Miquel Coll

The Bacterial Conjugation Protein Trwb Resembles Ring Helicases and F1-ATPase

The transfer of DNA across membranes and between cells is a central biological process; however, its molecular mechanism remains unknown. In prokaryotes, trans-membrane passage by bacterial conjugation, is the main route for horizontal gene transfer. It is the means for rapid acquisition of new genetic information, including antibiotic resistance by pathogens. Trans-kingdom gene transfer from bacteria to plants or fungi and even bacterial sporulation are special cases of conjugation. An integral membrane DNA-binding protein, called TrwB in the Escherichia coli R388 conjugative system, is essential for the conjugation process. This large multimeric protein is responsible for recruiting the relaxosome DNA–protein complex, and participates in the transfer of a single DNA strand during cell mating. Here we report the three-dimensional structure of a soluble variant of TrwB. The molecule consists of two domains: a nucleotide-binding domain of α/β topology, reminiscent of RecA and DNA ring helicases, and an all-α domain. Six equivalent protein monomers associate to form an almost spherical quaternary structure that is strikingly similar to F1-ATPase. A central channel, 20 Å in width, traverses the hexamer.

Bacterial conjugation: a two-step mechanism for DNA transport

Bacterial conjugation is a promiscuous DNA transport mechanism. Conjugative plasmids transfer themselves between most bacteria, thus being one of the main causal agents of the spread of antibiotic resistance among pathogenic bacteria. Moreover, DNA can be transferred conjugatively into eukaryotic host cells. In this review, we aim to address several basic questions regarding the DNA transfer mechanism. Conjugation can be visualized as a DNA rolling‐circle replication (RCR) system linked to a type IV secretion system (T4SS), the latter being macromolecular transporters widely involved in pathogenic mechanisms. The scheme ‘replication + secretion’ suggests how the mechanism would work on the DNA substrate and at the bacterial membrane. But, how do these two parts come into contact? Furthermore, how is the DNA transported? T4SS are known to be involved in protein secretion in different organisms, but DNA is a very different macromolecule. The so‐called coupling proteins could be the answer to both questions by performing a dual role in conjugation: coupling the two main components of the machinery (RCR and T4SS) and actively mediating DNA transport. We postulate that the T4SS is responsible for transport of the pilot protein (the relaxase) to the recipient. The DNA that is covalently linked to it is initially transported in a passive manner, trailing on the relaxase. We speculate that the pilus appendage could work as a needle, thrusting the substrate proteins to cross one or several membrane barriers into the recipient cytoplasm. This is the first step in conjugation. The second step is the active pumping of the DNA to the recipient, using the already available T4SS transport conduit. It is proposed that this second step is catalysed by the coupling proteins. Our ‘shoot and pump’ model solves the protein–DNA transport paradox of T4SS.

Tandem DNA Recognition by PhoB, a Two-Component Signal Transduction Transcriptional Activator

PhoB is a signal transduction response regulator that activates nearly 40 genes in phosphate depletion conditions in E. coli and closely related bacteria. The structure of the PhoB effector domain in complex with its target DNA sequence, or pho box, reveals a novel tandem arrangement in which several monomers bind head to tail to successive 11-base pair direct-repeat sequences, coating one face of a smoothly bent double helix. The protein has a winged helix fold in which the DNA recognition elements comprise helix alpha 3, penetrating the major groove, and a beta hairpin wing interacting with a compressed minor groove via Arg219, tightly sandwiched between the DNA sugar backbones. The transactivation loops protrude laterally in an appropriate orientation to interact with the RNA polymerase sigma(70) subunit, which triggers transcription initiation.

Structural Basis for DNA Binding Specificity by the Auxin-Dependent ARF Transcription Factors

Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response.

Crystal structure of a DNA Holliday junction

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 Å resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.

The structure of plasmid‐encoded transcriptional repressor CopG unliganded and bound to its operator

The structure of the 45 amino acid transcriptional repressor, CopG, has been solved unliganded and bound to its target operator DNA. The protein, encoded by the promiscuous streptococcal plasmid pMV158, is involved in the control of plasmid copy number. The structure of this protein repressor, which is the shortest reported to date and the first isolated from a plasmid, has a homodimeric ribbon–helix–helix arrangement. It is the prototype for a family of homologous plasmid repressors. CopG cooperatively associates, completely protecting several turns on one face of the double helix in both directions from a 13‐bp pseudosymmetric primary DNA recognition element. In the complex structure, one protein tetramer binds at one face of a 19‐bp oligonucleotide, containing the pseudosymmetric element, with two β‐ribbons inserted into the major groove. The DNA is bent 60° by compression of both major and minor grooves. The protein dimer displays topological similarity to Arc and MetJ repressors. Nevertheless, the functional tetramer has a unique structure with the two vicinal recognition ribbon elements at a short distance, thus inducing strong DNA bend. Further structural resemblance is found with helix–turn–helix regions of unrelated DNA‐binding proteins. In contrast to these, however, the bihelical region of CopG has a role in oligomerization instead of DNA recognition. This observation unveils an evolutionary link between ribbon–helix–helix and helix–turn–helix proteins.

The antimalarial and cytotoxic drug cryptolepine intercalates into DNA at cytosine-cytosine sites.

Cryptolepine, a naturally occurring indoloquinoline alkaloid used as an antimalarial drug in Central and Western Africa, has been found to bind to DNA in a formerly unknown intercalation mode. Evidence from competition dialysis assays demonstrates that cryptolepine is able to bind CG-rich sequences containing nonalternating CC sites. Here we show that cryptolepine interacts with the CC sites of the DNA fragment d(CCTAGG)2 in a base-stacking intercalation mode. This is the first DNA intercalator complex, from ∼90 solved by X-ray crystallography, to bind a nonalternating (pyrimidine-pyrimidine) DNA sequence. The asymmetry of the drug induces a perfect stacking with the asymmetric site, allowing for the stability of the complex in the absence of hydrogen bonding interactions. The crystal structure of this antimalarial drug–DNA complex provides evidence for the first nonalternating intercalation and, as such, provides a basis for the design of new anticancer or antimalarial drugs.

Molecular Structure of the Netropsin-d(CGCGATATCGCG) Complex: DNA Conformation in an Alternating AT Segment

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.

Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC

Relaxases are DNA strand transferases that catalyze the initial and final stages of DNA processing during conjugative cell-to-cell DNA transfer. Upon binding to the origin of transfer (oriT) DNA, relaxase TrwC melts the double helix. The three-dimensional structure of the relaxase domain of TrwC in complex with its cognate DNA at oriT shows a fold built on a two-layer α/β sandwich, with a deep narrow cleft that houses the active site. The DNA includes one arm of an extruded cruciform, an essential feature for specific recognition. This arm is firmly embraced by the protein through a β-ribbon positioned in the DNA major groove and a loop occupying the minor groove. It is followed by a single-stranded DNA segment that enters the active site, after a sharp U-turn forming a hydrophobic cage that traps the N-terminal methionine. Structural analysis combined with site-directed mutagenesis defines the architecture of the active site.

Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity.

Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N‐terminal activation segment of 95 amino acids. The three‐dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R‐factor of 0.169. The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled. The activation segment folds in a globular region with an open sandwich antiparallel‐alpha antiparallel‐beta topology and in a C terminal alpha‐helix which connects it to the enzyme moiety. The globular region (A7‐A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition. AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C‐terminal carboxyl group of substrate molecules. The connecting region occupies the putative extended substrate binding site. The scissile peptide bond cleaved by trypsin during activation is very exposed. Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site. An open‐sandwich folding has been observed in a number of other proteins and protein domains. One of them is the C‐terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase.