Structural Basis for the Enzymatic Activity of the HACE1 HECT-Type E3 Ligase Through N-Terminal Helix Dimerization.

HACE1 is an ankyrin repeat (AKR) containing HECT-type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well-known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo-EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N-terminal AKR domain, a middle (MID) domain, and a C-terminal HECT domain. Its unique G-shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N-terminal helix of one monomer restricts access to the C-terminal domain of the other. The in vitro ubiquitination assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition.

Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.

Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.

Structural and mechanistic insight into stem-loop RNA processing by yeast Pichia stipitis Dicer.

Dicer is a member of the ribonuclease III enzyme family and processes double-stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer among different species whilst preserving its biological dicing function is intriguing. Here, we describe the structure and function of a novel catalytically active RNase III protein, a non-canonical Dicer (PsDCR1), found in budding yeast Pichia stipitis. The structure of the catalytically active region (the catalytic RNase III domain and double-stranded RNA-binding domain 1 [dsRBD1]) of DCR1 showed that RNaseIII domain is structurally similar to yeast RNase III (Rnt1p) but uniquely presents dsRBD1 in a diagonal orientation, forming a catalytic core made of homodimer and large RNA-binding surface. The second dsRNA binding domain at C-terminus, which is absent in Rnt1, enhances the RNA cleavage activity. Although the cleavage pattern of PsDCR1 anchors an apical loop similar to Rnt1, the cleavage activity depended on the sequence motif at the lower stem, not the apical loop, of hairpin RNA. Through RNA sequencing and RNA mutations, we showed that RNA cleavage by PsDCR1 is determined by the stem-loop structure of the RNA substrate, suggesting the possibility that stem-loop RNA-guided gene silencing pathway exists in budding yeast.

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.

Crystal structure of Arabidopsis thaliana Dawdle forkhead-associated domain reveals a conserved phospho-threonine recognition cleft for dicer-like 1 binding.

Dawdle (DDL) is a microRNA processing protein essential for the development of Arabidopsis. DDL contains a putative nuclear localization signal at its amino-terminus and forkhead-associated (FHA) domain at the carboxyl-terminus. Here, we report the crystal structure of the FHA domain of Arabidopsis Dawdle, determined by multiple-wavelength anomalous dispersion method at 1.7-Å resolution. DDL FHA structure displays a seven-stranded ß-sandwich architecture that contains a unique structural motif comprising two long anti-parallel strands. Strikingly, crystal packing of the DDL FHA domain reveals that a glutamate residue from the symmetry-related DDL FHA domain, a structural mimic of the phospho-threonine, is specifically recognized by the structurally conserved phospho-threonine binding cleft. Consistently with the structural observations, co-immuno-precipitation experiments performed in Nicotiana benthamiana show that the DDL FHA domain co-immuno-precipitates with DCL1 fragments containing the predicted pThr+3(Ile/Val/Leu/Asp) motif. Taken together, we count the recognition of the target residue by the canonical binding cleft of the DDL FHA domain as the key molecular event to instate FHA domain-mediated protein-protein interaction in plant miRNA processing.

Molecular insights into miRNA processing by Arabidopsis thaliana SERRATE.

In plant, primary transcripts (pri-miRNAs) transcribed from miRNA genes by RNA polymerase II are first processed into stem-loop pre-miRNAs and further chopped into ∼21 nt long miRNAs by RNase III-like enzyme DCL1. SERRATE (SE) protein is an essential component for miRNA processing by assisting DCL1 for accurate cleavage. Here we report the crystal structure of Arabidopsis SE core (residues 194-543) at 2.7 Å. SE core adopts the 'walking man-like' topology with N-terminal α helices, C-terminal non-canonical zinc-finger domain and novel Middle domain resembling the leading leg, the lagging leg and the body, respectively. Pull-down assay shows that SE core provides the platform for HYL1 and DCL1 binding, whereas in vitro RNA binding and in vivo mutant rescue experiments suggest that the non-canonical zinc-finger domain coupled with C-terminal tail binds miRNA precursors. SE presumably works as a scaffold-like protein capable of binding both protein and RNA to guide the positioning of miRNA precursor toward DCL1 catalytic site within miRNA processing machinery in plant.

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.

Structure of Arabidopsis HYPONASTIC LEAVES1 and its molecular implications for miRNA processing.

The Arabidopsis HYPONASTIC LEAVES1 (HYL1) is a double-stranded RNA-binding protein that forms a complex with DICER-LIKE1 (DCL1) and SERRATE to facilitate processing of primary miRNAs into microRNAs (miRNAs). However, the structural mechanisms of miRNA maturation by this complex are poorly understood. Here, we present the crystal structures of double-stranded RNA binding domains (dsRBD1 and dsRBD2) of HYL1 and HYL1 dsRBD1 (HR1)/dsRNA complex as well as human TRBP2 dsRBD2 (TR2)/dsRNA complex for comparison analysis. Structural and functional study demonstrates that both HR1 and TR2 are canonical dsRBDs for dsRNA binding, whereas HR2 of HYL1 is a non-canonical dsRBD harboring a putative dimerization interface. Domain swapping within the context of HYL1 demonstrates that TR2 can supplant the function of HR1 in vitro and in vivo. Further biochemical analyses suggest that HYL1 probably binds to the miRNA/miRNA( *) region of precursors as a dimer mediated by HR2.

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.

Successive accumulation of two size classes of viroid-specific small RNA in potato spindle tuber viroid-infected tomato plants.

Like many plant RNA viruses, infection by potato spindle tuber viroid (PSTVd) is known to lead to RNA silencing and a marked reduction in visible disease. To examine the relationship between RNA silencing and this recovery phenomenon in greater detail, we have carried out time-course analyses of viroid-specific small RNA accumulation using several viroid-host combinations. These analyses revealed the presence of two size classes of viroid-specific small RNAs in infected plants, and sequence analysis subsequently demonstrated the presence of a previously undescribed cluster of small RNAs derived primarily from negative-strand PSTVd RNA. Although the clustering patterns were similar, the size distribution of PSTVd small RNAs isolated from symptomatic leaf tissue became more heterogeneous with time. The process by which viroid-specific small RNAs are generated appears to be more complicated than previously believed, possibly involving multiple DICER-LIKE activities, viroid RNA substrates and subcellular compartments.