Maria Solà

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.

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.

Structural basis of dynamic glycine receptor clustering by gephyrin

Gephyrin is a bi‐functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full‐length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C‐terminal region (gephyrin‐E), which binds a GlyR β‐subunit‐derived peptide with high and low affinity. The crystal structure of the tetra‐domain gephyrin‐E in complex with the β‐peptide bound to domain IV indicates how membrane‐embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full‐length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full‐length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.

Human mitochondrial transcription factor A induces a U-turn structure in the light strand promoter

Human mitochondrial transcription factor A, TFAM, is essential for mitochondrial DNA packaging and maintenance and also has a crucial role in transcription. Crystallographic analysis of TFAM in complex with an oligonucleotide containing the mitochondrial light strand promoter (LSP) revealed two high-mobility group (HMG) protein domains that, through different DNA recognition properties, intercalate residues at two inverted DNA motifs. This induced an overall DNA bend of ~180°, stabilized by the interdomain linker. This U-turn allows the TFAM C-terminal tail, which recruits the transcription machinery, to approach the initiation site, despite contacting a distant DNA sequence. We also ascertained that structured protein regions contacting DNA in the crystal were highly flexible in solution in the absence of DNA. Our data suggest that TFAM bends LSP to create an optimal DNA arrangement for transcriptional initiation while facilitating DNA compaction elsewhere in the genome.

Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain

During viral replication, herpesviruses package their DNA into the procapsid by means of the terminase protein complex. In human cytomegalovirus (herpesvirus 5), the terminase is composed of subunits UL89 and UL56. UL89 cleaves the long DNA concatemers into unit-length genomes of appropriate length for encapsidation. We used ESPRIT, a high-throughput screening method, to identify a soluble purifiable fragment of UL89 from a library of 18,432 randomly truncated ul89 DNA constructs. The purified protein was crystallized and its three-dimensional structure was solved. This protein corresponds to the key nuclease domain of the terminase and shows an RNase H/integrase-like fold. We demonstrate that UL89-C has the capacity to process the DNA and that this function is dependent on Mn2+ ions, two of which are located at the active site pocket. We also show that the nuclease function can be inactivated by raltegravir, a recently approved anti-AIDS drug that targets the HIV integrase.

X-ray crystal structure of the trimeric N-terminal domain of gephyrin.

Gephyrin is a ubiquitously expressed protein that, in the central nervous system, forms a submembraneous scaffold for anchoring inhibitory neurotransmitter receptors in the postsynaptic membrane. The N- and C-terminal domains of gephyrin are homologous to the Escherichia coli enzymes MogA and MoeA, respectively, both of which are involved in molybdenum cofactor biosynthesis. This enzymatic pathway is highly conserved from bacteria to mammals, as underlined by the ability of gephyrin to rescue molybdenum cofactor deficiencies in different organisms. Here we report the x-ray crystal structure of the N-terminal domain (amino acids 2–188) of rat gephyrin at 1.9-Å resolution. Gephyrin-(2–188) forms trimers in solution, and a sequence motif thought to be involved in molybdopterin binding is highly conserved between gephyrin and theE. coli protein. The atomic structure of gephyrin-(2–188) resembles MogA, albeit with two major differences. The path of the C-terminal ends of gephyrin-(2–188) indicates that the central and C-terminal domains, absent in this structure, should follow a similar 3-fold arrangement as the N-terminal region. In addition, a central β-hairpin loop found in MogA is lacking in gephyrin-(2–188). Despite these differences, both structures show a high degree of surface charge conservation, which is consistent with their common catalytic function.

Crystal structure of the GABAA‐receptor‐associated protein, GABARAP

The GABAA‐receptor‐associated protein (GABARAP) is a member of a growing family of intracellular membrane trafficking and/or fusion proteins and has been implicated in plasma membrane targeting and/or recycling of GABAA receptors. GABARAP is localized on intracellular membranes such as the trans‐Golgi network, binds to the γ 2 subunit of GABAA receptors and interacts with microtubules and the N‐ethylmaleimide‐sensitive factor. We report the X‐ray crystal structure of mammalian GABARAP at 2.0 Å resolution. GABARAP consists of an N‐terminal basic helical region, which has been implicated in tubulin binding, and a core structure with a conserved ubiquitin‐like fold. Consistent with the high extent of sequence conservation among GABARAP homologues from plants to mammals, one face of the core structure is absolutely conserved while the opposite face shows considerable divergence. These features are in agreement with the conserved surface mediating protein–protein interactions shared by all members of the family, whereas the non‐conserved surface region may play specific roles, such as docking to particular membrane receptors.

Picornavirus non-structural proteins as targets for new anti-virals with broad activity

Picornaviridae is one of the largest viral families and is composed of 14 genera, six of which include human pathogens. The best known picornaviruses are enteroviruses (including polio, PV, and rhinoviruses), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Although infections often are mild, certain strains may cause pandemic outbreaks accompanied with meningitis and/or paralysis. Vaccines are available for PV, HAV and FMDV. When the oral vaccines are given to immunocompromised individuals, they may be chronically infected, and remain secretors of vaccine-derived variants of virus for years. There is no effective prophylaxis available for these or other picornaviruses. So far, only the 3C protease from viruses in three genera has been fully characterized as an anti-viral target, whereas the mode of action of compounds targeting other non-structural proteins have remained largely unaddressed. Within the EU-supported FP6 project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication), the non-structural proteins were studied to identify conserved binding sites for broadly reactive anti-virals. The putative 2C helicase from echovirus-30 was shown to form ring-shaped hexamers typical for DNA-encoded SF3 helicases, and to possess ATPase activity. Hexamer formation of 2C from enterovirus 76 was in vitro shown to be dependent on the 44 N-terminal residues. Crystal structures of three enterovirus 3C proteases were solved and shown to be similar to those of other picornaviruses. A new binding site of VPg to the bottom of the thumb domain of CV-B3 3D polymerase was identified as a potential target. Broad anti-enterovirus compounds against 2C and 3A proteins were also identified, including thiazolobenzimidazoles (active against 2C) and TTP-8307 (targeting 3A). There is a need for more potent inhibitors against PV and other picornaviruses, which are potential silent reservoirs for re-emerging PV-like disease.

Unique Structure and Stability of HmuY, a Novel Heme-Binding Protein of Porphyromonas gingivalis

Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-β fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection.

Structural studies on the full-length LysR-type regulator TsaR from Comamonas testosteroni T-2 reveal a novel open conformation of the tetrameric LTTR fold

LysR‐type transcriptional regulators (LTTRs) constitute the largest family of regulators in prokaryotes. The full‐length structures of the LTTR TsaR from Comamonas testosteroni T‐2 and its complex with the natural inducer para‐toluensulfonate have been characterized by X‐ray diffraction. Both ligand‐free and complexed forms reveal a dramatically different quaternary structure from that of CbnR from Ralstonia eutropha, or a putative LysR‐type regulator from Pseudomonas aeruginosa, the only other determined full‐length structures of tetrameric LTTRs. Although all three show a head‐to‐head tetrameric ring, TsaR displays an open conformation, whereas CbnR and PA01‐PR present additional contacts in opposing C‐terminal domains that close the ring. Such large differences may be due to a broader structural versatility than previously assumed or either, reflect the intrinsic flexibility of tetrameric LTTRs. On the grounds of the sliding dimer hypothesis of LTTR activation, we propose a structural model in which the closed structures could reflect the conformation of a ligand‐free LTTR, whereas inducer binding would bring about local changes to disrupt the interface linking the two compact C‐terminal domains. This could lead to a TsaR‐like, open structure, where the pairs of recognition helices are closer to each other by more than 10 Å.