Molecular insights into plant cell proliferation disturbance by Agrobacterium protein 6b.

The Agrobacterium Ti plasmid (T-DNA) 6b proteins interact with many different host proteins implicated in plant cell proliferation. Here, we show that Arabidopsis plants overexpressing 6b display microRNA (miRNA) deficiency by directly targeting SERRATE and AGO1 via a specific loop fragment (residues 40-55). In addition, we report the crystal structures of Agrobacterium tumefaciens AK6b at 2.1 Å, Agrobacterium vitis AB6b at 1.65 Å, and Arabidopsis ADP ribosylation factor (ARF) at 1.8 Å. The 6b structure adopts an ADP-ribosylating toxin fold closely related to cholera toxin. In vitro ADP ribosylation analysis demonstrates that 6b represents a new toxin family, with Tyr 66, Thr 93, and Tyr 153 as the ADP ribosylation catalytic residues in the presence of Arabidopsis ARF and GTP. Our work provides molecular insights, suggesting that 6b regulates plant cell growth by the disturbance of the miRNA pathway through its ADP ribosylation activity.

Autoantigen La Regulates MicroRNA Processing from Stem-Loop Precursors by Association with DGCR8.

In humans, primary microRNA (pri-miRNA) processing starts from precise cleavage of the stem loop, which is catalyzed by the Drosha-DGCR8 complex. However, the significant inconsistencies in the expression levels among primary, precursor, and mature miRNAs clearly indicate that many other factors may be involved in this regulation. Here, we utilize a newly developed RNA affinity technique to isolate such factors. In this study, a tRNA-scaffolded aptamer (tRSA)-based RNA affinity tag, by directly fusing primary let-7 miRNA to the 3'-end of tRSA, is employed to pull down the protein components specifically binding to pri-let-7. We show that La protein binds to pri-let-7 via its La motif and significantly promotes the processing efficiency of pri-let-7 in vitro and in cells. In addition, we demonstrate that La protein is associated with DGCR8, but not Drosha, in an RNA-dependent manner. Interestingly, the RNA binding capacity of La motif is important for miRNA processing. Hence, we propose that La protein is an important microprocessor component regulating miRNA processing efficiency by association with DGCR8 to regulate formation of the DGCR8-Drosha complex for miRNA processing.

Prolonged suppression of HBV in mice by a novel antibody that targets a unique epitope on hepatitis B surface antigen.

OBJECTIVE: This study aimed to investigate the therapeutic potential of monoclonal antibody (mAb) against HBV as a novel treatment approach to chronic hepatitis B (CHB) in mouse models. METHODS: Therapeutic effects of mAbs against various epitopes on viral surface protein were evaluated in mice mimicking persistent HBV infection. The immunological mechanisms of mAb-mediated viral clearance were systematically investigated. RESULTS: Among 11 tested mAbs, a novel mAb E6F6 exhibited the most striking therapeutic effects in several HBV-persistent mice. Single-dose administration of E6F6 could profoundly suppress the levels of hepatitis B surface antigen (HBsAg) and HBV DNA for several weeks in HBV-transgenic mice. E6F6 regimen efficiently prevented initial HBV infection, and reduced viral dissemination from infected hepatocytes in human-liver-chimeric mice. E6F6-based immunotherapy facilitated the restoration of anti-HBV T-cell response in hydrodynamic injection (HDI)-based HBV carrier mice. Immunological analyses suggested that the Fcγ receptor-dependent phagocytosis plays a predominant role in E6F6-mediated viral suppression. Molecular analyses suggested that E6F6 recognises an evolutionarily conserved epitope (GPCK(R)TCT) and only forms a smaller antibody-viral particle immune complex with limited interparticle crosslinking when it binds to viral particles. This unique binding characteristic of E6F6 to HBV was possibly associated with its effective in vivo opsonophagocytosis for viral clearance. CONCLUSIONS: These results provided new insight into understanding the therapeutic role and mechanism of antibody against persistent viral infection. The E6F6-like mAbs may provide a novel immunotherapeutic agent against human chronic HBV infection.

Large-scale production of foot-and-mouth disease virus (serotype Asia1) VLP vaccine in Escherichia coli and protection potency evaluation in cattle.

BACKGROUND: Foot-and-mouth disease (FMD) is an acute, highly contagious disease that infects cloven-hoofed animals. Vaccination is an effective means of preventing and controlling FMD. Compared to conventional inactivated FMDV vaccines, the format of FMDV virus-like particles (VLPs) as a non-replicating particulate vaccine candidate is a promising alternative. RESULTS: In this study, we have developed a co-expression system in E. coli, which drove the expression of FMDV capsid proteins (VP0, VP1, and VP3) in tandem by a single plasmid. The co-expressed FMDV capsid proteins (VP0, VP1, and VP3) were produced in large scale by fermentation at 10 L scale and the chromatographic purified capsid proteins were auto-assembled as VLPs in vitro. Cattle vaccinated with a single dose of the subunit vaccine, comprising in vitro assembled FMDV VLP and adjuvant, developed FMDV-specific antibody response (ELISA antibodies and neutralizing antibodies) with the persistent period of 6 months. Moreover, cattle vaccinated with the subunit vaccine showed the high protection potency with the 50 % bovine protective dose (PD50) reaching 11.75 PD50 per dose. CONCLUSIONS: Our data strongly suggest that in vitro assembled recombinant FMDV VLPs produced from E. coli could function as a potent FMDV vaccine candidate against FMDV Asia1 infection. Furthermore, the robust protein expression and purification approaches described here could lead to the development of industrial level large-scale production of E. coli-based VLPs against FMDV infections with different serotypes.

Structural insights into specific crRNA G-rich sequence binding by Meiothermus ruber Cse2.

CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)-mediated defense against invading nucleic acids is a process recently discovered in prokaryotes, which includes recognition and incorporation of invading genetic elements, transcription and processing of CRISPR-RNA (crRNA) and targeting the invaders through base pair recognition. In the type I-E CRISPR-Cas system, Cse2 is proposed to provide a platform to facilitate the targeting of the invading dsDNA by crRNA. Here we report the crystal structure of Meiothermus ruber Cse2 at 2.8Å. M. ruber Cse2 adopts an α-helical bundle scaffold, harbors a positive surface for nucleic acid binding and a conserved dimer interface with strikingly low buried surface area. M. ruber Cse2 selectively binds to G-rich crRNA sequence, which is stripped off from the Cse2-crRNA and Cascade-crRNA complexes by ssDNA or dsDNA with complementary sequence. Stable M. ruber Cascade is readily formed by co-expression of M. ruber Cascade proteins together with G-rich crRNA in vitro. Docking of M. ruber Cse2 structures into the Escherichia coli Cascade Cryo-EM envelope reveals a curved elongated shallow groove for ssRNA binding, which adopts a similar dimer interface discovered by high-resolution crystal structure of Cse2 within E. Coli Cascade. Taken together, our data provides the structural insights into crRNA G-rich sequence recognition by M. ruber Cse2 and reveals the potential structural mechanism for M. ruber Cascade assembly and function.

Structure, mechanism, and enantioselectivity shifting of lipase LipK107 with a simple way.

Because of the complex mechanisms of enzymatic reactions, no precise and simple method of understanding and controlling the chiral selectivity of enzymes has been developed. However, structure-based rational design is a powerful approach to engineering enzymes with desired catalytic activities. In this work, a simple, structure-based, large-scale in silico design and virtual screening strategy was developed and successfully applied to enzyme engineering. We first performed protein crystallization and X-ray diffraction to determine the structure of lipase LipK107, which is a novel family I.1 lipase displaying activity for both R and S isomers in chiral resolution reactions. The catalytic mechanism of family I.1, which includes LipK107, was ascertained first through comparisons of the sequences and structures of lipases from other families. The binding states of LipK107, including the energy and the conformation of complexes with the R and S enantiomers, have been evaluated by careful biocomputation to figure out the reason for the higher S selectivity. Based on this study, a simple strategy for manipulating the chiral selectivity by modulating a crucial distance in the enzyme-substrate complex and judging virtual mutations in silico is recommended. Then, a novel electrostatic interaction analysis protocol was used to design LipK107 mutants to validate our strategy. Both positive and negative mutations determined using this theoretical protocol have been implemented in wet experiments and were proved to produce the desired enantioselectivity, showing a 176% increase or 50% decrease in enantioselectivity as desired. Because of its accuracy and versatility, the strategy is promising for practical applications.

An intracellular membrane junction consisting of flagellum adhesion glycoproteins links flagellum biogenesis to cell morphogenesis in Trypanosoma brucei.

African trypanosomes have a single, membrane-bounded flagellum that is attached to the cell cortex by membrane adhesion proteins and an intracellular flagellum attachment zone (FAZ) complex. The coordinated assembly of flagellum and FAZ, during the cell cycle and the life cycle development, plays a pivotal role in organelle positioning, cell division and cell morphogenesis. To understand how the flagellum and FAZ assembly are coordinated, we examined the domain organization of the flagellum adhesion protein 1 (FLA1), a glycosylated, transmembrane protein essential for flagellum attachment and cell division. By immunoprecipitation of a FLA1-truncation mutant that mislocalized to the flagellum, a novel FLA1-binding protein (FLA1BP) was identified in procyclic Trypanosoma brucei. The interaction between FLA1 on the cell membrane and FLA1BP on the flagellum membrane acts like a molecular zipper, joining flagellum membrane to cell membrane and linking flagellum biogenesis to FAZ elongation. By coordinating flagellum and FAZ assembly during the cell cycle, morphology information is transmitted from the flagellum to the cell body.

Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease.

In prokaryotes, the CRISPR/Cas system is known to target and degrade invading phages and foreign genetic elements upon subsequent infection. However, the structure and function of many Cas proteins remain largely unknown, due to the high diversity of Cas proteins. Here we report 3 crystal structures of Archaeoglobus fulgidus Csx3 (AfCsx3) in free form, in complex with manganese ions and in complex with a single-stranded RNA (ssRNA) fragment, respectively. AfCsx3 harbors a ferredoxin-like fold and forms dimer both in the crystal and in solution. Our structure-based biochemical analysis demonstrates that the RNA binding sites and cleavage sites are located at 2 separate surfaces within the AfCsx3 dimer, suggesting a model to bind, tether and cleave the incoming RNA substrate. In addition, AfCsx3 displays robust 3'-deadenylase activity in the presence of manganese ions, which strongly suggests that AfCsx3 functions as a deadenylation exonuclease. Taken together, our results indicate that AfCsx3 is a Cas protein involved in RNA deadenylation and provide a framework for understanding the role of AfCsx3 in the Type III-B CRISPR/Cas system.

Structural insights into stereospecific reduction of α, ß-unsaturated carbonyl substrates by old yellow enzyme from Gluconobacter oxydans.

We report the crystal structure of old yellow enzyme (OYE) family protein Gox0502 (a.a 1-315) in free form at 3.3 Å. Detailed structural analysis revealed the key residues involved in stereospecific determination of Gox0502, such as Trp66 and Trp100. Structure-based computational analysis suggested the bulky side chains of these tryptophan residues may play important roles in product stereoselectivity. The introduction of Ile or Phe or Tyr mutation significantly reduced the product diastereoselectivity. We hypothesized that less bulky side chains at these critical residues could create additional free space to accommodate intermediates with different conformations. Notably, the introduction of Phe mutation at residue Trp100 increased catalytic activity compared to wild-type Gox0502 toward a set of substrates tested, which suggests that a less bulky Phe side chain at residue W100F may facilitate product release. Therefore, Gox0502 structure could provide useful information to generate desirable OYEs suitable for biotechnological applications in industry.

Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9).

Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.

Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp. PCC6803.

Nitrilases are enzymes widely expressed in prokaryotes and eukaryotes that utilize a Cys­Glu­Lys catalytic triad to hydrolyze non-peptide carbon­nitrogen bonds. Nitrilase from Syechocystis sp. Strain PCC6803 (Nit6803) shows hydrolysis activity towards a broad substrate spectrum, ranging from mononitriles to dinitriles and from aromatic nitriles to aliphatic nitriles. Yet, the structural principle of the substrate specificity of this nitrilase is still unknown. We report the crystal structure of Nit6803 at 3.1 Å resolution and propose a structural mechanism of substrate selection. Our mutagenesis data exhibited that the aromaticity of the amino acid at position 146 of Nit6803 is absolutely required for its nitrilase activity towards any substrates tested. Moreover, molecular docking and dynamic simulation analysis indicated that the distance between the sulfhydryl group of the catalytic cysteine residue and the cyano carbon of the substrate plays a crucial role in determining the nitrilase catalytic activity of Nit6803 and its mutants towards different nitrile substrates.

Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans.

Gox2253 from Gluconobacter oxydans belongs to the short-chain dehydrogenases/reductases family, and catalyzes the reduction of heptanal, octanal, nonanal, and decanal with NADPH. To develop a robust working platform to engineer novel G. oxydans oxidoreductases with designed coenzyme preference, we adopted a structure based rational design strategy using computational predictions that considers the number of hydrogen bonds formed between enzyme and docked coenzyme. We report the crystal structure of Gox2253 at 2.6 Å resolution, ternary models of Gox2253 mutants in complex with NADH/short-chain aldehydes, and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between 2'-hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253 mutant after arginine at position 42 is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253-R42Y/K mutants can use both NADH and NADPH as a coenzyme. Hence, the strategies here could provide a practical platform to engineer coenzyme selectivity for any given oxidoreductase and could serve as an additional consideration to engineer substrate-binding pockets.

The archaeal COG1901/DUF358 SPOUT-methyltransferase members, together with pseudouridine synthase Pus10, catalyze the formation of 1-methylpseudouridine at position 54 of tRNA.

The methylation of pseudouridine (Ψ) at position 54 of tRNA, producing m(1)Ψ, is a hallmark of many archaeal species, but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m(1)Ψ was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m(1)Ψ minus phenotype of the ΔHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TΨ-arm (17-mer) fragments as substrates, the sequential pathway of m(1)Ψ54 formation in Archaea was reconstituted. The methylation reaction is AdoMet dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TΨ-loop. The presence of Ψ55 allowed the efficient conversion of Ψ54 to m(1)Ψ54, whereas in the presence of C55, the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins.

Structural implications into dsRNA binding and RNA silencing suppression by NS3 protein of Rice Hoja Blanca Tenuivirus.

Rice Hoja Blanca Tenuivirus (RHBV), a negative strand RNA virus, has been identified to infect rice and is widely transmitted by the insect vector. NS3 protein encoded by RHBV RNA3 was reported to be a potent RNAi suppressor to counterdefense RNA silencing in plants, insect cells, and mammalian cells. Here, we report the crystal structure of the N-terminal domain of RHBV NS3 (residues 21-114) at 2.0 Å. RHBV NS3 N-terminal domain forms a dimer by two pairs of α-helices in an anti-parallel mode, with one surface harboring a shallow groove at the dimension of 20 Å × 30 Å for putative dsRNA binding. In vitro RNA binding assay and RNA silencing suppression assay have demonstrated that the structural conserved residues located along this shallow groove, such as Arg50, His51, Lys77, and His85, participate in dsRNA binding and RNA silencing suppression. Our results provide the initial structural implications in understanding the RNAi suppression mechanism by RHBV NS3.

Crystal structure of Bombyx mori nucleopolyhedrovirus ORF75 reveals a pseudo-dimer of thiol oxidase domains with a putative substrate-binding pocket.

Bombyx mori nucleopolyhedrovirus (BmNPV) triggers the global shutdown of host silkworm gene expression and protein synthesis approximately 12-18 h post-infection. Genome sequence analysis suggests that BmNPV ORF75 could be a flavin adenine dinucleotide (FAD)-linked thiol oxidase essential for virion assembly and virus propagation. Here, we report the crystal structure of BmNPV ORF75 at 2.1 Å (0.21 nm). The structure of BmNPV ORF75 resembles that of the thiol oxidase domain of human quiescin thiol oxidase (QSOX), displaying a pseudo-dimer of canonical and non-canonical thiol oxidase domains. However, BmNPV ORF75 is further dimerized by its C-terminal canonical thiol oxidase domain. Within the unique quaternary structural arrangement, the FAD-binding pocket and the characteristic CXXC motif from each monomer is 35 Å (3.5 nm) away from that of its corresponding molecule, which suggests that BmNPV ORF75 might adopt a deviant mechanism from that of QSOX to catalyse disulfide bond formation. Our thiol oxidase activity assay on the point mutations of the conserved residues participating in FAD recognition reveals an aromatic cage next to the FAD isoalloxazine moiety for substrate binding. These data suggest that the thiol oxidase activity of BmNPV ORF75 could be critical to catalyse the formation of the disulfide bonds of certain BmNPV proteins essential for BmNPV virion assembly.

Structure of the Arabidopsis thaliana DCL4 DUF283 domain reveals a noncanonical double-stranded RNA-binding fold for protein-protein interaction.

Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) or small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of the unique DUF283 domain within dicer is largely unknown. Here we report the first structure of the DUF283 domain from the Arabidopsis thaliana DCL4. The DUF283 domain adopts an alpha-beta-beta-beta-alpha topology and resembles the structural similarity to the double-stranded RNA-binding domain. Notably, the N-terminal alpha helix of DUF283 runs cross over the C-terminal alpha helix orthogonally, therefore, N- and C-termini of DUF283 are in close proximity. Biochemical analysis shows that the DUF283 domain of DCL4 displays weak dsRNA binding affinity and specifically binds to double-stranded RNA-binding domain 1 (dsRBD1) of Arabidopsis DRB4, whereas the DUF283 domain of DCL1 specifically binds to dsRBD2 of Arabidopsis HYL1. These data suggest a potential functional role of the Arabidopsis DUF283 domain in target selection in small RNA processing.

Efficient inhibition of HIV-1 replication by an artificial polycistronic miRNA construct.

BACKGROUND: RNA interference (RNAi) has been used as a promising approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication for both in vitro and in vivo animal models. However, HIV-1 escape mutants after RNAi treatment have been reported. Expressing multiple small interfering RNAs (siRNAs) against conserved viral sequences can serve as a genetic barrier for viral escape, and optimization of the efficiency of this process was the aim of this study. RESULTS: An artificial polycistronic transcript driven by a CMV promoter was designed to inhibit HIV-1 replication. The artificial polycistronic transcript contained two pre-miR-30a backbones and one pre-miR-155 backbone, which are linked by a sequence derived from antisense RNA sequence targeting the HIV-1 env gene. Our results demonstrated that this artificial polycistronic transcript simultaneously expresses three anti-HIV siRNAs and efficiently inhibits HIV-1 replication. In addition, the biosafety of MT-4 cells expressing this polycistronic miRNA transcript was evaluated, and no apparent impacts on cell proliferation rate, interferon response, and interruption of native miRNA processing were observed. CONCLUSIONS: The strategy described here to generate an artificial polycistronic transcript to inhibit viral replication provided an opportunity to select and optimize many factors to yield highly efficient constructs expressing multiple siRNAs against viral infection.

A structural perspective of the protein-RNA interactions involved in virus-induced RNA silencing and its suppression.

Small RNAs, including small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-associated interfering RNAs (piRNAs), are powerful gene expression regulators. This RNA-mediated regulation results in sequence-specific inhibition of gene expression by translational repression and/or mRNA degradation. siRNAs and miRNAs are generated by RNase III enzymes and subsequently loaded into Argonaute protein, a key component of the RNA induced silencing complex (RISC), to form the core of the RNA silencing machinery. RNA silencing acts as an ancient cell defense system against molecular parasites, such as transgenes, viruses and transposons. RNA silencing also plays an important role in the control of development. In plants, RNA silencing serves as a potent antiviral defense system. In response, many viruses have developed strategies to suppress RNA silencing. The striking sequence diversity among viral suppressors suggests that different viral suppressors could target different components of the RNA silencing machinery at different steps in different suppressing modes. Significant progresses have been made in this field for the past 5 years on the basis of structural information derived from RNase III family proteins, Dicer fragments and homologs, Argonaute homologs and viral suppressors. In this paper, we will review the current progress on the understanding of molecular mechanisms of RNA silencing; highlight the structural principles determining the protein-RNA recognition events along the RNA silencing pathways and the suppression mechanisms displayed by viral suppressors.

Structural basis for RNA-silencing suppression by Tomato aspermy virus protein 2b.

The 2b proteins encoded by cucumovirus act as post-transcriptional gene silencing suppressors to counter host defence during infection. Here we report the crystal structure of Tomato aspermy virus 2b (TAV2b) protein bound to a 19 bp small interfering RNA (siRNA) duplex. TAV2b adopts an all alpha-helix structure and forms a homodimer to measure siRNA duplex in a length-preference mode. TAV2b has a pair of hook-like structures to recognize simultaneously two alpha-helical turns of A-form RNA duplex by fitting its alpha-helix backbone into two adjacent major grooves of siRNA duplex. The conserved pi-stackings between tryptophan and the 5'-terminal base of siRNA duplex from both ends enhance the recognition. TAV2b further oligomerizes to form a dimer of dimers through the conserved leucine-zipper-like motif at its amino-terminal alpha-helix. Biochemical experiments suggest that TAV2b might interfere with the post-transcriptional gene silencing pathway by directly binding to siRNA duplex.

Structural insights into histone H3 lysine 56 acetylation by Rtt109.

Histone acetylation plays important roles for the regulation of many fundamental cellular processes. Saccharomyces cerevisiae Rtt109 is an important class of histone acetyltransferases (HATs), which promote genome stability by directly acetylating newly synthesized histone H3 lysine 56 (H3-K56) through an unknown mechanism. Here, we report the crystal structures of Rtt109 at 2.2 A and Rtt109/Acetyl-CoA binary complex at 1.9 A. The structure displays a vise-like topology with mixed three-layered alpha/beta module forming the central module, whose core region resembles the structure of GCN5 HAT domain and P300/CBP HAT domain. Using structural and biochemical analyses, we have discovered the catalytic active site and have identified Asp288 as the deprotonation residue and Lys290 as the autoacetylation residue. We have further proposed the unique H3-K56 anchoring pocket and the potential H3alphaN binding groove. Our work has provided structural insights to understand the acetylation mechanism of H3-K56 by Rtt109.