutral' mechanisms of DNA binding substitute for base-specific hydrogen bonds made by equivalent residues of the sequence-specific HMG domain protein, lymphoid enhancer factor-1. The use of multiple intercalations and water-mediated DNA contacts may prove to be generally important mechanisms by which chromosomal proteins bind to DNA in the minor groove.

Co-crystallization and preliminary crystallographic analysis of the high mobility group domain of HMG-D bound to DNA.

Structural studies are essential to understand mechanisms of non-sequence-specific DNA binding used by chromosomal proteins. A non-histone high-mobility group (HMG) chromosomal protein from Drosophila melanogaster, HMG-D, binds duplex DNA in a non-sequence-specific fashion. The DNA-binding domain of HMG-D has been co-crystallized with a duplex DNA fragment in the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell dimensions a = 43.74, b = 53.80, c = 86.84 A. Data have been collected to 2.20 A at 99 K, with diffraction observed to at least 2.0 A. Heavy-atom derivative crystals have been obtained by co-crystallization with oligonucleotides halogenated at major-groove positions near the end of the DNA.

Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo.

Gcn5p is a transcriptional coactivator required for correct expression of various genes in yeast. Several transcriptional regulators, including Gcn5p, possess intrinsic histone acetyltransferase (HAT) activity in vitro. However, whether the HAT activity of any of these proteins is required for gene activation remains unclear. Here, we demonstrate that the HAT activity of Gcn5p is critical for transcriptional activation of target genes in vivo. Core histones are hyperacetylated in cells overproducing functional Gcn5p, and promoters of Gcn5p-regulated genes are associated with hyperacetylated histones upon activation by low-copy Gcn5p. Point mutations within the Gcn5p catalytic domain abolish both promoter-directed histone acetylation and Gcn5p-mediated transcriptional activation. These data provide the first in vivo evidence that promoter-specific histone acetylation, catalyzed by functional Gcn5p, plays a critical role in gene activation.

Structure prediction of a complex between the chromosomal protein HMG-D and DNA.

Non-histone chromosomal proteins are an important part of nuclear structure and function due to their ability to interact with DNA to form and modulate chromatin structure and regulate gene expression. However, the understanding of the function of chromosomal proteins at the molecular level has been hampered by the lack of structures of chromosomal protein-DNA complexes. We have carried out a molecular dynamics modeling study to provide insight into the mode of DNA binding to the chromosomal HMG-domain protein, HMG-D. Three models of a complex of HMG-D bound to DNA were derived through docking the protein to two different DNA fragments of known structure. Molecular dynamics simulations of the complexes provided data indicating the most favorable model. This model was further refined by molecular dynamics simulation and extensively analyzed. The structure of the corresponding HMG-D-DNA complex exhibits many features seen in the NMR structures of the sequence-specific HMG-domain-DNA complexes, lymphoid enhancer factor 1 (LEF-1) and testis determining factor (SRY). The model reveals differences from these known structures that suggest how chromosomal proteins bind to many different DNA sequences with comparable affinity.

Oxidation of a critical methionine modulates DNA binding of the Drosophila melanogaster high mobility group protein, HMG-D.

HMG-D is a major high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. During overexpression and purification of HMG-D from E. coli, a key DNA binding residue, methionine 13, undergoes oxidation to methionine sulfoxide. Oxidation of this critical residue decreases the affinity of HMG-D for DNA by three-fold, altering the structure of the HMG-D-DNA complex without affecting the structure of the free protein. This work shows that minor modification of DNA intercalating residues may be used to fine tune the DNA binding affinity of HMG domain proteins.

A purified mariner transposase is sufficient to mediate transposition in vitro.

Mariners are a widespread and diverse family of animal transposons. Extremely similar mariners of the irritans subfamily are present in the genomes of three divergent insect host species, which strongly suggests that species-specific host factors are unnecessary for mobility. We tested this hypothesis by examining the activity of a purified transposase from one of these elements (Himar1) present in the horn fly, Haematobia irritans. Himar1 transposase was sufficient to reproduce transposition faithfully in an in vitro inter-plasmid transposition reaction. Further analyses showed that Himar1 transposase binds to the inverted terminal repeat sequences of its cognate transposon and mediates 5' and 3' cleavage of the element termini. Independence of species-specific host factors helps to explain why mariners have such a broad distribution and why they are capable of horizontal transfer between species.

Modifying the helical structure of DNA by design: recruitment of an architecture-specific protein to an enforced DNA bend.

BACKGROUND: Proteins can force DNA to adopt distorted helical structures that are rarely if ever observed in naked DNA. The ability to synthesize DNA that contains defined helical aberrations would offer a new avenue for exploring the structural and energetic plasticity of DNA. Here we report a strategy for the enforcement of non-canonical helical structures through disulfide cross-linking; this approach is exemplified by the design and synthesis of an oligonucleotide containing a pronounced bend. RESULTS: A localized bend was site-specifically introduced into DNA by the formation of a disulfide cross-link between the 5' adenines of a 5'-AATT-3' region in complementary strands of DNA. The DNA bend was characterized by high-resolution NMR structure determination of a cross-linked dodecamer and electrophoretic mobility assays on phased multimers, which together indicate that the cross-linked tetranucleotide induces a helical bend of approximately 30 degrees and a modest degree of unwinding. The enforced bend was found to stimulate dramatically the binding of an architecture-specific protein, HMG-D, to the DNA. DNase I foot-printing analysis revealed that the protein is recruited to the section of DNA that is bent. CONCLUSIONS: The present study reports a novel approach for the investigation of non-canonical DNA structures and their recognition by architecture-specific proteins. The mode of DNA bending induced by disulfide cross-linking resembles that observed in structures of protein-DNA complexes. The results reveal common elements in the DNA-binding mode employed by sequence-specific and architecture-specific HMG proteins.

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region.

Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 26/9. Comparison of two different antibodies bound to the same peptide antigen.

The three-dimensional structure of the complex of a second anti-peptide antibody (Fab 26/9) that recognizes the same six-residue epitope of an immunogenic peptide from influenza virus hemagglutinin (HA1; 75-110) as Fab 17/9 with the peptide has been determined at 2.8 A resolution. The amino acid sequence of the variable region of the 26/9 antibody differs in 24 positions from that of 17/9, the first antibody in this series for which several ligand-bound and free structures have been determined and refined. Comparison of the 26/9-peptide with the 17/9-peptide complex structures shows that the two Fabs are very similar (r.m.s.d. 0.5 to 0.8 A) and that the peptide antigen (101-107) has virtually the same conformation (r.m.s.d. 0.3 to 0.8 A) when bound to both antibodies. A sequence difference in the 26/9 binding pocket (L94; His in 26/9, Asn in 17/9) results in an interaction with a bound water molecule that is not seen in the 17/9 structures. Epitope mapping shows that the relative specificity of 26/9 and 17/9 antibodies for individual positions of the peptide antigen are slightly different. Amino acid substitutions in the peptide, particularly at position SerP107, are tolerated to different extents by 17/9 and 26/9. Structural and sequence analysis suggests that amino acid differences near the peptide-binding site are responsible for altering slightly the specificity of 26/9 for three peptide residues and illustrates how amino acid substitutions can modify antibody-antigen interactions and thereby modulate antibody specificity.

The solution structure and dynamics of the DNA-binding domain of HMG-D from Drosophila melanogaster.

BACKGROUND: The HMG-box is a conserved DNA-binding motif that has been identified in many high mobility group (HMG) proteins. HMG-D is a non-histone chromosomal protein from Drosophila melanogaster that is closely related to the mammalian HMG-box proteins HMG-1 and HMG-2. Previous structures determined for an HMG-box domain from rat and hamster exhibit the same global topology, but differ significantly in detail. It has been suggested that these differences may arise from hinge motions which allow the protein to adapt to the shape of its target DNA. RESULTS: We present the solution structure of HMG-D determined by NMR spectroscopy to an overall precision of 0.85 A root mean squared deviation (rmsd) for the backbone atoms. The protein consists of an extended amino-terminal region and three alpha-helices that fold into a characteristic 'L' shape. The central core region of the molecule is highly stable and maintains an angle of approximately 80 degrees between the axes of helices 2 and 3. The backbone dynamics determined from 15N NMR relaxation measurements show a high correlation with the mean residue rmsd determined from the calculated structures. CONCLUSIONS: The structure determined for the HMG-box motif from HMG-D is essentially identical to the structure determined for the B-domain of mammalian HMG-1. Since these proteins have significantly different sequences our results indicate that the global fold and the mode of interaction with DNA are also likely to be conserved in all eukaryotes.

Detection of retinoblastoma gene deletions in spontaneous and radiation-induced mouse lung adenocarcinomas by polymerase chain reaction.

A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma gene using histological sections from radiation-induced and spontaneous tumors as the DNA source. Six mouse Rb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments relative to control PCR products on a Southern blot indicated a deletion of that portion of the mouse Rb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death. Spontaneous tumors as well as those from irradiated mice (5.69 Gy 60Co gamma rays or 0.6 Gy JANUS neutrons, which have been found to have approximately equal radiobiological effectiveness) were analyzed for mouse Rb deletions. Tumors in 6 neutron-irradiated mice had no mouse Rb deletions. However, 1 of 6 tumors from gamma-irradiated mice (17%) and 6 of 18 spontaneous tumors from unirradiated mice (33%) showed a deletion in one or both mouse Rb alleles. All deletions detected were in the 5' region of the mouse Rb gene.