Arthrobacter aurescens PafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA-binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA-binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA or other nucleic acid molecules. Using an in vivo mutational screen in Mycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determine the crystal structure of  Arthrobacter aurescens PafBC and suggest that PafBC might be activated by binding of a nucleic acid ligand, and bioinformatics analysis shows that its central WYL domain is a widespread feature in bacterial transcription factors.The transcriptional activator PafBC is a regulator of mycobacterial DNA damage response and upregulates genes involved in DNA repair. Here, the authors present the crystal structure of  DNA damage represents a threat to the integrity of genetic information and is therefore counteracted in all organisms by an arsenal of DNA repair processes that are activated by specific DNA damage response pathways. Mycobacteria and many other actinobacteria employ two distinct yet interconnected pathways in order to upregulate the expression of specific sets of genes required for repair and survival of DNA damage.2). Under normal conditions, LexA ensures low expression levels of the SOS genes by binding to a promoter motif called “SOS box”3. Single-stranded DNA (ssDNA) fragments accumulating under DNA damage conditions serve as DNA stress signal for the SOS response and are sensed by the ATPase RecA. RecA forms a filamentous complex with ssDNA that is able to induce autoproteolytic cleavage of the LexA repressor, leading to derepression of the SOS genes6. In Mycobacterium tuberculosis (Mtb), the LexA repressor controls about 25 genes8.The “SOS response”, the canonical pathway described in most bacterial species, relies on cleavage and removal of LexA, a transcriptional repressor of DNA repair genes (“SOS genes”)  that are encoded together in an operon that is tightly associated with the bacterial proteasome gene locus, suggesting a functional connection. Indeed, many DNA repair proteins are removed by proteasomal degradation after the DNA damage has been repaired, thereby helping to shut down the stress response and preventing negative impact of DNA-modifying activities under normal conditions12.In contrast, the second pathway regulates over 150 genes, including many of the LexA-controlled genes, amongst them also 12. However, PafBC protein levels are not changing in response to DNA stress11. Furthermore, specific interaction between PafBC and the identified DNA target regions could not be reconstituted in vitro. Taken together, these results suggest that an additional “response-producing” event must take place to initiate PafBC transcription activation.PafBC activates its target genes via a promoter motif called RecA-NDp (RecA-independent promoter), which was demonstrated by in vivo identification of PafBC binding sites using cell culture cross-linking followed by immunoprecipitation of PafBC-DNA complexesCaulobacter crescentus13 (2) Sll7009, Sll7062, Sll7078, transcriptional repressors of CRISPR/Cas system mature crRNA in Synechocystis 680314, (3) PIF1 helicase from Thermotoga elfii15 and (4) WYL domain-containing proteins stimulating RNA cleavage by Cas13d in Eubacterium siraeum and Ruminococcus sp16. However, structural information on WYL domain-containing transcriptional regulators is missing, and evidence as to how they exert their functions mechanistically has remained elusive.In order to establish the mechanistic principles employed by PafBC to activate transcription at the molecular level, understanding of the structural framework is crucial. Based on sequence similarity, PafBC belongs to a family of bacterial regulators featuring a winged helix-turn-helix (HTH) domain at the N-terminus, followed by a C-terminal domain of unknown function named WYL domain after a consecutive W-Y-L sequence motif. It has been suggested that the WYL domain might play the role of a ligand-binding domain in the context of this class of transcription factors. A handful of other WYL domain-containing proteins were studied to date: (1) DriD, an SOS response-independent transcriptional activator of a cell division inhibitor protein in Arthrobacter aurescens in its non-activated, DNA-free state. The structure reveals that the WYL domain exhibits an Sm-fold, commonly encountered in RNA-binding proteins, and is followed by an additional C-terminal extension (WCX) domain featuring a ferredoxin-like fold. Based on the structure of the PafBC WYL-domain, we carry out a comprehensive computational analysis of WYL domain-containing proteins, and demonstrate that the WYL domain is a widespread feature of bacterial transcription factors present in almost all bacterial taxa. Our study shows that Sm-fold proteins are a much more frequent occurrence in bacteria than previously thought. Based on the high structural similarity of the WYL motif-containing domain to the bacterial RNA chaperone Hfq and the known binding sites of Hfq, we identify functionally essential residues in the WYL domain of PafBC, which are likely involved in binding of a response-producing ligand in this distinct class of transcriptional regulators.In this study, we determine the crystal structure of PafBC from Kocuria rhizophila, Thermobifida fusca, and Arthrobacter aurescens). Three-dimensional crystals suitable for data collection were ultimately obtained using an A. aurescens PafBC construct (AauPafBCΔNC), which was shortened by 17 amino acids at the N-terminus and 7 amino acids at the C-terminus based on sequence alignment, since these residues are not conserved amongst the orthologs and not even present in most of them. Indeed, it is likely that the start site for the A. aurescens protein was misassigned, since a valine (encoded by GTG) is present at the position where the other PafBC proteins feature the conserved initiator methionine, and GTG is a frequent start codon in actinobacteria18  involved in forming the hydrophobic core of the three-helix bundle  is accessible in the structure, since only helix H1 and a small part of H3 make hydrophobic contacts to the rest of the molecule  with an ancillary C-terminal α-helix. Interestingly, a number of RNA-binding proteins such as human hnRNP A1  and certain spliceosomal proteins. Proteins containing an Sm-fold are very abundant in eukaryotes, while only few examples (amongst them Hfq) have been described in bacteria. Closer comparison of the WYL domain with Hfq shows that the WYL domain Sm-fold features a slightly longer N-terminal helix, longer β2, and β3 strands, as well as longer loops between strands β1/β2 and β3/β4   Fig. , we alsoM. smegmatis cells grow in presence of up to 100 ng/ml MMC, while the ΔpafBC strain shows growth only up to about 8 ng/ml MMC, which is in agreement with the previously determined minimal inhibitory concentrations for these strains  cells to resorufin, giving rise to a color change. Wild type ins Fig. 11.pafBC knockout strain with wild type PafBC restores the viability to the level observed for the wild type. Deletion of either the HTH domain of PafB or PafC leads to the same reduced viability as observed for the ΔpafBC strain  to alanines, which resulted in the same phenotype as for the ΔpafBC strain  domain. Based on the domain boundaries of the WYL domains in our structure and sequence alignment with other PafBC orthologs, we generated a WYL domain HMM and used it to retrieve all WYL domain-containing proteins from the UniProt reference proteomes yielding 15,079 entries In order to identify domain families associated with WYL domain proteins, the retrieved WYL domain-containing bacterial sequences were annotated based on all Pfam HMMs and additional HMM profiles we generated for the WCX domain and the PafBC N-terminal winged HTH domain that was not recognized by any of the existing Pfam HMMs. Two observations can immediately be made from the final set of domain architecture classes Fig. : First, About two thirds of all sequences are found in class A, which also comprises all PafB and PafC sequences. The second largest group, class B , contains proteins with only an HTH and a WYL domain, lacking the WCX domain. Notably, class C could also be viewed as a subgroup of class A, as it is made up of natural fusion proteins of actinobacterial PafB and PafC homologs. A significant number of sequences contain a Helicase C3 domain in combination with the WYL domain, but lacking the WCX domain.Actinobacteria and Firmicutes exhibit on average roughly 5.2 and 2.8 WYL domain-containing proteins per organism, respectively, while the gram-negative phyla of Proteobacteria and Bacteroidetes show only 2.0 and 2.1 average WYL domain-containing proteins per organism, respectively lum Fig. . It is pThe crystal structure of PafBC provides the framework for understanding the mechanism by which PafBC connects the signal of DNA stress with a transcriptional response through use of its WYL/WCX domains. These results will also help us to better understand WYL domain-containing proteins in general.AauPafBC was amplified from genomic DNA of Arthrobacter aurescens strain 579 (DSM-20116) using the primers aaubc-fw and aaubc-rv (Supplementary Table AauPafBC (AauPafBCΔNC) missing the first 17 amino acids at the N-terminus and the last 7 amino acids at the C-terminus was amplified from this vector using primers aaubcdNC-fw and aaubcdNC-rv -inducible expression vector with a cleavable His6-TEV tag at the N-terminus. Selenomethionine-labeled protein was expressed according to a procedure adapted from ref. 46: E. coli Rosetta (DE3) cells harboring the expression vector were grown as shaking cultures at 37 °C in M9 medium . At an OD600 of 0.5, 100 mg/ml of phenylalanine, lysine, and threonine, 50 mg/ml of isoleucine, leucine, and valine, as well as 80 mg/ml of selenomethionine (Chemie Brunschwig) were added as solid powder to the cultures, which were further incubated for 30 min. Expression was then induced with 0.5 mM IPTG and cells were further incubated at 16 °C overnight. Cells were harvested , 10 min, 4 °C) and pellets were resuspended in lysis buffer . The cell suspension was homogenized using a Heidolph DIAX600 mixer and cells were lysed by high pressure shear force using a Microfluidizer M110-L device . After removal of cell debris , 4 °C, 30 min), the cleared lysate was supplemented with 1 mM PMSF, 1× c0mplete EDTA-free protease inhibitors (Roche), 50 U/ml DNase I, 10 mM imidazole and incubated for 30 min on ice. The lysate was passed over a self-packed Ni2+-charged IMAC Sepharose 6 Fast Flow  column, and bound protein was eluted step-wise with lysis buffer containing 80 mM to 250 mM imidazole. After pooling protein-containing elution fractions, His-tagged TEV protease was added to a 1:30 molar ratio and the protein sample was dialyzed against 25 mM HEPES-NaOH pH 7.8/4 °C, 150 mM NaCl, 2 mM DTT, 1 mM EDTA at 4 °C overnight. TEV protease was removed by affinity chromatography and the protein sample was dialyzed against 25 mM HEPES-NaOH pH7.8/4 °C, 40 mM NaCl, 2 mM DTT, 1 mM EDTA at 4 °C overnight. The protein was further loaded on a Source 30Q column and eluted with a 50 mM to 400 mM NaCl gradient in 25 mM HEPES-NaOH pH 7.8/4 °C, 1 mM TCEP. Protein-containing elution fractions were pooled and concentrated using an Amicon Ultra 30 K centrifugal filter . The concentrated protein was run on a self-packed 100 ml Superose 12 prep grade column in crystallization buffer . Protein was concentrated as above to 22 mg/ml, aliquotted, frozen in liquid nitrogen and stored at −80 °C until use.Full-length 79 DSM-2016 using AauPafBCΔNC were grown in sitting drop vapor diffusion plates (Hampton Research) at 4 °C by mixing 1–2 μl protein solution (5.6–7.5 mg/ml) and 1 μl reservoir solution. Crystals appeared after 2–3 days using reservoir solutions containing 100 mM Bis-Tris-propane pH 8.8 to 9.2 (20 °C), 80–160 mM KSCN, 18–21% PEG-2000 (Sigma). PEG-2000 was added in 5% steps to reach a final concentration of 36% w/v in drops containing crystals before flash cooling the crystals in liquid nitrogen.Crystals of 47 and scaled using AIMLESS48. Determination of heavy atom sites, initial phases, and crude main chain tracing were carried out using the SHELX programs49. The resulting experimental electron density map displayed easily discernible protein features. The initial model from SHELX was further extended with PHENIX AutoBuild50 and subsequent iterative model building and refinement was carried out using Coot51 and phenix.refine52, respectively.Reflection image data was collected at the X06SA beamline of the Swiss Light Source  at 100 K and 12,670 eV beam energy (0.978561 Å). Diffraction images were processed using XDS53. Amino acid residue conservation was calculated using AL2CO 54. For that, PafBC protein sequences of 23 different organisms spanning the entire actinobacterial phylum  were aligned against the A. aurescens strain 579 PafBC protein sequence. To be able to calculate conservation across the entire length of naturally fused PafBC proteins, sequences of separately encoded PafB/PafC proteins were concatenated prior to alignment.Graphical representations of protein structures were prepared using UCSF Chimera v1.12 build 41623pafBC was amplified from Mycobacterium smegmatis mc2-155 SMR555 genomic DNA and cloned into a pMyNT-derived integrative plasmid , and viability in presence of mitomycin C was assessed with the resazurin assay as described previously12. In addition, viability was assessed by a spotting assay: Cells were grown as shaking cultures in 7H9 medium supplemented with 50 μg/ml hygromycin at 37 °C. At an OD600 of 1.0 to 1.3, cultures were split and mitomycin C was added to one half to a final concentration of 80 ng/ml. Cultures were incubated for another 4 h at 37 °C. Subsequently, cultures were diluted to an OD600 of 0.8 based on the OD600 value measured at the time of splitting and a 1:5 serial dilution was prepared in 7H9 supplemented with 50 μg/ml hygromycin. 10 μl were spotted onto 7H10 agar plates and the plates were incubated facing upwards at 37 °C for 3 days.The coding sequence of M. smegmatis strains carrying complementation plasmids or empty control vectors were grown as shaking cultures to an OD600 of about 1.0 in 7H9 medium supplemented with 50 μg/ml hygromycin at 37 °C. Cells were harvested by centrifugation  and were washed once in 700 μl PBS. Pellets were then resuspended in 700 μl PBS supplemented with 1 mM DTT, 1 mM EDTA, 1 mM PMSF, 1× cOmplete EDTA-free protease inhibitor (Roche) and lysed by bead beating in 2 ml screw cap tubes containing 500–700 mg 0.15 mm zirconium oxide beads using an MP Biomedicals FastPrep-24 bead beater . After removing insoluble material by centrifugation , protein content of the cleared lysate was determined by Bradford assay. Equal amounts of total protein were separated on a 10% SDS-containing polyacrylamide gel and transferred onto PVDF membrane Immobilon-P (Merck) using a BioRad Trans-Blot SD semidry transfer cell . Membranes were blocked in PBS containing 0.05% Tween-20 and 1% polyvinylpyrrolidone 40  for 1 h at room temperature. Immunoblotting for RecA, RpoB, or PafBC was carried out using commercially available anti-RecA antibody  raised in mouse, commercially available anti-RpoB antibody  raised in mouse, or previously described anti-PafBC antibody (1:5000) raised in rabbit11. The blocked membrane was incubated with primary antibodies in blocking buffer for 1 h at room temperature. Detection was achieved by incubation with horseradish peroxidase conjugated anti-mouse IgG antibody  or anti-rabbit IgG antibody  in blocking buffer for 1 h at room temperature and using ECL substrate (BioRad Clarity Western Substrate).56. Manual analyses of alignments were performed in Jalview v2.10.557. Steps involving hidden Markov-models (HMMs) were conducted using the programs “hmmbuild”, “hmmsearch”, and “hmmscan” from the software suite HMMER v3.2.1 (hmmer.org)28. A local search database was created from all UniProt reference proteomes 58.All alignments were generated using ClustalO v1.2.4 with default settings59 using Mycobacterium smegmatis PafB or PafC as input. The identity of the obtained PafBC homologs was cross-checked on the genome level for the operon organization of the genes and association with the Pup-proteasome system gene locus. In total, ten PafB/PafC sequence pairs were retrieved . A global alignment of these sequences was used to generate an HMM with “hmmbuild” (default settings), which was subsequently used to search against the reference proteomes database with “hmmsearch” (command line options were -E 1 -domE 1 -incE 0.01 -incdomE 0.03). A list of domains contained in the retrieved sequences was obtained with the “hmmscan” module (command line options were -E 0.1 -domE 0.1 -incE 0.01 -incdomE 0.03) using all HMMs from the Pfam database release 32.060. Domain lengths were analyzed based on the envelope boundaries given by “hmmscan” for sequences with a domain score above 30.0 .For the initial analysis, PafBC homologs from other actinobacteria were identified by BLAST AauPafBC and the length distribution of C-terminal region of the retrieved Pfam-WYL-containing proteins. Sequences with obvious defects in the WYL region were discarded manually resulting in 477 entries that were used for alignment. From this alignment, the WYL domain boundaries were established using the N-terminal boundary of the Pfam-WYL, while the C-terminal boundary was chosen by the C-termini of short Pfam-WYLs and the crystal structure. The alignment was then trimmed to the WYL boundaries, sequences with a pairwise identity above 70% were clustered using CD-HIT v4.6.8 (command line options -n 4 -c 0.7)61 and an HMM was generated as above. To mature the WYL HMM, an iterative approach similar to the Pfam HMM generation was chosen. Sequences were retrieved from the reference proteomes using the WYL HMM to generate a full alignment, which was then again trimmed to the WYL domain boundaries to make up a new seed alignment used to build a new HMM (gathering threshold: domain score > 27.0). The process was repeated for a total of three iterations.Because the Pfam HMM profile of the WYL domain (Pfam-WYL) includes both WYL and WCX domain, we had to define a new WYL domain HMM in order to annotate it correctly for our analysis. To build the WYL HMM, 250 sequences were randomly sampled from sequences with a Pfam-WYL length greater than 127 residues and another 250 sequences were randomly sampled from sequences with a Pfam-WYL length less than 127 residues . The threshold was chosen based on the domain boundaries seen in the crystal structure of To build the HMM for the unrecognized (winged) HTH domains in PafBC and many other WYL domain-containing proteins, 500 sequences were randomly sampled from the group of sequences with a median length of 326 amino acids and containing only a WYL domain . The sequences were aligned and curated based on the presence of the conserved blocks representing the helices of the HTH fold. The sequences were further split into groups exhibiting a PafB-like or PafC-like wing sequence of 184 and 235 sequences, respectively. For each group, an HMM was built and searched against reference proteomes database as described above, but using an identity threshold of 90% and iterating only once in order to keep a separation to other HTH-type domains.To build the HMM for the C-terminal extension found in many WYL domains (WCX), 500 sequences were randomly sampled from the group containing a Pfam-WYL with a length greater than 127 amino acids . Sequences missing the C-terminal conserved block were removed, leaving 434 sequences for HMM generation. The HMM generated from the manually curated seed alignment was used without iteration.The custom HMMs were then added to the local search database of the Pfam HMM database .To establish the domain architecture classes of the WYL domain-containing proteins, the WYL HMM was used to search against the reference proteome database with “hmmsearch” (command line options as above). Obtained sequences with a domain score > 27.0 were analyzed for the presence of other domains using “hmmscan” together with the local Pfam HMM database including the custom HMMs as described above. Protein sequences were then categorized according to their sequence length, their identified domains  and the domain length. Domain architecture classes with large regions apparently containing no domain were checked manually by alignment for presence of conserved features, by alignment to other classes and by alignment to the manually curated PafBC seed sequences (see above). They were then grouped together with other classes, where appropriate. Due to low overall abundance and being mostly candidate species, all non-bacterial sequences were excluded from the main analysis.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting SummarySource Data


Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue ‘cystatin motif’ (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two β-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse β-hairpin proteins. Designing interfaces that can induce protein-protein interactions is a challenging problem. Here the authors show that a five amino acid sequence known to mediate domain swapping in cystatins can drive oligomerization when grafted onto functionally and structurally unrelated host proteins, providing a simple approach to the design of protein assemblies. The formation of a dimer is a basic step in building such assemblies2, and diverse methods have been invented to engineer protein homodimers and heterodimers3. However, the required protein manipulation is generally difficult because of the presence of a complex array of cooperative and long-range interactions in proteins. This structural complexity and the marginal stability of proteins necessitate the optimization of each design approach in a protein-specific manner. Thus, the number of proteins and protein sites amenable to a given design strategy is typically low.Rational design of protein–protein interactions can be used to build supramolecular assemblies capable of performing both biological and bio-inspired functions4). In domain swapping, two protein molecules exchange “domains” or structural units connected by a hinge loop, such that intermolecular interactions replace the intramolecular interactions at the dimer interface of each monomer8 , leading to the formation of different protein assemblies from a single protein10. Moreover, nature uses domain swapping not only as a mechanism for oligomer assembly, but also to encode for novel functions and for the evolution of novel protein folds21.Several proteins dimerize (or oligomerize) naturally through domain swapping  proteins35 into the β-turns of proteins could be a general strategy for designing domain swapping. In the domain-swapping cystatins, the loop connecting β2–β3 has a QXVXG consensus motif, which has been implicated in both protease inhibition and domain swapping39 . MNEI is similar structurally to stefin B , in which the β-sheet forms the dimer interface. It was important to determine if the dimers observed in this study were formed by domain swapping or through the β-sheet interface. Despite a variety of conditions being tested, both L2MN and L3MN dimers did not yield good-quality crystals for diffraction. Moreover, L2MN dimers precipitated at high sample concentration, and could not be studied by nuclear magnetic resonance (NMR) spectroscopy. It should be noted that even though it was difficult to concentrate the L2MN dimer for NMR studies, the L2MN monomer to dimer ratio was unchanged over a concentration range of 10–200 μM.21. A Val to Asn mutation has been shown to disfavor domain swapping in human cystatin C (hCC)43. Asn is a polar residue that is preferred in β-turns and substitution of the solvent-exposed hydrophobic Val in the “QIVAG” motif of hCC by Asn stabilized its monomeric conformation. We recently observed a similar effect in MNEI with a shortened loop134. In an analogous manner, the monomeric conformation of L2MN could be stabilized by mutating QVVAG to QVNAG in loop2  spectra of the monomer and dimer overlapped well  of the L3MN dimer were similar to those of the L3MN monomer, except in loop3 . Finally, similar to L2MN, the QVVAG to QVNAG mutation in loop3 of L3MN (L3MN*) led to the disappearance of the observed dimer . MK-Ctd was chosen because it is unrelated to the cystatins, folds to a different topology45 , a protein domain derived from the protein MK0293 from 15N-edited HSQC spectrum of the L1MK-Ctd dimer showed well-dispersed peaks  and human ubiquitin (UBQ). These proteins were chosen because they adopt folds that are structurally unrelated to the monellin/cystatin fold, and their purification protocols, biochemical properties, and structural details have already been characterized in our laboratories.The QVVAG motif was then introduced into a β-hairpin motif of Sso7d  has an SH3-like foldogy Fig. . We usedfold see . This mufold see . L1Sso7dfold see . The SECfold see . To conf10-β5 topology  motif is not found naturally in structural contexts similar to those in which it was engineered into the variants L2MN and L3MN . The induction of domain-swapped dimerization in L2MN and L3MN suggests that the QVVAG motif can drive domain swapping in diverse β-hairpin containing proteins, which may fold to topologies/folds different from the monellin/cystatin fold. Experiments with structurally and functionally unrelated proteins, MK-Ctd, Sso7dv, and UBQ, lend support to this claim because dimerization/oligomerization of these proteins was observed when the QVVAG motif was introduced into their β-turn-β loops as well. Thus, the QVVAG stretch appears to be a versatile motif for engineering domain-swapped oligomerization in proteins. However, given how intricately the phenomenon of domain swapping is linked to protein folding54 shows that the configuration of the two QXVXG motifs which enable dimerization are variable across the structures with both stacked  and staggered  arrangements being present. The angle between the two subunits of the domain-swapped structures is also variable. For example, L1MN has a wider angle (~160°) between its two subunits as compared to stefin B (~42°)  domain-swapped cystatins35. One way to stabilize such solvent-exposed loop hydrophobic residues is domain-swapped dimerization54. The stacked arrangement of the QVVAG stretches observed in the L1MN and L13MN crystal structures and L1MK-Ctd NMR structure reduces the solvent-exposure of these hydrophobic residues, but is also likely to contribute to stability due to the formation of a new anti-parallel β-sheet between the two polypeptide chains in the crossover region (secondary interface). However it is difficult to delineate the relative contribution of these two factors in driving domain swapping because Val is both hydrophobic and has a high propensity to form β-sheets due to its β-branched side chain.Earlier studies had indicated that the reason behind QVVAG-induced domain swapping is the presence of hydrophobic residues (VVA) in a solvent-exposed loop. It has been hypothesized that the solvent exposure of the hydrophobic residues creates strain and does not allow the loop to fold back upon itself. Support for this hypothesis comes from the fact that the central V of the QVVAG motif in the monomeric conformation of Stefin B Fig.  is strai43 reduces the destabilization due to solvent exposure, shifts the N to a “generously allowed” region of the Ramachandran plot and stabilizes the monomer. A similar monomer stabilization is seen in the QVVAG to QVNAG or QVVAG to QNNAG mutants in our experiments ts Figs. .57.The second approach to monomer stabilization is through a reduction in the solvent-exposure of the hydrophobic QVVAG loop. The monomeric conformation of L1UBQ was not destabilized enough to induce domain swapping in it  < loop2 in L2MN (2 residues long) < loop3 in L3MN (6 residues long); calculated using60) and the extent of observed dimerization in the resultant variant  peptide display scaffold Adhiron61 (PDB ID: 4N6T), derived from a photocystatin62 consensus sequence, is monomeric despite the presence of the QVVAG motif. Nevertheless, a correlation between protein stability and efficiency of dimerization was not observed for the four proteins studied here  observed at the secondary interface in L1MN is 15  and the domain-swapped dimer of a ubiquitin-like plant protein, ATG1265 (PDB ID: 1WZ3), where loop4 exists in an extended conformation, suggests that it is plausible that there are two distinct modes of domain swapping possible for L4UBQ, a phenomenon which could also increase the probability of generating higher-order oligomers. Structural characterization of the L4UBQ multimers should aid in further understanding the factors that contribute to the observed distinct domain-swapping behavior of L4UBQ and the rational design of higher-order oligomerization.The induction of multimerization by engineered open-ended domain swapping has been achieved earlier for a few proteins66. The similarity of the swapped structure with the monomer is expected to reduce the inadvertent loss of protein function that can occur when many amino acid residues are mutated to design a new protein-protein interaction interface. The lack of a clear strategy for inducing domain swapping has hindered its use in the design of protein oligomerization and assembly. Here, we provide a possible strategy, the insertion of the QVVAG stretch into tight and solvent-exposed β-turns of proteins. The design of domain swapping using the QVVAG stretch does not require the use of computational tools, and given the minimal perturbation to a sequence that this strategy presents, it could be easily integrated with existing methods for the design of protein assemblies and to yield a rich complexity of protein nanostructures.In summary, the design of dimerization (or oligomerization) through domain swapping is advantageous because it requires the mutation of only a few amino-acid residues67. In L1MN, residues 51–56 (EGFREI) were deleted (MNEIΔ6Asn)34 followed by mutation of the flanking YENK stretch to QVVA (48–51) to convert residues number 48–52 to QVVAG. L1MN is 91 residues long. In L2MN, residues 66–70 were mutated from YASDK to QVVAG. In L3MN, residues 79–83 were mutated from DYKTR to QVVAG. Both L2MN and L3MN are 97 amino acid residues long. In L13MN, residues 73–77 (which correspond to residues 79–83 in wt MNEI) in L1MN were mutated from DYKTR to QVVAG. L13MN is 91 residues long. In L1MK-Ctd, residues 13–17 were mutated from YGERE to QVVAG. In L1Sso7dv, residues 7–12 were mutated from KYKGEE to QVVAG. In L1UBQ, residues 6–9 were mutated from KTLT to QVVA to convert residues number 6–10 to QVVAG. In L4UBQ, residues 44–48 were mutated from IFAGK to QVVAG. Primers for SDM were obtained from BioServe, India. The purification methods for MNEI, MK-Ctd, Sso7dv, and UBQ loop variants are described in detail in the Loop variants of MNEI, MK-Ctd, Sso7dv, and UBQ with the QVVAG motif engineered into different loops were constructed by site-directed mutagenesis (SDM), using a single mutagenic (forward) primerAll the chemicals used for the study were of high purity grade, and were procured from Sigma. Experiments with MNEI and its loop variants were carried out using 50 mM phosphate buffer, containing 250 μM EDTA and 1 mM DTT, at pH 7. Experiments with MK-Ctd and its loop variants were carried out using 20 mM Tris-HCl buffer, containing 500 mM GdnHCl, at pH 8. Experiments with Sso7dv and its loop variants were carried out using 50 mM phosphate buffer, containing 200 mM NaCl, at pH 7.5. Experiments with UBQ and its loop variants were carried out using 20 mM Tris-HCl buffer, containing 150 mM NaCl, at pH 8.3.-1, and protein elution was monitored at 280 nm. The apparent molecular weight of different proteins was estimated from a calibration curve generated using a Bio-Rad gel filtration standard. For all the protein variants, a representative SEC profile is reported. Proteins were lyophilized immediately after purification and stored at −20 °C. These proteins were later dissolved in appropriate buffers and their oligomeric status was analyzed by SEC. Each variant was expressed and purified at least three times to verify the reproducibility of the observed trends.The oligomeric status of different proteins was determined using a Superdex 75 10/300 GL size exclusion column (which can resolve proteins in the molecular weight range 3–70 kDa) on an ÄKTA FPLC. The column was run at 0.5 ml min−1, and a digital integration time of 1 s. Fifteen scans were averaged for each sample.The far-UV circular dichroism (CD) spectra of MNEI, MK-Ctd, Sso7dv, and UBQ, and their different loop variants, were acquired on a Jasco J-815 spectropolarimeter, using a protein concentration of 10–20 μM in a 1 mm path length quartz cuvette, with a bandwidth of 1 nm, a scan speed of 50 nm min−1 of protein  was mixed with 2 μl of reservoir solution, and set for crystallization using the hanging drop vapor diffusion method at 4 °C. The reservoir solution contained 8–12% (wt/vol) PEG 8000 and 50 mM sodium phosphate at pH 6.4–6.8. Crystals of L1MN dimer and L3MN monomer grew to their maximum size in 4–5 days. Crystals for L13MN appeared only after a week. Crystals were cryoprotected by soaking them in a solution containing 15% (wt/vol) PEG 8000 and 50 mM sodium phosphate pH 6.4–6.8, supplemented with glycerol that was increased in steps of 5% from 0 to 30% (vol/vol). At each incremental step, crystals were dehydrated at 4 °C for 6–12 h. Crystals were then flash frozen in liquid N2.2 μl of 2–4 mg ml68. Data collection for L13MN crystals was carried out at 100 K at the ID30A-1/MASSIF-1 beamline in ESRF Synchrotron France, on a PILATUS3 2 M detector using a beam of wavelength 0.966 Å69. Data collection for L3MN monomer was carried out on a Rigaku FR-X machine (1.5418 Å wavelength) at 100 K. Data were indexed and integrated using the XDS software70. Scaling and merging of diffraction intensities were carried out using the POINTLESS and AIMLESS software in the CCP4 package71. The structures of the different MNEI variants were solved by molecular replacement using wt MNEI (PDB ID: 1IV7) as a search model, and the program MOLREP72. The model was refined iteratively using the program PHENIX73, and manually rebuilt using the program COOT74. Water molecules were modeled using COOT. The model was partitioned into multiple groups, identified from the TLSMD server75, and was subjected to TLS (Translation/Libration/Screw) vibrational motion refinement in PHENIX. Simulated Annealing Composite Omit 2Fo-Fc electron density map was generated using PHENIX. Cartesian dynamics with starting temperature of 5000 K was used for simulated annealing. The map was calculated over the entire unit cell and about 5% of the structure was omitted at a time. The map was then used to manually rebuild and correct the structure model of any discrepancy. The Fo-Fc map was generated similarly. All the structures had good geometries, with 93–95% residues in the favored region and 0–0.5% residues in the outlier regions of the Ramachandran Plot. Clashscores, calculated by Molprobity76, were <6 for all structures. Diffraction data and refinement statistics are summarized in Supplementary Table Data collection for L1MN crystals was carried out at 100 K at the Proxima-1 beamline of the Soleil Synchrotron France, on a PILATUS 6 M detector using a beam of wavelength 0.97857 Å13C, 15N-labeled L3MN were separated using size exclusion chromatography. Each fraction was concentrated to ~400 μM in 20 mM phosphate buffer at pH 7.2, containing 0.03% sodium azide. For the backbone assignment of the L3MN dimer, NMR spectra were recorded at 298 K on an 800 MHz Bruker Avance III HD spectrometer, equipped with a cryo-probe head. All NMR samples contained 10% D2O (vol/vol). Standard HN(CO)CACB, HNCACB, HNCO, HNCA and HN(CO)CA NMR triple resonance 3D experiments were used for backbone assignments. All NMR data were processed using NMRPipe77, and analyzed by the Sparky software78. Following peak picking of the backbone experimental data in Sparky, the data were assigned by the PINE NMR-server79 and then verified, corrected and completed manually. The backbone assignments are 97% complete. The TALOS+ software46 was used to predict the ϕ, ψ torsion angles from the assigned 1Hα, 15N, 13Cα, 13Cβ, and 13CO chemical shifts, for both the monomer and the dimer. The backbone resonance assignment of 13C, 15N-labeled monomeric wt MK-Ctd, and the 13C, 15N-labeled L1MK-Ctd dimer were carried out by standard triple resonance 3D experiments, as mentioned above. The torsion angles and secondary structure were calculated as described above. The side chain resonances of wt MK-Ctd were assigned by triple resonance 13C-edited HSQC, (H)CC(CO)NH and H(CCCO)NH experiments. Some side chain resonances of L1MK-Ctd dimer were assigned by comparing peaks between 13C-edited HSQC of the mutant dimer and the wt MK-Ctd. The rest of the side chain resonances were assigned by using HAHB(CO)NH and HCCH-TOCSY experiments. 13C-edited and 15N-edited NOESY-HSQC experiments were carried out on uniformly 13C, 15N-labeled L1MK-Ctd. A 12C/14N,13C/15N heterolabeled dimeric sample was prepared by unfolding the unlabeled (12C/14N) and uniformly 13C/15N labeled dimeric proteins separately, by incubating them in 7 M GdnHCl  for 3 h at a concentration of ~0.7 mM. Following this, equal volumes of the two proteins were mixed and left at room temperature for 15 min. Finally, refolding was initiated by reducing the denaturant concentration to ~0.5 M by 15-fold dilution in the refolding buffer , which decreased the total protein concentration to ~50 μM. The mixture was incubated overnight for equilibration. The SEC profile of the refolded mixture prepared in this manner showed that >90% of the total protein existed as a dimer .The monomeric and dimeric fractions of 80. Data were analyzed using the Primus and Gnom software in the ATSAS suite (EMBL Hamburg). The radius of gyration (Rg) was averaged for all concentrations to obtain the mean value. The Rg values were found to be 26.5 ± 2.2 Å and 27.4 ± 2.8 Å  for the L2MN and L3MN dimers, respectively.SAXS measurements were carried out using a BIOSAXS-1000 small-angle X-ray scattering Kratky camera system, installed on a Rigaku microfocus X-ray generator (1.5418 Å wavelength). Purified L2MN and L3MN dimer fractions at 2–3 different concentrations were subjected to X-rays for 30 min each at 25 °C. Buffer scattering, recorded under identical conditions, was subtracted from the scattering of the protein sample. The scattering curve was fitted to structural models using the software FoXS−1 was applied to a glow discharged Quantifoil 1.2/1.3 holey carbon grids. Grids were frozen in liquid ethane with a Vitrobot (FEI) at 100% humidity and 18 °C for 3 s. The grids were transferred to Krios Autogrids and images were acquired on a Titan Krios with a Falcon 3 detector at a nominal magnification of 47,000×  resulting in 1.78 Å per pixel. The total exposure was 3 s, and dose was ~70 e- per Å2.Three microliter of the L4UBQ void fraction at 1 mg mlFurther information on experimental design is available in the Supplementary InformationReporting SummarySource Data


DNA strand displacement, which plays a fundamental role in DNA computing, has been widely applied to many biological computing problems, including biological logic circuits. However, there are many biological cascade logic circuits with domain labels based on DNA strand displacement that have not yet been designed. Thus, in this paper, cascade 8-bit adder/subtractor with a domain label is designed based on DNA strand displacement; domain t and domain f represent signal 1 and signal 0, respectively, instead of domain t and domain f are applied to representing signal 1 and signal 0 respectively instead of high concentration and low concentration high concentration and low concentration. Basic logic gates, an amplification gate, a fan-out gate and a reporter gate are correspondingly reconstructed as domain label gates. The simulation results of Visual DSD show the feasibility and accuracy of the logic calculation model of the adder/subtractor designed in this paper. It is a useful exploration that may expand the application of the molecular logic circuit. In recent years, biological computing has become a new hotspot due to DNA molecules having the advantages of parallelism, low energy consumption, and high storability in dealing with massive information; therefore, DNA nanotechnology stands out DNA nanotechnology has potential applications in biological calculations. A range of information circuits and bio-computing models have been implemented in DNA by using strand displacement. Examples include DNA strand displacement reactions ,2, molecAlthough Winfree  solved tIn this paper, DNA signals are marked with domain labels based on the freedom of DNA hybridization and the high sensitivity of domain labels, and then logic gates with domain labels are constructed by redesigning the special DNA structure, where logic value 1 and logic value 0 are represented by domain t and domain f, respectively, which solves the instability issue of NOT Gate in dual-rail logic. Based on this, the first domain label, cascade 8-bit adder/subtractor is designed. The innovation of this paper is that the design of the molecular domain label is used in the logic gates, where logical results are detected through the labeling of the domain label with a fluorescent label in the reaction solution. This was not seen in the previous design of the molecular logic circuit, and this paper broadens the range of input signals with DNA molecules to construct the logic circuit. The detection method of the logic gate model has high sensitivity and simple operation. It has less stringent requirements for base mismatches, reducing the impact of hybrid competition in the experimental results to a certain extent. In addition, the domain label cascade 8-bit adder/subtractor can be used to design large scale biochemical circuits to allow good encapsulation.This paper is arranged as follows: the development of DNA molecule logic circuits is introduced in the first part; the background of DNA strand displacement and logic gates is presented in the second part; a brief method for building domain label logic gates is presented in the third part; the simulation of the domain label cascade 8-bit adder/subtractor by Visual DSD is presented in the fourth part; and the fifth part presents the conclusions of this paper.Utilizes the characteristics of the free energy of the molecular hybridization system to stabilize the state and control or induce downstream strand displacement reactions by changing the sequence and length of the input signal. Intuitively, DNA strand displacement is the process of replacing a shorter hybridization region with a longer, double-stranded hybridization region. The process of this is shown in The process of the reversible DNA strand displacement reaction is shown in On the basis of the existing DNA logic model, the domain label is used to realize the operation of the domain label logic module which can react spontaneously at room temperature. Domain t and domain f respectively represent signal 1 and the signal 0, which correspond to the regions of high concentration and low concentration in the solution, which are shown in As can be seen from The AND Gate with a domain label, the OR Gate with a domain label and the NOT Gate with a domain label are the most elementary logic modules, the logic gates of which are shown in Whether a DNA single strand represents logic 1 or logic 0 depends on its presence . When two strands with domain t are input to the AND Gate with a domain label at the same time, the DNA molecule reaction depends on K operation: Let A be a non-empty set. The Cartesian product In this paper, the logic circuit domain label and double-dual are briefly compared in terms of the stability of the molecular reaction process. In the double-dual logic circuit, the Seesaw module contains threshold gates which react with the upstream DNA signal strands irreversibly. The next step of the molecular reaction can be carried out only the threshold gate is completely consumed, so theoretically, the smaller the concentration of the threshold is, the faster the speed of reaction is. The significance of the threshold is to distinguish between the high and low concentrations of the reaction process so that the logical values 1 and 0 are correctly expressed. The double-dual logic circuit corresponds to logic 1 and logic 0 via the high concentration and low concentration, respectively. In general, a DNA signal strand with a unit concentration of 0.9~1  represents logic value 1, and a DNA signal strand with a unit density of 0~0.1 represents logic value 0. It is worth noting that the threshold can correctly express the logical value at the average concentration value, but the molecular reaction is extremely unstable at the threshold concentrations of 0.1~0.2 units and 0.8~0.9 units. In such cases, the threshold cannot strictly distinguish between high concentration and low concentration in the reaction process, resulting in the output of the wrong signal, while logic circuits with domain t and domain f are determined throughout the reaction process and will not output the wrong signal, thereby avoiding the instability of the molecular circuit in the reaction process. On this basis, the paper constructed an N-mapping module with a domain label, an amplification gate with a domain label, a fan-out gate with a domain label, and a reporter gate with a domain label. This formed the basis for constructing the 8-bit addition and subtraction with a domain label.The 1-input mapping module consists of strands with the domain label {T^*} [mL^ f mR^ T^] <nL^ n1 nR^> and {T^*} [mL^ t mR^ T^] <nL^ n2 nR>, which note , . As shown in As shown in Therefore, when double strands with domain labels n1 and n2 are inverted , the NOT operations are performed with each of them at the same time, which makes the reaction proceed simultaneously and reduces the time consumed by the reaction. The advantages of the modules are demonstrated here.The K-input mapping module consists of The 2-input mapping module consists of strands with domain labels {T^*} [mL^ f mR^ T^]: [nL^ f nR^ T^] <hL^ n1 hR^>, {T^*} [mL^ f mR^ T^]: [nL^ t nR^ T^] <hL^ n2 hR^>, {T^*} [mL^ t mR^ T^]: [nL^ f nR^ T^] <hL^ n3 hR^>, and {T^*} [mL^ t mR^ T^]: [nL^ t nR^ T^] <hL^ n4 hR^> Among them , , , and . As shown in Therefore, when n1 = n4 = f, n2 = n4 = t, XOR logic can be realized, which is used as an n-bit adder with a domain label. Generally, the reactions proceed simultaneously, reducing the time consumed by the reaction. The advantages of the modules are demonstrated again.DNA signals will be attenuated during the reaction process, and lower concentrations will affect the rate of the DNA reaction and the final detection accuracy, so an amplification gate with a domain label was designed in this paper, consisting of two DNA double strands with domain labels and two DNA single strands with domain labels, namely, {T^*} [mL^ f mR^ T^] <nL^ f nR^>, {T^*} [mL^ t mR^ T^] <nL^ t nR^>, and <mL^ f mR^ T^ iL^ jiR^>, <mL^ t mR^ T^ iL^ jiR^>, which corresponds to amplifier strand-3, amplifier strand-4, fuel strand-1, and fuel strand-2. As shown in It is noteworthy that the domain label is a long domain so only two DNA strands with domain labels are involved in the reaction, amplifying the concentration of the upstream DNA signal strand with a domain label to a set value (one-unit concentration in this paper). Namely, fuel strand-2 and amplifier strand-4 will react with the upstream DNA signal strand that represents logic value 1, and the other two strands do not participate in the reaction. Similarly, fuel strand-1 and amplifier strand-3 will react with the upstream DNA signal strand that represents logic value 0, and the other two strands do not participate in the reaction. In summary, one and only one of the above situations occurs when the DNA system is being reacted. More specifically, for DNA signal strands with a domain label that represents a logical value of 0, the DNA single strands <mL^ t mR^ T^ nL^ t nR^> cannot appear in the output strands. For DNA signal strands with a domain label that represents a logical value of 1, it is also unlikely that the DNA single strands <mL^ f mR^ T^ nL^ fnR^> will appear in the output strands. The reaction between the amplification gate with a domain label and the DNA signal strand with a domain label is shown in The functions of the fan-out gate with a domain label and the fan-out gate with a dual-rail logic circuit are the same principle, so they can transform the DNA signal strand into several DNA signal strands representing the same logic signal (the specific quantity can be set). The N fan-out gate’s function is to convert a domain-labeled DNA signal strand into an identical domain-labeled DNA signal strands, with a concentration N times that of the original signal strand. Through the fan-out gate with domain label conversion, the output strands can react with different encapsulated logic modules which are relatively independently packaged. This ensures that the logic inside the DNA system is encapsulated, as are the combined DNA systems, and the entire DNA system is at a steady state. The 2-fan out gate with a domain label is shown in It can be seen from In the reaction shown in In this paper, the experimental results are tested via the reporter gate with a domain label. The detection gate is composed of fluorophore and quenchers, which converts the DNA signal strands into fluorescent signal strands. Fluorescent signal strands are released when the reporter gate with a domain label reacts with DNA signal strands, and the fluorescent signal strands do not react with other logic gates. As shown in As can be seen in In The one-bit full adder takes the adjacent low-order carry into account. The one-bit full adder has DNA input strands denoted by c1 (low-order carry), x1, and x2, respectively, and has two DNA output strands denoted by y (sum) and c2 (carry). It can implement the addition of three binary logic values and simulate an electronic one-bit full adder. The logic circuit is shown in The single-rail one-bit full adder consisting of six AND gates, three OR gates, and four NOT gates, as shown in The 1-bit binary adder constructed by two 3-input mapping modules, which consists of 1 XOR gates with domain labels, 3 amplification gates with domain labels, one 2-fan out gate with a domain label, and reporter gates with domain labels. It can implement an addition between the 1-bit binary The reporter gates with domain labels are <S1L^ _ S1R^ fluor> and <S2L^ _ S2R^ fluor>, which correspond to the summation-bit and carry-bit respectively. Specifically, when the 1-bit binary numbers are From To further verify the advantages of the adder with a domain label, we simulate the double-rail 1-bit adder when the threshold concentration in the double-rail logic is at the extreme edge, as shown in In In theory, when the 1-bit binary numbers are However, the actual logic operations in The reason for the logic error in the reactions shown in The concentration of the eight domain-labeled DNA signal strands rapidly decreases to less than 0.1 times the unit concentration within the initial 60 s, and then slowly decreases to almost zero over the remaining time period; the concentration of the domain-labeled fluorescent signal chain rapidly rose to 0.9 times the unit concentration or more within 2100 s, and then it slowly rose until it was very close to a concentration of 1 unit. The domain-labeled binary DNA 4 × 4 multiplier can realize the multiplication of 4-bit binary number and 4-bit binary number. The whole reaction is very stable, which indicates that the designed domain-labeled binary DNA 4 × 4 multiplier has good stability and encapsulation, which further demonstrates that the AND gate, OR gate, NOT gate, amplifier, fan-out gate, and detection gate with the domain label have good stability and encapsulation, which lays the foundation for the realization of DNA computers.Similar to the 1-bit full adder, the 8-bit adder takes the adjacent low-order carry into account. It is constructed by 1-mapping modules, 2-mapping modules, and 3-mapping modules, and consists of eight XOR gates with a domain label, eight one-bit full adders with a domain label, 24 amplification gates with a domain label and one 9-fan out gate with a domain label. It can implement an adder or subtractor between the 8-bit binary The single logic circuit discussed below has eight inputs and eight outputs. In In The logic circuit constructed by a 1-mapping module, 2-mapping module and 3-mapping module has a lower design complexity, a shorter calculation time, and higher stability than the dual-rail logic circuit. Regarding the complexity of the design, firstly, the 1-mapping module and the 2-mapping module are combined to simulate the correctness of the one-bit full adder with the domain label, and then, the n-bit stable logic circuit is constructed. The fewer layers used, the higher the parallelism is, and the shorter the computing time is. Regarding the stability, logic values of 1 and 0 in the dual-rail logic circuit are represented by a higher concentration and a lower concentration, respectively; however, thresholding strands may not distinguish DNA signal strands with higher concentrations from DNA signal strands with lower concentrations, which introduces an error signal into the DNA reaction. Logic values of 1 and 0 in the logic circuit constructed by mapping modules are represented by the domain labels t and f. This is deterministic and does not make DNA reaction produce an error signal. In addition, the standard deviation of the computation time of the logic circuit constructed by mapping modules is far less than that of the dual-rail logic circuit, indicating that mapping modules possess stability and logic circuits constructed by them are more stable.Basic logic gates, an amplification gate, a fan-out gate, a reporter gate with a domain label (domains t and f), and N-mapping modules with domain labels were designed in this paper. The mapping modules included a 1-mapping module, a 2-mapping module, a 3-mapping module and an N-mapping module according to how many inputs they operated on. DNA logic circuits constructed with a 1-mapping module, a 2-mapping module, a 3-mapping module and an N-mapping module were shown to possess a lower design complexity, fewer layers, higher parallelism, higher stability, and shorter time complexity, which was verified through a comparison with a one-bit full adder with a domain label. A DNA 8-bit adder/subtractor was designed with mapping modules; this could be applied to design more stable and faster DNA computers in the future, so that more and more NP-complete problems can be solved with shorter time complexity.


Cyclin-dependent kinase 5 (CDK5) plays a pivotal role in neural development and neurodegeneration. CDK5 activity can be regulated by posttranslational modifications, including phosphorylation and S-nitrosylation. In this study, we demonstrate a novel mechanism by which the acetylation of CDK5 at K33 (Ac-CDK5) results in the loss of ATP binding and impaired kinase activity. We identify GCN5 and SIRT1 as critical factor controlling Ac-CDK5 levels. Ac-CDK5 achieved its lowest levels in rat fetal brains but was dramatically increased during postnatal periods. Intriguingly, nuclear Ac-CDK5 levels negatively correlated with neurite length in embryonic hippocampal neurons. Either treatment with the SIRT1 activator SRT1720 or overexpression of SIRT1 leads to increases in neurite length, whereas SIRT1 inhibitor EX527 or ectopic expression of acetyl-mimetic (K33Q) CDK5 induced the opposite effect. Furthermore, the expression of nuclear-targeted CDK5 K33Q abolished the SRT1720-induced neurite outgrowth, showing that SIRT1 positively regulates neurite outgrowth via deacetylation of nuclear CDK5. The CDK5 activity-dependent increase of neurite length was mediated by enhanced transcriptional regulation of BDNF via unknown mechanism(s). Our findings identify a novel mechanism by which acetylation-mediated regulation of nuclear CDK5 activity plays a critical role in determining neurite length in embryonic neurons. However, CDK5 kinase activity is detected only in postmitotic neurons via its association with the neuron-specific activators p35 or p395. In contrast to other CDKs that promote cell proliferation, CDK5 plays a role in regulating proper brain development, neuronal maturation and neuronal death6. For example, CDK5 is hyperactivated by the conversion of p35 to p25 by the calcium-dependent protease calpain under pathological conditions8. Dysregulation of CDK5 kinase activity is linked to an array of neurodegenerative diseases9. During early brain development, CDK5 expression and kinase activity closely correlate with the extent of neuronal differentiation11. More specifically, CDK5 is involved in axonal and dendritic growth, neuronal migration and synapse development via the phosphorylation of specific substrates in different cellular compartments15. Consequently, CDK5 knock-out (KO) or p35/p39 double-KO mice exhibit perinatal lethality characterized by cortical layering, lamination and fasciculation failure17.Cyclin-dependent kinase 5 (CDK5), a proline-directed serine/threonine kinase, is closely related to other cyclin-dependent kinases and is expressed in various tissues, although its highest level is found in the brainin vitro18. In contrast, phosphorylation of the adjacent Y15 by c-Abl, Fyn and EPHA4 leads to increased kinase activity21, although a contradicting report is available22. Interestingly, the c-Abl-induced phosphorylation of CDK5 is enhanced by Cables, a CDK5-interacting protein in the brain, and promotes neurite outgrowth in cultured cortical neurons21. Furthermore, Fyn-mediated phosphorylation of CDK5 in response to Sema3A signaling results in growth-cone collapse, suggesting that Y15 phosphorylation is potentially involved in axon and dendrite guidance20. Similarly, S-nitrosylation also plays an important role in regulating CDK5 activity24. C83 and C157 of CDK5 are S-nitrosylated and these modifications lead to kinase activity inhibition24. Consequently, mutant CDK5 lacking these cysteine residues delays dendritic development in hippocampal neurons. In contrast, another group reported an opposing outcome for the S-nitrosylation of CDK5 in Aβ1–42 and NMDA-induced dendritic spine loss23.In addition to activation by virtue of association with p35 (or its truncated form p25) and p39, post-translational modification of CDK5 itself is an additional determinant that influences kinase activity. Although CDK5 is not needed to be phosphorylated to become active, it has been reported that phosphorylation at T14 in a glycine-rich loop  of CDK5 by one or more unknown kinase down-regulates kinase activity 25. Prompted by this observation, we specifically investigated (i) whether CDK5 acetylation affects its kinase activity and (ii) whether it is involved in neuronal differentiation. Here, we found that acetylation of CDK5 at K33 inhibited its kinase activity via the loss of ATP binding. We further demonstrated that (de)acetylated CDK5 levels and concomitant kinase activity were controlled by SIRT1 in developing hippocampal neurons. Based on both pharmacological and genetic evidence, acetylated CDK5 levels inversely correlated with the extent of total neurite length and the length of the longest neurite. Transcriptional regulation of BDNF involved CDK5 kinase activity-dependent neurite extension via one or more unknown mechanism independent of MeCP2 and CREB phosphorylation. Collectively, our findings suggest a novel mechanism to regulate CDK5 activity in the nucleus and highlight the crucial role of CDK5 in determining neurite length in the developing brain.While searching for additional posttranslational modifications of CDK5, we previously found that general control non-derepressible 5  acetylates CDK5 at K3325. The K33 residue in CDK5 is highly conserved among various species and is necessary for ATP binding25. We thus speculated that the acetylation of CDK5 at K33 might alter its kinase activity. To address this question, HEK293 cells were transfected with CDK5 wild type (WT) or CDK5 mutants in which K33 was substituted with arginine  or glutamine  in combination with p35 or p25. The kinase activities of the immunoprecipitates were assessed using histone H1 (H1) protein as a CDK5 substrate. The in vitro kinase assays showed that immunoprecipitated CDK5 WT significantly phosphorylated H1 in the presence of p35 or p25 , CDK5 WT and Ac-CDK5 proteins were purified for use in in vitro kinase assays utilizing a protocol to perform site-directed acetylation in a bacterial system  familyp25 Fig. . In conttro Fig. , suggestin vivo, we considered highest efficacy of acetylation by GCN5 and a predominant expression of GCN5 over PCAF in the brain28. Therefore, we focused to test whether the expression of GCN5 altered CDK5 kinase activity. Co-expression of GCN5 plus p35 in HEK293 cells decreased CDK5 kinase activity of CDK5 by 20%  are responsible for CDK5 acetylation at K33, we transfected HEK293 cells with CDK5 plus one of the KATs  and measured K33-acetylation levels using an anti-Ac-CDK5 antibody. Although the expression levels of several KATs varied in HEK293 cells, PCAF, GCN5 and p300 KATs effectively acetylated K33 of CDK5, and GCN5 demonstrated the highest efficacy Fig. . Althoug20% Fig. . Interes20% Fig. , and thi20% Fig. . Unexpec20% Fig. . In cont20% Fig. . Again, 20% Fig. . When HE20% Fig.  but not 20% Fig.  or with 20% Fig. 29. Becauity Fig. .Figure 231. Thus, we experimentally searched for HDACs responsible for Ac-CDK5 deacetylation. First, CDK5-transfected HEK293 cells were treated with inhibitors targeting two distinct classes of HDAC: nicotinamide -dependent sirtuin family inhibitor) or trichostatin A . Nicotinamide blocked a deacetylation step performed by sirtuins and consequently increased Ac-CDK5 levels in a dose-dependent manner  and is a critical modification step for numerous histone and non-histone proteinsner Fig. . To idenner Fig. . In contner Fig. , althougner Fig. . We thenner Fig. . In in vAD+ Fig. . TreatmeAD+ Fig. . ConcomiAD+ Fig. . Similar32. GCN5 expression gradually decreased with age  and persisted until postnatal day 0 (P0) Fig. . Intriguage Fig. . Althoug17. In addition, SIRT1 is involved in axon growth and dendritic arborization via the deacetylation of its substrates34. Given the overlapping effects of SIRT1 and CDK5 on neurite outgrowth, we examined whether the pharmacological regulation of SIRT1 activity affects the extent of neurite outgrowth by regulating the kinase activity of CDK5. We chose primary cultures of rat hippocampal neurons established from E18 embryos in which several hallmarks of neurite extension are easily monitored and reproducible35. In our cultures, the extent of neurite outgrowth was augmented over 7 days in vitro (DIV)  Fig. . ImmunocIV) Fig. . Using tIV) Fig. . In contIV) Fig. . CleavagIV) Fig. .Figure 536. In the SH-SY5Y neuroblastoma cell line, RA induces cell cycle arrest and neural differentiation, and therefore it has been adopted as a simple experimental system to study molecular signaling pathways associated with neuronal differentiation37. Treatment with 10 μM RA caused dramatic neurite extension  the endogenous Ac-CDK5 is strictly accumulated in the nucleus Fig.  and (ii)40. Recent studies have demonstrated that CDK5 is involved in brain-derived neurotrophic factor (BDNF)-induced dendritic growth12 and accumulates in the nucleus upon KCl-induced depolarization of cultured neurons to increase phosphorylation of methyl-CpG-binding protein 2 (MeCP2) at S421, which is released from its repressive association with the bdnf promoter41. Thus, we tested whether bdnf expression patterns correlate with SRT1720-triggered increases in neurite length and CDK5 kinase activity. The expression of either all bdnf transcripts or bdnf mRNA containing exon IV  binding protein (CREB) at S13343, a transcriptional activator of bdnf were not affected  in embryonic hippocampal neurons  family, and the AMP-activated protein kinase (AMPK) family. Indeed, a subset of these kinases including CDK2, CDK9, p38 and salt-inducible kinase 2 (SIK2), undergoes acetylation at positions equivalent to CDK5 K3332. Supporting this notion, SIRT1 is predominantly present in the nucleus in proximity to Ac-CDK5, whereas SIRT2 preferentially resides in the cytosol, although both can shuttle between the cytosol and the nucleus under different cellular circumstances, such as during differentiation, the cell cycle or apoptosis50. Furthermore, pharmacological inhibition of SIRT1 stimulates the accumulation of Ac-CDK5 in the nuclei of hippocampal neurons. Conversely, SIRT1 activation increases neurite length in association with increased CDK5 deacetylation. This effect is abolished by either CDK5 knockdown or the overexpression of nuclear-targeted acetyl-mimetic CDK5, indicating that SIRT1-mediated deacetylation of nuclear CDK5 at K33 is essential for neurite outgrowth in differentiating neurons. Therefore, our findings reveal a crucial role for nuclear CDK5, which is regulated by its (de)acetylation status via SIRT1 activity. Interestingly, SIRT1 itself promotes neuritogenesis and dendritic arborization in differentiating neurons. For example, SIRT1 overexpression further stimulates NGF-induced neurite outgrowth in PC12 cells51, and treatment with resveratrol, a natural SIRT1 activator, promotes neuritogenesis and neurite elongation in hippocampal neurons34. The mechanism underlying SIRT1-mediated neurogenesis involve protein kinase signaling pathways such as Akt, Rho-associated protein kinase (ROCK) and mammalian target of rapamycin (mTOR)52. Thus, SIRT1 contributes to normal neurogenesis by modulating differential phosphorylating activities of a subset of kinases, although further investigation is required to determine whether deacetylation of these kinases occurs directly via SIRT1.We consider SIRT1, but not SIRT2, an upstream deacetylase of CDK5 because SIRT1 expression levels are inversely correlated with CDK5 acetylation levels in developing brain, although SIRT2 also effectively deacetylates CDK5 in HEK293 cells. In contrast, our data and a previous study revealed that SIRT2 is maintained at lower levels during development and exponentially accumulates after the juvenile phase bdnf, a neurotrophic factor that activates TrkB-PI3K/MAPK cascade in a cell-non-autonomous fashion39. The increased bdnf transcript levels are dramatically abolished by co-treatment with the CDK5 inhibitor roscovitine, suggesting that CDK5 functions downstream of SIRT1 to induce bdnf transcription. Although we could not detect a correlation between changes in phosphorylated MeCP2 or CREB levels and altered bdnf transcription, other cis-elements on the bdnf exon IV promoter are able to recognize transcription factors. These cis-elements include calcium responsive factor (CaRF), upstream stimulatory factor (USF) 1/2, nuclear factor κB (NF-κb) and NFATc454. However, we do not exclude the possibility that bdnf mRNA may be regulated in a transcription independent manner including an alteration of its stability or editing55. Therefore, it would be of great interest to identify transcriptional regulators responsive to CDK5 activation and its associated pathways and determine whether SIRT1-mediated nuclear CDK5 deacetylation and the subsequent phosphorylation of one or more unidentified factor participate in neurite outgrowth via autocrine or paracrine actions of bdnf.How does SIRT1-activated nuclear CDK5 affect neurite outgrowth? SRT1720-mediated neurite outgrowth is accompanied by increased mRNA levels of 56. SIRT1 is recruited to DNA double strand break sites and is required to stabilize HDAC1 and activate ATM autophosphorylation at S1981 to ameliorate DNA damage in cultured neurons57. Intriguingly, CDK5 likely participates in this process because CDK5-mediated ATM phosphorylation at S794 precedes and is crucial for ATM autophosphorylation upon DNA damage58. It would be of great interest to investigate whether CDK5 (de)acetylation is involved in SIRT1/2-mediated signaling in various neurodegenerative diseases characterized by aberrant CDK5 activation8, and to determine whether the modulation of acetylated CDK5 levels is a viable therapeutic target.Because acetylation directly targets the critical residue for ATP binding to CDK5, modified CDK5 is fully “locked” to prevent activation, even after p35 is cleaved into the potent activator p25 when neurons are exposed to pathological conditions. Given its temporal expression pattern, SIRT2 may be the major deacetylating enzyme for CDK5 in aged brains. Furthermore, Ac-CDK5 in aged brains is maintained at substantially higher levels than in embryonic brains, indicating that acetylation may be critical to regulate CDK5 and avoid excessive activation in the nucleus. Although it is unclear whether nuclear SIRT2 levels are sufficient to affect CDK5 acetylation status under these conditions, it would be intriguing to explore whether the aberrant activation of CDK5 in aging-related neuronal disorders is modulated by the inhibition of SIRT2 activity. In this context, SIRT2 inhibition exerts beneficial effects on neurodegeneration. For example, the SIRT2 inhibitor AGK2 effectively rescues neurons from α-synuclein-induced neurotoxicity in Parkinson’s disease modelsIn conclusion, our findings provide evidence supporting a novel regulatory mechanism for nuclear CDK5 activity via (de)acetylation and the emerging role of SIRT1 as a determinant in this process. Given the broad spectrum of CDK5 action in neurons under physiological and pathological conditions, our findings lay the foundation to further understand brain development and the etiology of neurological disorders.2 atmosphere. For transient transfections, predetermined amounts of a eukaryotic expression plasmid were used to transfect HEK293 cells in combination with polyethylenimine  as previously described25. Twenty-four hours after transfection, cells were treated with a pharmacological reagent for an additional 24 hrs. These reagents included nicotinamide , trichostatin A , SRT1720 , EX-527  and AGK2 . Primary cultures of hippocampal or cortical neurons were prepared from hippocampi or cerebral cortices derived form E17-18 Sprague-Dawley (SD) rat embryos as previously described with certain modifications59. Briefly, dissociated neurons were plated at a density of 5 × 103 cells/cm2 on 100 μg/ml poly-D-lysine  and 1 μg/ml laminin -coated glass coverslips in MEM supplemented with 0.6% D-glucose , 1 mM sodium pyruvate , 2 mM L-glutamine , 100 U/ml penicillin-streptomycin  and 10% FBS . Twenty-four hours after plating, the medium was changed to a neurobasal medium  supplemented with 2% B27  and 0.5 mM L-glutamine. Phase-contrast photomicrographs were captured with a Zeiss Axiovert 100 microscope (Carl Zeiss). Hippocampal neurons at DIV3 were treated with SRT1720 or Ex527 for 48 hrs. For transient expression studies, hippocampal neurons at DIV3 were transfected with the indicated plasmids using Lipofectamine 2000  according to the manufacturer’s protocol. All experimental animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committees of Yonsei University.Human embryonic kidney 293 (HEK293) cells originally purchased from the ATCC were routinely tested for mycoplasma contamination and independently maintained in the laboratory. HEK293 cells were cultivated in DMEM  supplemented with 10% heat-inactivated FBS  at 37 °C in a 5% CObdnf was purchased from Origene (#SR510549) and transfected into neurons using RNAiMAX  as recommended by the manufacturer’s protocol.To carry out the epitope-tagging of desired proteins, FLAG, HA and Myc sequences were inserted into the Nhe1 site of a pCI-neo mammalian expression vector . Mouse CDK5, p25 and p35 sequences were generated by PCR. All constructs harboring CDK5, p35 or p25 vectors were generated via subcloning into one of these modified vectors. The K33R and K33Q mutants of mouse CDK5 were generated using a site-directed mutagenesis kit . DsRed-tagged mouse CDK5 WT and its mutants were constructed by inserting PCR products into the pDsRed2-N1 vector . FLAG-tagged human PCAF and HA-tagged human HBO1 were provided by Dr. Hyunsook Lee of Seoul National University and by Dr. Jacques Côte of Laval University, respectively. Protein S-FLAG-streptavidin binding protein (SFP)-tagged human SIRT1 and SIRT6 were kindly provided by Dr. Ja-Eun Kim of Kyunghee University. All other plasmids containing KATs and SIRTs were purchased from Addgene (Cambridge). GFP-tagged human GCN5 was constructed by introducing the appropriate PCR product into the pAcGFP-C1 vector . Nuclear localization signal (NLS) -tagged CDK5 WT and K33Q, and nuclear export signal (NES)-tagged CDK5 K33Q were generated by subcloning into the pEF/myc/nuc/GFP vectors . For knockdown experiments, shRNAs against rat CDK5 were cloned into pRNAT-U6.1/Neo . The target sequences are: shCDK5#1, 5′-gaagctcacattggtgtttga-3′; shCDK5#2, 5′-gaatctgctcattaacaggaa-3′. siRNA against rat 26. The pCDF-PylT and pBK-AcKRS-3 vectors were generously provided by Dr. Jason W. Chin of the University of Cambridge. Briefly, E. coli was transformed with the pBK-AcRS3 vector encoding acetyl-lysyl-tRNA synthetase, and the pCDF-PylT vector harboring His-tagged mouse CDK5 mutated into the amber codon (TAG) at the K33 site and tRNACUA. The native stop codon (TAG) of CDK5 was also mutated into TAA to avoid undesired acetylation. After IPTG induction, acetyl-lysine was incorporated into the K33 site to replace native lysine via recognition of the mutated amber codon by tRNACUA. Nicotinamide (NA) was added to the culture to inhibit the endogenous bacterial deacetylase CobB. Recombinant His-tagged Ac-CDK5 and -CDK5 were purified with Ni-NTA beads, and the eluted protein was concentrated in 50 mM Tris (pH 7.5), 150 mM NaCl, 5% glycerol, 20 mM NA, and a protease inhibitor cocktail  using an Amicon® Ultra-0.5 ml centrifugal filter . To purify the His-tagged p25 protein, mouse p25 was cloned into a pET-28a(+) vector  and purified as described above. All proteins were stored at −80 °C until use.To produce a His-tagged recombinant CDK5 protein acetylated at K33 (abbreviated as Ac-CDK5 below), a method to genetically apply site-specific acetylation on recombinant proteins was performed as previously described RVRLD, which corresponds to sequences surrounding the K33 residue within human CDK5. The antibody production process was performed by Abmart . Antibody specificity was determined as described in Supplementary Fig. 25. To prepare tissue lysates, male rats of different ages were obtained from Orient  and euthanized in a CO2 chamber. Rat cerebral cortices were rapidly isolated and homogenized via sonication in RIPA buffer containing PIC. For these experiments, rats were randomized and investigators were not blinded. The nuclear fraction was prepared according to a protocol provided by Abcam. The following commercially available antibodies were used for immunoblot analysis and immunoprecipitation: mouse monoclonal anti-FLAG-peroxidase ; anti-hemagglutinin (HA)-peroxidase ; anti-V5-peroxidase ; anti-GAPDH antibody ; anti-GCN5 ; and rabbit polyclonal anti-acetylated-lysine . All other antibodies were purchased from Santa Cruz Biotechnology, including rabbit polyclonal anti-CDK5 , anti-His probe , anti-SIRT1 , anti-SIRT2 , anti-p35 , mouse monoclonal anti-CDK5 , anti-CHIP , and goat polyclonal anti-SOD-1  and anti-Lamin A/C . Rabbit polyclonal anti-MeCP2 , anti-phospho-MeCP2 (S421) , anti-phospho-MeCP2 (S80) , rabbit monoclonal anti-CREB  and anti-phospho-CREB (S133)  were also used. Mouse monoclonal antibodies against for total tau  and paired helical filaments  were kindly provided by Dr. Virginia M. Lee at University of Pennsylvania. Rabbit polyclonal antibody for phospho-CHIP (Ser20) was produced by Abmart60. Membranes were incubated at RT for 1 hr with HRP-conjugated goat anti-rabbit , goat anti-mouse  or donkey anti-goat antibody IgG . For immunoprecipitation, protein (1–2 mg) was pre-incubated with protein A agarose beads  for pre-clearance and then further incubated with 1–2 µg of corresponding antibodies or 10 µl of anti-Flag M2 affinity gel beads  in RIPA buffer at 4 °C overnight. Immunocomplexes were collected with protein A agarose beads followed by centrifugation at 800 × g at 4 °C for 2 min. Proteins were eluted from the beads with 1 × protein sample buffer, denatured by boiling, separated by 11.5% SDS-PAGE and subjected to immunoblot analysis employing corresponding antibodies. Specific bands were detected with enhanced chemiluminescence . Band intensity was quantified using Image-J software provided by the National Institutes of Health (NIH). Original images of the uncropped blots are shown in the Supplementary material.Cellular lysate preparation and immunoblot analysis were performed as describedImmunofluorescence microscopy was performed with rabbit polyclonal anti-Ac-CDK5 (1:200), anti-neuron specific beta-III-tubulin , and mouse monoclonal anti-NeuN  antibodies. The following secondary antibodies were used: Alexa-488-conjugated goat anti-rabbit IgG  or anti-mouse IgG  and Alexa-594-conjugated goat anti-rabbit IgG  or anti-mouse IgG . Nuclei were counterstained with 2 μg/ml Hoechst 33258 . Glass coverslips were mounted on methanol-soaked glass slides with Vectashield mounting medium . Fluorescence images were observed under an LSM 700 confocal microscope equipped with epifluorescence and LSM image browser (Carl Zeiss).Purified His-CDK5 WT or His-Ac-CDK5 (10 μg) diluted in PBS containing PIC and 10 mM NA was incubated with 30 μl of Ni-NTA resin  at 4 °C overnight. Cell lysates (2 mg) transfected with p35 or p25-FLAG were pre-incubated with 30 μl of Ni-NTA resin at 4 °C for 2 hrs to avoid non-specific protein binding. The pre-cleared cell lysates were then incubated with His-CDK5-bound Ni-NTA resin at 4 °C overnight under gentle rotation. CDK5/p35 or p25 complexes were washed with PBS and subjected to SDS-PAGE followed by immunoblotting with antibodies targeting FLAG, His and Ac-CDK5.In vitro phosphorylation assays were performed as previously described60. Briefly, purified recombinant His-CDK5 WT or His-Ac-CDK5 (1 µg) was incubated at 30 °C for 30 min with 1 µg of histone H1 protein  and 1 µg of p25-His in 1 × kinase buffer  in the presence of 5 µCi [γ-32P]ATP or cold ATP. Phospho-H1 signals were visualized by autoradiography or immunoblotting with a rabbit polyclonal anti-phospho-histone H1 antibody . Transiently transfected HEK293 cells and hippocampal neurons were lysed, immunoprecipitated with anti-CDK5 antibody and processed for in vitro phosphorylation assays. To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose  as previously described61. For the in vitro deacetylation assay, immunoprecipitated FLAG-tagged SIRT1 or SIRT2 was incubated with 0.5 µg of purified recombinant Ac-CDK5 supplemented with 5 mM β-nicotinamide adenine dinucleotide  at 30 °C for 2 hrs as previously described62.65, purified CDK5 proteins (500 ng) were incubated with varying concentration of mant-ATP (2′/3′-O-(N-methylanthraniloyl)-adenosine-5′-triphosphate, triethylammonium salt, Jena Bioscience, #NU-202) in a binding buffer  at 37 °C. Mant-ATP binding to either CDK5 WT or Ac-CDK5 was monitored by following fluorescence at λex 355/λem 460 nm (threshold λ455 nm) on a FlexStation 3 multi-mode microplate reader (Molecular Devices). Nonlinear regression was performed to obtain a best-fit curve for a specific binding [Y = Bmax*X/(Kd + X)] to the fluorescence raw data using GraphPad Prism 7 . Representative data were presented as the mean ± SEM from at least three to four independent biological replicate experiments with triplicate measurements. For testing the differences in specific binding to mant-ATP between CDK5 WT and Ac-CDK5, Wilcoxon matched-pairs rank test was employed, unless otherwise noted. The effectiveness of pairing was also analyzed by calculating nonparametric Spearman correlation coefficient. Data from CDK5 binding experiments using mant-ADP as well as mant-ATP were evaluated by one-way analysis of variance (Friedman test) with Dunn’s post-hoc analysis for multiple comparisons. Statistical calculations were carried out using Prism 7 (GraphPad). P < 0.05 was considered statistically significant.For fluorescence measurements66, the ratio of total neurite length or the length of longest neurite to soma size was determined to minimize the effects of soma size on neurite length. The representative experimental data were provided from at least 2–3 independent experiments performed.To evaluate neurite outgrowth in hippocampal neurons, more than 35 neurons were randomly selected from a minimum of 3–5 glass coverslips per experiment and imaged by Z-stacking by an individual who was blind to the treatment conditions. After image processing using LSM image browser, fluorescent signals from beta-III-tubulin, GFP- or Ds-Red-containing vectors were measured to assess total neurite length, the length of the longest neurite, the number of primary neurites and soma size using Image-J software. Only processes longer than the soma size were considered neurites. The number of primary neurites was defined as the number of neurite segments originating directly from the soma. As previously described7 cells per P-150 dish were treated at DIV 3 with 100 nM SRT1720 for 24 hrs followed by 10 μM roscovitine for an additional 24 hrs. As directed by the protocol provided by Santa Cruz Biotechnology, DNA-protein complexes were crosslinked, quenched, lysed and then immunoprecipitated with 5 μl of anti-MeCP2 antibody or rabbit IgG at 4 °C overnight, followed by incubation with 20 μl of protein A agarose for an additional 2 hrs. MeCP2/DNA complexes were eluted in 300 μl of elution buffer  at 65 °C overnight, reverse cross-linked by adding 5 μl of RNase A  and incubated for 4 hrs. The following PCR primers were used for the ChIP assay: BDNF promoter (exon IV) sense 5′- GCGCGGAATTCTGATTCTGGTAAT-3′, and antisense 5′-GAGAGGGCTCCACGCTGCCTTGACG-3′.Real-time PCR was performed using a SensiFAST SYBR Hi-Rox kit  according to the manufacturer’s protocol. The following oligonucleotide primers were used for real-time PCR: rat BDNF  sense, 5′-TTGAGCACGTGATCGAAGAG-3′, and antisense, 5′-CCAGCAGAAAGAGCAGAGGA-3′; rat BDNF (exon IV) sense, 5′-CTGCCTTGATGTTTACTTTGACAAG-3′, and antisense, 5′-ACCATAGTAAGGAAAAGGATGGTCAT-3′; rat GAPDH sense, 5′-GACATGCCGCCTGGAGAAAC-3′, and antisense, 5′-AGCCCAGGATGCCCTTTAGT-3′. For the ChIP assay, rat primary cortical neurons plated at 2 × 10t-tests were also used when necessary . Two-tailed Student’s ary Figs  and 8. VSupplementary information


After publication of abstract S 2‑06 in supplement , it was S 2‑06 Crystal structure of PKG Iβ holoenzyme reveals a trans‑inhibiting dimer assembly1,2, Rajesh Sharma1, Darren E. Casteel3Choel Kim1Baylor College of Medicine, Pharmacology and Chemical Biology, Houston, TX, USA; 2Baylor College of Medicine, Biochemistry and Molecular Biology, Houston, TX, USA; 3University of California, San Diego, Medicine, La Jolla, CA, USACorrespondence: Choel Kim - ckim@bcm.eduJournal of Translational Medicine 2019, 17(2): S2-06Introduction: As the major molecular switch for regulation of smooth muscle/vascular tone and nociception, mammalian cGMP dependent protein kinase I is a promising therapeutic target for hypertensive diseases and chronic pain. The lack of structural information on the PKG holoenzyme has hindered a detailed understanding of its regulation, though the holoenzyme structure for cAMP dependent protein kinase (PKA) suggests plausible models for PKG regulatory (R) and catalytic (C) domain interactions in the inhibited state.Methods: We determined a crystal structure of PKG Iβ holoenzyme complex at 2.3 Å that enables us to visualize the R–C interface of PKG Iβ of the inhibited state for the first time. Results: We crystallized a monomeric PKG Iβ that lacks the dimerization domain, but the asymmetric unit of the crystal contains a twofold symmetric dimer. The interfaces formed between PKG R and C domains are similar to those seen in the PKA Iα holoenzyme with the inhibitor sequence docked to the active site cleft. However, the overall topology unexpectedly reveals that the R domain of each PKG monomer binds the C domain of the other monomer, giving rise to inhibition in trans (Fig. ans Fig. .Fig. 1CrConclusions: In the light of previous PKG structural biology, this structure suggests that the PKG inhibited and activated states are stabilized by mutually exclusive domain–domain contacts that either occlude or expose the active site. Our previous structure of the activated state of the isolated PKG Iβ regulatory domain [y domain  identifiy domain –5 sugges