Uwe H. Sauer

Structural Basis for Thermophilic Protein Stability: Structures of Thermophilic and Mesophilic Malate Dehydrogenases

The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions.

Redox-mediated mechanisms regulate DNA binding activity of the G-group of basic region leucine zipper (bZIP) transcription factors in Arabidopsis.

Background: The G-box cis-element is enriched in promoters of genes responding to light and to high light. Results: DTT induces DNA binding activity of bZIP transcription factors by reducing a disulfide bond. Conclusion: Redox regulation is crucial for DNA binding of the G-group of Arabidopsis bZIP transcription factors. Significance: Redox-dependent mechanisms modulate the activity of plant bZIPs in response to environmental signals. Plant genes that contain the G-box in their promoters are responsive to a variety of environmental stimuli. Bioinformatics analysis of transcriptome data revealed that the G-box element is significantly enriched in promoters of high light-responsive genes. From nuclear extracts of high light-treated Arabidopsis plants, we identified the AtbZIP16 transcription factor as a component binding to the G-box-containing promoter fragment of light-harvesting chlorophyll a/b-binding protein2.4 (LHCB2.4). AtbZIP16 belongs to the G-group of Arabidopsis basic region leucine zipper (bZIP) type transcription factors. Although AtbZIP16 and its close homologues AtbZIP68 and AtGBF1 bind the G-box, they do not bind the mutated half-sites of the G-box palindrome. In addition, AtbZIP16 interacts with AtbZIP68 and AtGBF1 in the yeast two-hybrid system. A conserved Cys residue was shown to be necessary for redox regulation and enhancement of DNA binding activity in all three proteins. Furthermore, transgenic Arabidopsis lines overexpressing the wild type version of bZIP16 and T-DNA insertion mutants for bZIP68 and GBF1 demonstrated impaired regulation of LHCB2.4 expression. Finally, overexpression lines for the mutated Cys variant of bZIP16 provided support for the biological significance of Cys330 in redox regulation of gene expression. Thus, our results suggest that environmentally induced changes in the redox state regulate the activity of members of the G-group of bZIP transcription factors.

The Runx1 Runt Domain at 1.25 A Resolution: A Structural Switch and Specifically Bound Chloride Ions Modulate DNA Binding

The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

OnD-CRF: predicting order and disorder in proteins conditional random fields

Motivation: Order and Disorder prediction using Conditional Random Fields, OnD-CRF, is a new method for accurately predicting the transition between structured and mobile or disordered regions in proteins. OnD-CRF applies CRFs relying on features which are generated from the amino acids sequence and from secondary structure prediction. Benchmarking results based on CASP7 targets, and evaluation with respect to several CASP criteria, rank the OnDCRF model highest among the fully automatic server group. Availability: http://babel.ucmp.umu.se/ond-crf/

Three-dimensional structure of the bifunctional protein PCD/DCoH, a cytoplasmic enzyme interacting with transcription factor HNF1.

The bifunctional protein pterin‐4a‐carbinolamine dehydratase (PCD)/dimerization cofactor of HNF1 (DCoH) is a cytoplasmic enzyme involved in the tetrahydrobiopterin regeneration and is found in complex with the transcription factor HNF1 in liver cell nuclei. An atypical hyperphenylalaninemia and the depigmentation disorder vitiligo are related to a deficiency of PCD/DCoH activity. The crystal structure of PCD/DCoH was solved by multiple isomorphous replacement and refined to a crystallographic R‐factor of 20.5% at 2.7 A resolution. The single domain monomer comprises three alpha‐helices packed against one side of a four‐stranded, antiparallel beta‐sheet. The functional enzyme is a homo‐tetramer of 222 symmetry where each of the monomers contributes one helix to a central four helix bundle. In the tetramer two monomers form an eight‐stranded, antiparallel beta‐sheet with six helices packing against it from one side. The concave, hydrophobic surface of the eight‐stranded beta‐sheet with its two protruding loops at either end is reminiscent of the saddle‐like shape seen in the TATA‐box binding protein. PCD/DCoH binds as a dimer to the helical dimerization domain of dimeric HNF1 forming a hetero‐tetramer possibly through a mixed four helix bundle.

Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria.

Significance Infection by the human bacterial pathogen Listeria monocytogenes is controlled mainly by the transcriptional activator PrfA, a member of the Crp/Fnr family. Here we report the crystal structures of PrfA in complex with glutathione (GSH) and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. The structures provide detailed information and insight into how GSH interacts with PrfA and thus induces the correct fold of the HTH motif promoting PrfA DNA binding. Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.

FISH—family identification of sequence homologues using structure anchored hidden Markov models

The FISH server is highly accurate in identifying the family membership of domains in a query protein sequence, even in the case of very low sequence identities to known homologues. A performance test using SCOP sequences and an E-value cut-off of 0.1 showed that 99.3% of the top hits are to the correct family saHMM. Matches to a query sequence provide the user not only with an annotation of the identified domains and hence a hint to their function, but also with probable 2D and 3D structures, as well as with pairwise and multiple sequence alignments to homologues with low sequence identity. In addition, the FISH server allows users to upload and search their own protein sequence collection or to quarry public protein sequence data bases with individual saHMMs. The FISH server can be accessed at .

Chloride binding by the AML1/Runx1 transcription factor studied by NMR

It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl− ions. In this paper we have determined Cl− binding affinities of AML1 by 35Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl− with a dissociation constant (K d,Cl) of 34 mM. If CBFβ is added to form a 1:1 complex, the K d,Cl value increases to 56 mM. Homology modeling suggests that a high occupancy Cl− binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl− ion. Possible biological roles of Cl− binding are discussed based on these findings.

Attenuating Listeria monocytogenes Virulence by Targeting the Regulatory Protein PrfA

Summary The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes cellular uptake by reducing the expression of virulence genes. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA-binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds at two separate sites on the protein: one within a hydrophobic pocket or tunnel, located between the C- and N-terminal domains of PrfA, and the second in the vicinity of the DNA-binding helix-turn-helix motif. At both sites the compound interacts with residues important for PrfA activation and helix-turn-helix formation. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.

Purification, crystallization and preliminary X-ray analysis of PPD6, a PsbP-domain protein from Arabidopsis thaliana.

The PsbP protein is an extrinsic component of photosystem II that together with PsbO and PsbQ forms the thylakoid lumenal part of the oxygen-evolving complex in higher plants. In addition to PsbP, the thylakoid lumen contains two PsbP-like proteins (PPLs) and six PsbP-domain proteins (PPDs). While the functions of the PsbP-like proteins PPL1 and PPL2 are currently under investigation, the function of the PsbP-domain proteins still remains completely unknown. PPD6 is unique among the PsbP family of proteins in that it contains a conserved disulfide bond which can be reduced in vitro by thioredoxin. The crystal structure determination of the PPD6 protein has been initiated in order to elucidate its function and to gain deeper insights into redox-regulation pathways in the thylakoid lumen. PPD6 has been expressed, purified and crystallized and preliminary X-ray diffraction data have been collected. The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 64.3, c = 62.0 Å, β = 94.2°, and diffracted to a maximum d-spacing of 2.1 Å.