Biomedical subjects
Publications and source records attributed to Matthias Wilmanns.
Revealing the molecular principles of eukaryotic transcription factor assembly on specific DNA sites is pivotal to understanding how genes are differentially expressed. By analyzing structures of transcription factor complexes bound to specific DNA elements we demonstrate how protein and DNA regulators manage gene expression in a combinatorial fashion.
Members of the POU and SOX transcription factor families exemplify the partnerships established between various transcriptional regulators during early embryonic development. Although functional cooperativity between key regulator proteins is pivotal for milestone decisions in mammalian development, little is known about the underlying molecular mechanisms. In this study, we focus on two transcription factors, Oct4 and Sox2, as their combination on DNA is considered to direct the establishment of the first three lineages in the mammalian embryo. Using experimental high-resolution structure determination, followed by model building and experimental validation, we found that Oct4 and Sox2 were able to dimerize onto DNA in distinct conformational arrangements. We demonstrate that the DNA enhancer region of their target genes is responsible for the correct spatial alignment of glue-like interaction domains on their surface. Interestingly, these surfaces frequently have redundant functions and are instrumental in recruiting various interacting protein partners.
The POU transcription factors Oct1 and Oct2 bind to DNA in various monomer and dimer configurations. Depending on the DNA sequence to which they bind, the dimers are arranged in configurations that are either accessible (PORE sequence) or inaccessible (MORE sequence) to the B-cell-specific cofactor OBF1 (OcaB, Bob1). As shown previously, the MORE and related sequences (such as the heptamer/octamer motif) are found in immunoglobulin heavy chain promoters. Here we show that the expression of Osteopontin, which contains a PORE sequence in its enhancer region, depends on the presence of OBF1 in B cells. OBF1 alleviates DNA sequence requirements of the Oct1 dimer on PORE-related sequences in vitro. Furthermore, OBF1 stabilizes POU dimer-DNA interactions and overrides Oct1 interface mutations, which abolish PORE-mediated dimerization without OBF1. Our data indicate that the PORE-type Oct1 or Oct2 dimer, rather than the monomer, is the primary target of the cofactor OBF1. Based on our biochemical data, we propose a mode of OBF1-Oct1 dimer interaction, suggesting a novel arrangement of the subdomain connectivities.
Enzymes with the (beta/alpha)(8)-barrel fold are involved in the catalysis of a wide variety of biochemical reactions. The active sites of these enzymes are located on the C-terminal face of the central beta-barrel. Conserved amino acid sequence, as well as secondary, tertiary and quaternary structure patterns are providing a rich body of data to support the premise of a common ancestry of many members of the (beta/alpha)(8)-barrel fold family of enzymes. Recent data indicate that there is at least one example of a bienzyme that functions as an ammonia channel, adding a new level of functional diversity to the (beta/alpha)(8)-barrel fold. These proteins have become ideal tools that can be used in conjunction with directed evolution techniques to engineer novel catalytic activities.
The crystal structure of the alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei was solved in the presence of three inhibitors: acarbose, Tris, and zinc. In the absence of exogenous metals, this alpha-amylase bound 1 and 4 molar eq of zinc and calcium, respectively. The structure reveals a novel, activating, two-metal (Ca,Zn)-binding site and a second inhibitory zinc-binding site that is found in the -1 sugar-binding pocket within the active site. The data resolve the apparent paradox between the zinc requirement for catalytic activity and its strong inhibitory effect when added in molar excess. They provide a rationale as to why this alpha-amylase, in contrast to commercially available alpha-amylases, does not require the addition of metal ions for full catalytic activity, suggesting it as an ideal target to maximize the efficiency of industrial processes like liquefaction of starch.
Telethonin interacts specifically with the two Z-disk IG-like domains (Z1Z2) at the N terminus of titin, the largest presently known protein. Analytical ultracentrifugation and synchrotron radiation x-ray scattering were employed to study the solution structures of Z1Z2 and its complexes with telethonin, and low resolution models were constructed ab initio from the scattering data. A seven residues-long polyhistidine tag was localized at the tip of the Z1 domain by comparison of independent models of native and His-tagged versions of Z1Z2. The stoichiometry and shape of the complex between the telethonin construct lacking the C terminus and Z1Z2 indicate antiparallel association of two Z1Z2 molecules with telethonin acting as a central linker. The complex of full-length telethonin with Z1Z2 appears to also have a 1:2 stoichiometry at concentrations below 1 mg/ml but dimerizes at higher concentrations. These results suggest a possible role of telethonin in linking titin filaments at the Z-disk periphery.
Twinfilin is an evolutionarily conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. It is composed of two actin-depolymerization factor homology (ADF-H) domains that show approximately 20% sequence identity to ADF/cofilin proteins. In contrast to ADF/cofilins, which bind both G-actin and F-actin and promote filament depolymerization, twinfilin interacts only with G-actin. To elucidate the molecular mechanisms of twinfilin-actin monomer interaction, we determined the crystal structure of the N-terminal ADF-H domain of twinfilin and mapped its actin-binding site by site-directed mutagenesis. This domain has similar overall structure to ADF/cofilins, and the regions important for actin monomer binding in ADF/cofilins are especially well conserved in twinfilin. Mutagenesis studies show that the N-terminal ADF-H domain of twinfilin and ADF/cofilins also interact with actin monomers through similar interfaces, although the binding surface is slightly extended in twinfilin. In contrast, the regions important for actin-filament interactions in ADF/cofilins are structurally different in twinfilin. This explains the differences in actin-interactions (monomer versus filament binding) between twinfilin and ADF/cofilins. Taken together, our data show that the ADF-H domain is a structurally conserved actin-binding motif and that relatively small structural differences at the actin interfaces of this domain are responsible for the functional variation between the different classes of ADF-H domain proteins.
The crystal structure of the dimeric anthranilate phosphoribosyltransferase (AnPRT) reveals a new category of phosphoribosyltransferases, designated as class III. The active site of this enzyme is located within the flexible hinge region of its two-domain structure. The pyrophosphate moiety of phosphoribosylpyrophosphate is co-ordinated by a metal ion and is bound by two conserved loop regions within this hinge region. With the structure of AnPRT available, structural analysis of all enzymatic activities of the tryptophan biosynthesis pathway is complete, thereby connecting the evolution of its enzyme members to the general development of metabolic processes. Its structure reveals it to have the same fold, topology, active site location and type of association as class II nucleoside phosphorylases. At the level of sequences, this relationship is mirrored by 13 structurally invariant residues common to both enzyme families. Taken together, these data imply common ancestry of enzymes catalysing reverse biological processes--the ribosylation and deribosylation of metabolic pathway intermediates. These relationships establish new links for enzymes involved in nucleotide and amino acid metabolism.
The NAD(+)-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from the hyperthermophilic archaeum Thermoproteus tenax represents an archaeal member of the diverse superfamily of aldehyde dehydrogenases (ALDHs). GAPN catalyzes the irreversible oxidation of d-glyceraldehyde 3-phosphate to 3-phosphoglycerate. In this study, we present the crystal structure of GAPN in complex with its natural inhibitor NADP(+) determined by multiple anomalous diffraction methods. The structure was refined to a resolution of 2.4 A with an R-factor of 0.21. The overall fold of GAPN is similar to the structures of ALDHs described previously, consisting of three domains: a nucleotide-binding domain, a catalytic domain, and an oligomerization domain. Local differences in the active site are responsible for substrate specificity. The inhibitor NADP(+) binds at an equivalent site to the cosubstrate-binding site of other ALDHs and blocks the enzyme in its inactive state, possibly preventing the transition to the active conformation. Structural comparison between GAPN from the hyperthermophilic T. tenax and homologs of mesophilic organisms establishes several characteristics of thermostabilization. These include protection against heat-induced covalent modifications by reducing and stabilizing labile residues, a decrease in number and volume of empty cavities, an increase in beta-strand content, and a strengthening of subunit contacts by ionic and hydrophobic interactions.
Recent structural studies on transcription factors from the POU family in complex with multiple cognate DNA enhancer elements have established a novel concept in DNA-mediated formation of distinct conformations of transcription regulator assemblies. Two crystal structures of the Oct-1 transcription factor in the presence of two different DNA sites have demonstrated how its POU DNA-binding segment is capable in forming two unrelated dimer arrangements, which is DNA motif dependent. While one arrangement allows binding of the Oct-1 specific coactivator OBF-1, binding of this coactivator is blocked in the second arrangement because the binding site is involved in its own dimer assembly. Conversely, two crystal structures of another POU transcription factor, Pit-1, have demonstrated how the same overall assembly is maintained in the presence of two different DNA response elements. However, since the distance of the two Pit-1 half-binding sites on these elements differ by two base pairs, the overall dimensions of the two complexes vary, allowing binding of a specific represssor (N-CoR) in one conformation but not in the other. Thus, despite the occurrence of different DNA-mediated molecular mechanisms, the net result, conformation-dependent binding of further regulators, is equivalent. These data introduce a concept where the DNA motif not only serves as binding site for specific transcription factors but also regulates their function by mediating specific transcription factor assemblies, which determine binding to conformation-dependent coregulators.
The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging beta-strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B beta-strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A'- and G-strands. The disulfide bond also limits the maximum extension of I1 to approximately 220 A. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.
Since reactive ammonia is not available under physiological conditions, glutamine is used as a source for the incorporation of nitrogen in a number of metabolic pathway intermediates. The heterodimeric ImGP synthase that links histidine and purine biosynthesis belongs to the family of glutamine amidotransferases in which the glutaminase activity is coupled with a subsequent synthase activity specific for each member of the enzyme family. Its X-ray structure from the hyperthermophile Thermotoga maritima shows that the glutaminase subunit is associated with the N-terminal face of the (beta alpha)(8) barrel cyclase subunit. The complex reveals a putative tunnel for the transfer of ammonia over a distance of 25 A. Although ammonia tunneling has been reported for glutamine amidotransferases, the ImGP synthase has evolved a novel mechanism, which extends the known functional properties of the versatile (beta alpha)(8) barrel fold.
While the function of most small signaling domains is confined to binary ligand interactions, the peroxisomal Pex13p SH3 domain has the unique capacity of binding to two different ligands, Pex5p and Pex14p. We have used this domain as a model to decipher its structurally independent ligand binding sites. By the combined use of X-ray crystallography, NMR spectroscopy, and circular dichroism, we show that the two ligands bind in unrelated conformations to patches located at opposite surfaces of this SH3 domain. Mutations in the Pex13p SH3 domain that abolish interactions within the Pex13p-Pex5p interface specifically impair PTS1-dependent protein import into yeast peroxisomes.
The aim of this study was to increase the stability of the thermolabile (betaalpha)8-barrel enzyme indoleglycerol phosphate synthase from Escherichia coli by the introduction of disulfide bridges. For the design of such variants, we selected two out of 12 candidates, in which newly introduced cysteines potentially form optimal disulfide bonds. These variants avoid short-range connections, substitutions near catalytic residues, and crosslinks between the new and the three parental cysteines. The variant linking residues 3 and 189 fastens the N-terminus to the (betaalpha)8-barrel. The rate of thermal inactivation at 50 degrees C of this variant with a closed disulfide bridge is 65-fold slower than that of the reference dithiol form, but only 13-fold slower than that of the parental protein. The near-ultraviolet CD spectrum, the reactivity of parental buried cysteines with Ellman's reagent as well as the decreased turnover number indicate that the protein structure is rigidified. To confirm these data, we have solved the X-ray structure to 2.1-A resolution. The second variant was designed to crosslink the terminal modules betaalpha1 and betaalpha8. However, not even the dithiol form acquired the native fold, possibly because one of the targeted residues is solvent-inaccessible in the parental protein.
Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the Abp1-SH3 domain. None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction projects. Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for their localization to actin cortical patches. The Abp1-SH3 domain has a rather unusual binding specificity, because its target peptides contain the tetrapentapeptide +XXXPXXPX+PXXL with positive charges flanking the polyproline core on both sides. Here we present the structure of the Abp1-SH3 domain solved at 1.3-A resolution. The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon at those positions in the SH3 domain family. We have shown by site-directed mutagenesis that one of these negatively charged side chains may be the key determinant for the preference for non-classical ligands.