Discovery of a non-canonical GRHL1 binding site using deep convolutional and recurrent neural networks.

BACKGROUND: Transcription factors regulate gene expression by binding to transcription factor binding sites (TFBSs). Most models for predicting TFBSs are based on position weight matrices (PWMs), which require a specific motif to be present in the DNA sequence and do not consider interdependencies of nucleotides. Novel approaches such as Transcription Factor Flexible Models or recurrent neural networks consequently provide higher accuracies. However, it is unclear whether such approaches can uncover novel non-canonical, hitherto unexpected TFBSs relevant to human transcriptional regulation. RESULTS: In this study, we trained a convolutional recurrent neural network with HT-SELEX data for GRHL1 binding and applied it to a set of GRHL1 binding sites obtained from ChIP-Seq experiments from human cells. We identified 46 non-canonical GRHL1 binding sites, which were not found by a conventional PWM approach. Unexpectedly, some of the newly predicted binding sequences lacked the CNNG core motif, so far considered obligatory for GRHL1 binding. Using isothermal titration calorimetry, we experimentally confirmed binding between the GRHL1-DNA binding domain and predicted GRHL1 binding sites, including a non-canonical GRHL1 binding site. Mutagenesis of individual nucleotides revealed a correlation between predicted binding strength and experimentally validated binding affinity across representative sequences. This correlation was neither observed with a PWM-based nor another deep learning approach. CONCLUSIONS: Our results show that convolutional recurrent neural networks may uncover unanticipated binding sites and facilitate quantitative transcription factor binding predictions.

Mapping the Transglycosylation Relevant Sites of Cold-Adapted ß-d-Galactosidase from Arthrobacter sp. 32cB.

ß-Galactosidase from Arthrobacter sp. 32cB (ArthßDG) is a cold-adapted enzyme able to catalyze hydrolysis of ß-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthßDG: D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to shed light onto the structural basis of the transglycosylation reaction. The catalytic assays proved loss of function mutation E517 into glutamine and a significant drop of activity for mutation of D207 into alanine. Solving crystal structures of two new mutants, and new complex structures of previously presented mutant E441Q enables description of introduced changes within active site of enzyme and determining the importance of mutated residues for active site size and character. Furthermore, usage of mutants with diminished and abolished enzymatic activity enabled solving six complex structures with galactose, lactulose or sucrose bounds. As a result, not only the galactose binding sites were mapped on the enzyme's surface but also the mode of lactulose, product of transglycosylation reaction, and binding within the enzyme's active site were determined and the glucopyranose binding site in the distal of active site was discovered. The latter two especially show structural details of transglycosylation, providing valuable information that may be used for engineering of ArthßDG or other analogous galactosidases belonging to GH2 family.

Active Site Architecture and Reaction Mechanism Determination of Cold Adapted ß-d-galactosidase from Arthrobacter sp. 32cB.

ArthßDG is a dimeric, cold-adapted ß-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthßDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthßDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthßDG_E441Q/LACs and ArthßDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthßDG_E441Q/LACd, ArthßDG/ONPG (ligands bound in deep mode), and galactose ArthßDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 ßDGs, in ArthßDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.

Insights into the substrate specificity, structure, and dynamics of plant histidinol-phosphate aminotransferase (HISN6).

Histidinol-phosphate aminotransferase is the sixth protein (hence HISN6) in the histidine biosynthetic pathway in plants. HISN6 is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of imidazole acetol phosphate into L-histidinol phosphate (HOLP). Here, we show that plant HISN6 enzymes are closely related to the orthologs from Chloroflexota. The studied example, HISN6 from Medicago truncatula (MtHISN6), exhibits a surprisingly high affinity for HOLP, which is much higher than reported for bacterial homologs. Moreover, unlike the latter, MtHISN6 does not transaminate phenylalanine. High-resolution crystal structures of MtHISN6 in the open and closed states, as well as the complex with HOLP and the apo structure without PLP, bring new insights into the enzyme dynamics, pointing at a particular role of a string-like fragment that oscillates near the active site and participates in the HOLP binding. When MtHISN6 is compared to bacterial orthologs with known structures, significant differences arise in or near the string region. The high affinity of MtHISN6 appears linked to the particularly tight active site cavity. Finally, a virtual screening against a library of over 1.3 mln compounds revealed three sites in the MtHISN6 structure with the potential to bind small molecules. Such compounds could be developed into herbicides inhibiting plant HISN6 enzymes absent in animals, which makes them a potential target for weed control agents.

Structural Evidence of Active Site Adaptability towards Different Sized Substrates of Aromatic Amino Acid Aminotransferase from Psychrobacter Sp. B6.

Aromatic amino acid aminotransferases present a special potential in the production of drugs and synthons, thanks to their ability to accommodate a wider range of substrates in their active site, in contrast to aliphatic amino acid aminotransferases. The mechanism of active site adjustment toward substrates of psychrophilic aromatic amino acid aminotransferase (PsyArAT) from Psychrobacter sp. B6 is discussed based on crystal structures of complexes with four hydroxy-analogs of substrates: phenylalanine, tyrosine, tryptophan and aspartic acid. These competitive inhibitors are bound in the active center of PsyArAT but do not undergo transamination reaction, which makes them an outstanding tool for examination of the enzyme catalytic center. The use of hydroxy-acids enabled insight into substrate binding by native PsyArAT, without mutating the catalytic lysine and modifying cofactor interactions. Thus, the binding mode of substrates and the resulting analysis of the volume of the catalytic site is close to a native condition. Observation of these inhibitors' binding allows for explanation of the enzyme's adaptability to process various sizes of substrates and to gain knowledge about its potential biotechnological application. Depending on the character and size of the used inhibitors, the enzyme crystallized in different space groups and showed conformational changes of the active site upon ligand binding.

Technologies for profiling the impact of genomic variants on transcription factor binding.

Transcription factors (TFs) bind DNA in a sequence-specific manner and thereby regulate target gene expression. TF binding and its regulatory activity is highly context dependent, and is not only determined by specific cell types or differentiation stages but also relies on other regulatory mechanisms, such as DNA and chromatin modifications. Interactions between TFs and their DNA binding sites are critical mediators of phenotypic variation and play important roles in the onset of disease. A continuously growing number of studies therefore attempts to elucidate TF:DNA interactions to gain knowledge about regulatory mechanisms and disease-causing variants. Here we summarize how TF-binding characteristics and the impact of variants can be investigated, how bioinformatic tools can be used to analyze and predict TF:DNA binding, and what additional information can be obtained from the TF protein structure.

Co-expression with chaperones can affect protein 3D structure as exemplified by loss-of-function variants of human prolidase.

Prolidase catalyzes the cleavage of dipeptides containing proline on their C terminus. The reduction in prolidase activity is the cause of a rare disease named 'Prolidase Deficiency'. Local structural disorder was indicated as one of the causes for diminished prolidase activity. Previous studies showed that heat shock proteins can partially recover prolidase activity in vivo. To analyze this mechanism of enzymatic activity rescue, we compared the crystal structures of selected prolidase mutants expressed in the absence and in the presence of chaperones. Our results confirm that protein chaperones facilitate the formation of more ordered structures by their substrate protein. These results also suggest that the protein expression system needs to be considered as an important parameter in structural studies. DATABASES: The reported crystal structures and their associated structure factor amplitudes were deposited in the Protein Data Bank under the accession codes 6SRE, 6SRF, and 6SRG, respectively.

Structural features of cold-adapted dimeric GH2 ß-D-galactosidase from Arthrobacter sp. 32cB.

Crystal structures of cold-adapted ß-d-galactosidase (EC 3.2.1.23) from the Antarctic bacterium Arthrobacter sp. 32cB (ArthßDG) have been determined in an unliganded form resulting from diffraction experiments conducted at 100 K (at resolution 1.8 Å) and at room temperature (at resolution 3.0 Å). A detailed comparison of those two structures of the same enzyme was performed in order to estimate differences in their molecular flexibility and rigidity and to study structural rationalization for the cold-adaptation of the investigated enzyme. Furthermore, a comparative analysis with structures of homologous enzymes from psychrophilic, mesophilic, and thermophilic sources has been discussed to elucidate the relationship between structure and cold-adaptation in a wider context. The performed studies confirm that the structure of cold-adapted ArthßDG maintains balance between molecular stability and structural flexibility, which can be observed independently on the temperature of conducted X-ray diffraction experiments. Obtained information about proper protein function under given conditions provide a guideline for rational engineering of proteins in terms of their temperature optimum and thermal stability.