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Despite their popularity as enzyme engineering targets structural information about Sucrose Phosphorylases remains scarce. We recently clarified that the Q345F variant of Bifidobacterium adolescentis Sucrose Phosphorylase is able to accept large polyphenolic substrates like resveratrol via a domain shift. Here we present a crystal structure of this variant in a conformation suitable for the accommodation of the donor substrate sucrose in excellent agreement with the wild type structure. Remarkably, this conformation does not feature the previously observed domain shift which is therefore reversible and part of a dynamic process rather than a static phenomenon. This crystallographic snapshot completes our understanding of the catalytic cycle of this useful variant and will allow for a more rational design of further generations of Sucrose Phosphorylase variants.
Meiotic chromosomes undergo rapid prophase movements, which are thought to facilitate the formation of inter-homologue recombination intermediates that underlie synapsis, crossing over and segregation. The meiotic telomere complex (MAJIN, TERB1, TERB2) tethers telomere ends to the nuclear envelope and transmits cytoskeletal forces via the LINC complex to drive these rapid movements. Here, we report the molecular architecture of the meiotic telomere complex through the crystal structure of MAJIN-TERB2, together with light and X-ray scattering studies of wider complexes. The MAJIN-TERB2 2:2 hetero-tetramer binds strongly to DNA and is tethered through long flexible linkers to the inner nuclear membrane and two TRF1-binding 1:1 TERB2-TERB1 complexes. Our complementary structured illumination microscopy studies and biochemical findings reveal a telomere attachment mechanism in which MAJIN-TERB2-TERB1 recruits telomere-bound TRF1, which is then displaced during pachytene, allowing MAJIN-TERB2-TERB1 to bind telomeric DNA and form a mature attachment plate.
The covalent modification of target proteins with ubiquitin or ubiquitin-like modifiers is initiated by E1 activating enzymes, which typically transfer a single modifier onto cognate conjugating enzymes. UBA6 is an unusual E1 since it activates two highly distinct modifiers, ubiquitin and FAT10. Here, we report crystal structures of UBA6 in complex with either ATP or FAT10. In the UBA6-FAT10 complex, the C-terminal domain of FAT10 binds to where ubiquitin resides in the UBA1-ubiquitin complex, however, a switch element ensures the alternate recruitment of either modifier. Simultaneously, the N-terminal domain of FAT10 interacts with the 3-helix bundle of UBA6. Site-directed mutagenesis identifies residues permitting the selective activation of either ubiquitin or FAT10. These results pave the way for studies investigating the activation of either modifier by UBA6 in physiological and pathophysiological settings.
Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain
(2018)
The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsinguanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio > 1000. After light excitation the putative signaling state forms with tau = 31 ms and decays with tau = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 angstrom) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.
The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.
RNA-catalysed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyses the site-specific synthesis of 1-methyladenosine (m\(^1\)A) in RNA, using O\(^6\)-methylguanine (m\(^6\)G) as methyl group donor. Here we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution.
Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO+ and DMHBI+ ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.
Cesium based phasing of macromolecules: a general easy to use approach for solving the phase problem
(2021)
Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a "quick-soak"-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy.
The reduction of a cyclic alkyl(amino)carbene (CAAC)-stabilized organoberyllium chloride yields the first neutral beryllium radical, which was characterized by EPR, IR, UV/Vis spectroscopy and X-ray crystallography. DFT calculations show significant spin density at beryllium and confirm donor–acceptor bonding between an alkylberyllium radical fragment and a neutral CAAC ligand.
The biogenesis of spliceosomal UsnRNPs is a highly elaborate cellular process that occurs both in the nucleus and the cytoplasm. A major part of the process is the assembly of the Sm-core particle, which consists of a ring shaped heptameric unit of seven Sm proteins (SmD1•D2•F•E•G•D3•B) wrapped around a single stranded RNA motif (termed Sm-site) of spliceosomal UsnRNAs. This process occurs mainly in the cytoplasm by the sequential action of two biogenesis factors united in PRMT5- and SMN-complexes, respectively. The PRMT5-complex composed of the three proteins PRMT5, WD45 and pICln is responsible for the symmetric dimethylation of designated arginine residues in the C-terminal tails of some Sm proteins. The action of the PRMT5- complex results in the formation of assembly incompetent Sm-protein intermediates sequestered by the assembly chaperone pICln (SmD1•D2•F•E•G•pICln and pICln•D3•B). Due to the action of pICln, the Sm proteins in these complexes fail to interact with UsnRNAs to form the mature Sm-core. This kinetic trap is relieved by the action of the SMN-complex, which removes the pICln subunit and facilitates the binding of the Sm-core intermediates to the UsnRNA, thus forming the mature Sm-core particle. The human SMN complex consists of 9 subunits termed SMN, Gemin2-8 and Unrip. So far, there are no available atomic structures of the whole SMN-complex, but structures of isolated domains and subunits of the complex have been reported by several laboratories in the past years. The lack of structural information about the entire SMN complex most likely lies in the biophysical properties of the SMN complex, which possesses an oligomeric SMN core, and many unstructured and flexible regions. These were the biggest roadblocks for its structural elucidation using traditional methods such as X-ray crystallography, NMR or CryoEM. To circumvent these obstacles and to obtain structural insight into the SMN-complex, the Schizosaccharomyces pombe SMN complex was used as a model system in this work. In a collaboration with the laboratory of Dr. Remy Bordonne (IGMM, CNRS, France), we could show that the SpSMN complex is minimalistic in its composition, consisting only of SpSMN, SpGemin2, SpGemin8, SpGemin7 and SpGemin6. Using biochemical experiments, an interaction map of the SpSMN complex was established which was found to be highly similar to the reported map of the human SMN complex. The results of this study clearly show that SpSMN is the oligomeric core of the complex and provides the binding sites for the rest of the subunits. Through biochemical and X-ray scattering experiments, the properties of the SpSMN subunit such as oligomerization viii and intrinsic disorder, were shown to determine the overall biophysical characteristics of the whole complex. The structural basis of SpSMN oligomerization is presented in atomic detail which establishes a dimeric SpSMN as the fundamental unit of higher order SpSMN oligomers. In addition to oligomerization, the YG-box domain of SpSMN serves as the binding site for SpGemin8. The unstructured region of SpSMN imparts an unusual large hydrodynamic size, intrinsic disorder, and flexibility to the whole complex. Interestingly, these biophysical properties are partially mitigated by the presence of SpGemin8•SpGemin7•SpGemin6 subunits. These results classify the SpSMN complex as a multidomain entity connected with flexible linkers and characterize the SpSMN subunit to be the central oligomeric structural organizer of the whole complex.