Clemens Grimm

The crystal structure of 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase/reductase family.

The crystal structure of 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3α-HSDH) as well as the structure of its binary complex with NAD+ have been solved at 1.68-Å and 1.95-Å resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix αG and strand βG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3α-HSDH by the presence of a predominantly α-helical subdomain which is missing in all other SDRs of known structure.

Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins

The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA‐binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven‐helix fold that binds DNA as a cooperative dimer, forming base‐specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFYs effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix‐turn‐helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.

Molecular recognition of histone lysine methylation by the Polycomb group repressor dSfmbt.

Polycomb group (PcG) proteins repress transcription by modifying chromatin structure in target genes. dSfmbt is a subunit of the Drosophila melanogaster PcG protein complex PhoRC and contains four malignant brain tumour (MBT) repeats involved in the recognition of various mono‐ and dimethylated histone peptides. Here, we present the crystal structure of the four‐MBT‐repeat domain of dSfmbt in complex with a mono‐methylated histone H4 peptide. Only a single histone peptide binds to the four‐MBT‐repeat domain. Mutational analyses show high‐affinity binding with low peptide sequence selectivity through combinatorial interaction of the methyl‐lysine with an aromatic cage and positively charged flanking residues with the surrounding negatively charged surface of the fourth MBT repeat. dSfmbt directly interacts with the PcG protein Scm, a related MBT‐repeat protein with similar methyl‐lysine binding activity. dSfmbt and Scm co‐occupy Polycomb response elements of target genes in Drosophila and they strongly synergize in the repression of these target genes, suggesting that the combined action of these two MBT proteins is crucial for Polycomb silencing.

Structural and functional analyses of methyl‐lysine binding by the malignant brain tumour repeat protein Sex comb on midleg

Sex comb on midleg (Scm) is a member of the Polycomb group of proteins involved in the maintenance of repression of Hox and other developmental control genes in Drosophila. The two malignant brain tumour (MBT) repeats of Scm form a domain that preferentially binds to monomethylated lysine residues either as a free amino acid or in the context of peptides, while unmodified or di‐ or trimethylated lysine residues are bound with significantly lower affinity. The crystal structure of a monomethyl‐lysine‐containing histone tail peptide bound to the MBT repeat domain shows that the methyl‐lysine side chain occupies a binding pocket in the second MBT repeat formed by three conserved aromatic residues and one aspartate. Insertion of the monomethylated side chain into this pocket seems to be the main contributor to the binding affinity. Functional analyses in Drosophila show that the MBT domain of Scm and its methyl‐lysine‐binding activity are required for repression of Hox genes.

Structural Basis of Assembly Chaperone- Mediated snRNP Formation

Small nuclear ribonucleoproteins (snRNPs) represent key constituents of major and minor spliceosomes. snRNPs contain a common core, composed of seven Sm proteins bound to snRNA, which forms in a step-wise and factor-mediated reaction. The assembly chaperone pICln initially mediates the formation of an otherwise unstable pentameric Sm protein unit. This so-called 6S complex docks subsequently onto the SMN complex, which removes pICln and enables the transfer of pre-assembled Sm proteins onto snRNA. X-ray crystallography and electron microscopy was used to investigate the structural basis of snRNP assembly. The 6S complex structure identifies pICln as an Sm protein mimic, which enables the topological organization of the Sm pentamer in a closed ring. A second structure of 6S bound to the SMN complex components SMN and Gemin2 uncovers a plausible mechanism of pICln elimination and Sm protein activation for snRNA binding. Our studies reveal how assembly factors facilitate formation of RNA-protein complexes in vivo.

Structural Basis of Tbx5-DNA Recognition: The T-Box Domain in its DNA-Bound and -Unbound Form.

TBX5, a member of the T-box transcription factor family, plays an important role in heart and limb development. More than 60 single point or deletion mutations of human TBX5 are associated with Holt-Oram syndrome that manifests itself as heart and limb malformations in 1 out of 100,000 live births. The majority of these mutations are located in the TBX5 T-box domain. We solved the crystal structures of the human TBX5 T-box domain in its DNA-unbound form and in complex with a natural DNA target site allowing for the first time the comparison between unbound and DNA-bound forms. Our analysis identifies a 3(10)-helix at the C-terminus of the T-box domain as an inducible recognition element, critically required for the interaction with DNA, as it only forms upon DNA binding and is unstructured in the DNA-unbound form. Using circular dichroism, we characterized the thermal stability of six TBX5 mutants containing single point mutations in the T-box domain (M74V, G80R, W121G, G169R, T223M, and R237W) and compared them with wild-type protein. Mutants G80R and W121G show drastically reduced thermal stability, while the other mutants only show a marginal stability decrease. For all TBX5 mutants, binding affinities to specific and nonspecific DNA sequences were determined using isothermal titration calorimetry. All TBX5 mutants show reduced binding affinities to a specific DNA target site, although to various degrees. Interestingly, all tested TBX5 mutants differ in their ability to bind unspecific DNA, indicating that both sequence-specific and unspecific binding might contribute to the misregulation of target gene expression.

Crystal structures and enzymatic properties of three formyltransferases from archaea: Environmental adaptation and evolutionary relationship

Formyltransferase catalyzes the reversible formation of formylmethanofuran from N5‐formyltetrahydromethanopterin and methanofuran, a reaction involved in the C1 metabolism of methanogenic and sulfate‐reducing archaea. The crystal structure of the homotetrameric enzyme from Methanopyrus kandleri (growth temperature optimum 98°C) has recently been solved at 1.65 Å resolution. We report here the crystal structures of the formyltransferase from Methanosarcina barkeri (growth temperature optimum 37°C) and from Archaeoglobus fulgidus (growth temperature optimum 83°C) at 1.9 Å and 2.0 Å resolution, respectively. Comparison of the structures of the three enzymes revealed very similar folds. The most striking difference found was the negative surface charge, which was −32 for the M. kandleri enzyme, only −8 for the M. barkeri enzyme, and −11 for the A. fulgidus enzyme. The hydrophobic surface fraction was 50% for the M. kandleri enzyme, 56% for the M. barkeri enzyme, and 57% for the A. fulgidus enzyme. These differences most likely reflect the adaptation of the enzyme to different cytoplasmic concentrations of potassium cyclic 2,3‐diphosphoglycerate, which are very high in M. kandleri (>1 M) and relatively low in M. barkeri and A. fulgidus. Formyltransferase is in a monomer/dimer/tetramer equilibrium that is dependent on the salt concentration. Only the dimers and tetramers are active, and only the tetramers are thermostable. The enzyme from M. kandleri is a tetramer, which is active and thermostable only at high concentrations of potassium phosphate (>1 M) or potassium cyclic 2,3‐diphosphoglycerate. Conversely, the enzyme from M. barkeri and A. fulgidus already showed these properties, activity and stability, at much lower concentrations of these strong salting‐out salts.

ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space

Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules with a variety of functions. Structure determination of these complexes in a purified state is often tedious owing to their compositional complexity and the associated relative structural instability. To improve the stability of macromolecular complexes in vitro, we present a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behavior in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a biophysical unfolding model for complexes. We found that under stabilizing conditions, even large multicomponent complexes reveal an almost ideal two-state unfolding behavior. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and cryo-electron microscopy.

Mutations in SNRPE, which Encodes a Core Protein of the Spliceosome, Cause Autosomal-Dominant Hypotrichosis Simplex

Hypotrichosis simplex (HS) comprises a group of hereditary isolated alopecias that are characterized by a diffuse and progressive loss of hair starting in childhood and shows a wide phenotypic variability. We mapped an autosomal-dominant form of HS to chromosome 1q31.3-1q41 in a Spanish family. By direct sequencing, we identified the heterozygous mutation c.1A>G (p.Met1?) in SNRPE that results in loss of the start codon of the transcript. We identified the same mutation in a simplex HS case from the UK and an additional mutation (c.133G>A [p.Gly45Ser]) in a simplex HS case originating from Tunisia. SNRPE encodes a core protein of U snRNPs, the key factors of the pre-mRNA processing spliceosome. The missense mutation c.133G>A leads to a glycine to serine substitution and is predicted to disrupt the structure of SNRPE. Western blot analyses of HEK293T cells expressing SNRPE c.1A>G revealed an N-terminally truncated protein, and therefore the mutation might result in use of an alternative in-frame downstream start codon. Subcellular localization of mutant SNRPE by immunofluorescence analyses as well as incorporation of mutant SNRPE proteins into U snRNPs was found to be normal, suggesting that the function of U snRNPs in splicing, rather than their biogenesis, is affected. In this report we link a core component of the spliceosome to hair loss, thus adding another specific factor in the complexity of hair growth. Furthermore, our findings extend the range of human phenotypes that are linked to the splicing machinery.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: biological significance, three-dimensional structure and gene regulation

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) catalyses the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralisation of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. The enzyme was found to be functional towards a variety of steroid substrates, including the steroid antibiotic fusidic acid. The enzyme also catalyses the carbonyl reduction of non-steroidal aldehydes and ketones such as a novel insecticide. It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. The 3alpha-HSD/CR gene (hsdA) is 774 base pairs long and the deduced amino acid sequence comprises 258 residues with a calculated molecular mass of 26.4 kDa. A homology search revealed 3alpha-HSD/CR as a new member of the short-chain dehydrogenase/reductase (SDR) superfamily. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein indicating a dimeric nature of 3alpha-HSD/CR. The protein was crystallised and the structure solved by X-ray analysis. The crystal structure reveals one homodimer per asymmetric unit, thereby verifying its dimeric nature. Dimerisation takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far, this type of intermolecular contact has exclusively been observed in homotetrameric SDRs, but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSD/CR by the presence of a predominantly alpha-helical subdomain, which is missing in all other SDRs of known structure. The promoter domain was localised within the 93 bp region upstream of hsdA and the transcriptional start site was identified at 28 bp upstream of the translation start site. Interestingly, hsdA expression was found to be under negative control by two repressor proteins, the genes of which were found in opposite direction downstream or overlapping with hsdA. Based on our results, we propose that induction of hsdA expression in C. testosteroni by steroids actually appears to be a de-repression by preventing the binding of repressor proteins to regulatory regions.