Serial-femtosecond crystallography reveals how a phytochrome variant couples chromophore and protein structural changes.

The photoreaction and commensurate structural changes of a chromophore within biological photoreceptors elicit conformational transitions of the protein promoting the switch between deactivated and activated states. We investigated how this coupling is achieved in a bacterial phytochrome variant, Agp2-PAiRFP2. Contrary to classical protein crystallography, which only allows probing (cryo-trapped) stable states, we have used time-resolved serial femtosecond x-ray crystallography (tr-SFX) and pump-probe techniques with various illumination and delay times with respect to photoexcitation of the parent Pfr state.

CrysFormer: Protein structure determination via Patterson maps, deep learning, and partial structure attention.

Determining the atomic-level structure of a protein has been a decades-long challenge. However, recent advances in transformers and related neural network architectures have enabled researchers to significantly improve solutions to this problem. These methods use large datasets of sequence information and corresponding known protein template structures, if available.

Heterotrimeric collagen helix with high specificity of assembly results in a rapid rate of folding.

The most abundant natural collagens form heterotrimeric triple helices. Synthetic mimics of collagen heterotrimers have been found to fold slowly, even compared to the already slow rates of homotrimeric helices. These prolonged folding rates are not understood.

Crystal structure of the photosensory module from a PAS-less cyanobacterial phytochrome as Pr shows a mix of dark-adapted and photoactivated features.

Phytochromes (Phys) are a diverse collection of photoreceptors that regulate numerous physiological and developmental processes in microorganisms and plants through photointerconversion between red-light-absorbing Pr and far-red light-absorbing Pfr states. Light is detected by an N-terminal photo-sensing module (PSM) sequentially comprised of Period/ARNT/Sim (PAS), cGMP-phosphodiesterase/adenylyl cyclase/FhlA (GAF), and Phy-specific (PHY) domains, with the bilin chromophore covalently-bound within the GAF domain. Phys sense light via the Pr/Pfr ratio measured by the light-induced rotation of the bilin D-pyrrole ring that triggers conformational changes within the PSM, which for microbial Phys reaches into an output region.

Biochemical and Structural Studies of the Carminomycin 4--Methyltransferase DnrK.

Structural and functional studies of the carminomycin 4--methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides.

A deep learning solution for crystallographic structure determination.

The general de novo solution of the crystallographic phase problem is difficult and only possible under certain conditions. This paper develops an initial pathway to a deep learning neural network approach for the phase problem in protein crystallography, based on a synthetic dataset of small fragments derived from a large well curated subset of solved structures in the Protein Data Bank (PDB). In particular, electron-density estimates of simple artificial systems are produced directly from corresponding Patterson maps using a convolutional neural network architecture as a proof of concept.

An NmrA-like enzyme-catalysed redox-mediated Diels-Alder cycloaddition with anti-selectivity.

The Diels-Alder cycloaddition is one of the most powerful approaches in organic synthesis and is often used in the synthesis of important pharmaceuticals. Yet, strictly controlling the stereoselectivity of the Diels-Alder reactions is challenging, and great efforts are needed to construct complex molecules with desired chirality via organocatalysis or transition-metal strategies. Nature has evolved different types of enzymes to exquisitely control cyclization stereochemistry; however, most of the reported Diels-Alderases have been shown to only facilitate the energetically favourable diastereoselective cycloadditions.

Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid.

Genetic code expansion technology allows for the use of noncanonical amino acids (ncAAs) to create semisynthetic organisms for both biochemical and biomedical applications. However, exogenous feeding of chemically synthesized ncAAs at high concentrations is required to compensate for the inefficient cellular uptake and incorporation of these components into proteins, especially in the case of eukaryotic cells and multicellular organisms. To generate organisms capable of autonomously biosynthesizing an ncAA and incorporating it into proteins, we have engineered a metabolic pathway for the synthesis of O-methyltyrosine (OMeY).

The Structure of the PEX4-PEX22 Peroxin Complex-Insights Into Ubiquitination at the Peroxisomal Membrane.

Peroxisomes are eukaryotic organelles that sequester critical oxidative reactions and process the resulting reactive oxygen species into less toxic byproducts. Peroxisome function and formation are coordinated by peroxins (PEX proteins) that guide peroxisome biogenesis and division and shuttle proteins into the lumen and membrane of the organelle. Despite the importance of peroxins in plant metabolism and development, no plant peroxin structures have been reported.

The crystal structure of DynF from the dynemicin-biosynthesis pathway of Micromonospora chersina.

Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin.

Structure and Function of a Dual Reductase-Dehydratase Enzyme System Involved in -Terphenyl Biosynthesis.

We report the identification of the gene cluster responsible for the formation of the -terphenyl derivatives terfestatins B and C and echoside B from the Appalachian strain RM-5-8. We characterize the function of TerB/C, catalysts that work together as a dual enzyme system in the biosynthesis of natural terphenyls. TerB acts as a reductase and TerC as a dehydratase to enable the conversion of polyporic acid to a terphenyl triol intermediate.

Structural characterization of DynU16, a START/Bet v1-like protein involved in dynemicin biosynthesis.

The 1.5 Å resolution crystal structure of DynU16, a protein identified in the dynemicin-biosynthetic gene cluster, is reported. The structure adopts a di-domain helix-grip fold with a uniquely positioned open cavity connecting the domains.

Moving beyond static snapshots: Protein dynamics and the Protein Data Bank.

Proteins are the molecular machines of living systems. Their dynamics are an intrinsic part of their evolutionary selection in carrying out their biological functions. Although the dynamics are more difficult to observe than a static, average structure, we are beginning to observe these dynamics and form sound mechanistic connections between structure, dynamics, and function.

A collagen glucosyltransferase drives lung adenocarcinoma progression in mice.

Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen's amino- and carboxy-terminal telopeptides to create stable collagen cross-links.

Molecular-replacement phasing using predicted protein structures from .

The phase problem in X-ray crystallography arises from the fact that only the intensities, and not the phases, of the diffracting electromagnetic waves are measured directly. Molecular replacement can often estimate the relative phases of reflections starting with those derived from a template structure, which is usually a previously solved structure of a similar protein. The key factor in the success of molecular replacement is finding a good template structure.

The crystal structure of AbsH3: A putative flavin adenine dinucleotide-dependent reductase in the abyssomicin biosynthesis pathway.

Natural products and natural product-derived compounds have been widely used for pharmaceuticals for many years, and the search for new natural products that may have interesting activity is ongoing. Abyssomicins are natural product molecules that have antibiotic activity via inhibition of the folate synthesis pathway in microbiota. These compounds also appear to undergo a required [4 + 2] cycloaddition in their biosynthetic pathway.

Covalent Capture of Collagen Triple Helices Using Lysine-Aspartate and Lysine-Glutamate Pairs.

Collagen mimetic peptides (CMPs) self-assemble into a triple helix reproducing the most fundamental aspect of the collagen structural hierarchy. They are therefore important for both further understanding this complex family of proteins and use in a wide range of biomaterials and biomedical applications. CMP self-assembly is complicated by a number of factors which limit the use of CMPs including their slow rate of folding, relatively poor monomer-trimer equilibrium, and the large number of competing species possible in heterotrimeric helices.

Methionine Adenosyltransferase Engineering to Enable Bioorthogonal Platforms for AdoMet-Utilizing Enzymes.

The structural conservation among methyltransferases (MTs) and MT functional redundancy is a major challenge to the cellular study of individual MTs. As a first step toward the development of an alternative biorthogonal platform for MTs and other AdoMet-utilizing enzymes, we describe the evaluation of 38 human methionine adenosyltransferase II-α (hMAT2A) mutants in combination with 14 non-native methionine analogues to identify suitable bioorthogonal mutant/analogue pairings. Enabled by the development and implementation of a hMAT2A high-throughput (HT) assay, this study revealed hMAT2A K289L to afford a 160-fold inversion of the hMAT2A selectivity index for a non-native methionine analogue over the native substrate l-Met.

Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain.

Biochemical and Structural Characterization of TtnD, a Prenylated FMN-Dependent Decarboxylase from the Tautomycetin Biosynthetic Pathway.

Tautomycetin (TTN) is a polyketide natural product featuring a terminal alkene. Functional characterization of the genes within the ttn gene cluster from Streptomyces griseochromogenes established the biosynthesis of the TTN polyketide backbone, its dialkylmaleic anhydride moiety, the coupling of the two moieties to form the nascent intermediate TTN F-1, and the tailoring steps converting TTN F-1 to TTN. Here, we report biochemical and structural characterization of TtnD, a prenylated FMN (prFMN)-dependent decarboxylase belonging to the UbiD family that catalyzes the penultimate step of TTN biosynthesis.

Author Correction: Pro-metastatic collagen lysyl hydroxylase dimer assemblies stabilized by Fe-binding.

In the originally published version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to also include support from the National Institutes of Health grant T32GM008280 to Sarah Alvarado.

Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography.

Background: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases.

Results: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC).