Wei-Zen Yang

DNA binding and cleavage by the periplasmic nuclease Vvn: a novel structure with a known active site

The Vibrio vulnificus nuclease, Vvn, is a non‐specific periplasmic nuclease capable of digesting DNA and RNA. The crystal structure of Vvn and that of Vvn mutant H80A in complex with DNA were resolved at 2.3 Å resolution. Vvn has a novel mixed α/β topology containing four disulfide bridges, suggesting that Vvn is not active under reducing conditions in the cytoplasm. The overall structure of Vvn shows no similarity to other endonucleases; however, a known ‘ββα–metal’ motif is identified in the central cleft region. The crystal structure of the mutant Vvn–DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by ∼20°. Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting a structural basis for its sequence‐independent recognition of DNA and RNA. Based on the enzyme–substrate and enzyme–product structures observed in the mutant Vvn–DNA crystals, a catalytic mechanism is proposed. This structural study suggests that Vvn hydrolyzes DNA by a general single‐metal ion mechanism, and indicates how non‐specific DNA‐binding proteins may recognize DNA.

Structural and functional insights into human Tudor-SN, a key component linking RNA interference and editing

Human Tudor-SN is involved in the degradation of hyper-edited inosine-containing microRNA precursors, thus linking the pathways of RNA interference and editing. Tudor-SN contains four tandem repeats of staphylococcal nuclease-like domains (SN1–SN4) followed by a tudor and C-terminal SN domain (SN5). Here, we showed that Tudor-SN requires tandem repeats of SN domains for its RNA binding and cleavage activity. The crystal structure of a 64-kD truncated form of human Tudor-SN further shows that the four domains, SN3, SN4, tudor and SN5, assemble into a crescent-shaped structure. A concave basic surface formed jointly by SN3 and SN4 domains is likely involved in RNA binding, where citrate ions are bound at the putative RNase active sites. Additional modeling studies provide a structural basis for Tudor-SNs preference in cleaving RNA containing multiple I·U wobble-paired sequences. Collectively, these results suggest that tandem repeats of SN domains in Tudor-SN function as a clamp to capture RNA substrates.

The transactivation region of the fis protein that controls site-specific DNA inversion contains extended mobile beta-hairpin arms.

The Fis protein regulates site‐specific DNA inversion catalyzed by a family of DNA invertases when bound to a cis‐acting recombinational enhancer. As is often found for transactivation domains, previous crystal structures have failed to resolve the conformation of the N‐terminal inversion activation region within the Fis dimer. A new crystal form of a mutant Fis protein now reveals that the activation region contains two β‐hairpin arms that protrude over 20 Å from the protein core. Saturation mutagenesis identified the regulatory and structurally important amino acids. The most critical activating residues are located near the tips of the β‐arms. Disulfide cross‐linking between the β‐arms demonstrated that they are highly flexible in solution and that efficient inversion activation can occur when the β‐arms are covalently linked together. The emerging picture for this regulatory motif is that contacts with the recombinase at the tip of the mobile β‐arms activate the DNA invertase in the context of an invertasome complex.

Crystal structure of human polynucleotide phosphorylase: insights into its domain function in RNA binding and degradation

Human polynucleotide phosphorylase (hPNPase) is a 3′-to-5′ exoribonuclease that degrades specific mRNA and miRNA, and imports RNA into mitochondria, and thus regulates diverse physiological processes, including cellular senescence and homeostasis. However, the RNA-processing mechanism by hPNPase, particularly how RNA is bound via its various domains, remains obscure. Here, we report the crystal structure of an S1 domain-truncated hPNPase at a resolution of 2.1 Å. The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore. Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase. Our studies thus provide structural and functional insights into hPNPase, which uses a KH pore to trap a long RNA 3′ tail that is further delivered into an RNase PH channel for the degradation process. Structural RNA with short 3′ tails are, on the other hand, transported but not digested by hPNPase.

Structural insights into apoptotic DNA degradation by CED-3 Protease Suppressor-6 (CPS-6) from Caenorhabditis elegans*

Background: CPS-6 (EndoG) degrades chromosomal DNA during apoptosis. Results: The crystal structure of C. elegans CPS-6 was determined, and the DNA binding and cleavage mechanisms by CPS-6 were revealed. Conclusion: The DNase activity of CPS-6 is positively correlated with its pro-cell death activity. Significance: This study improves our general understanding of DNA hydrolysis by ββα-metal finger nucleases and the process of apoptotic DNA fragmentation. Endonuclease G (EndoG) is a mitochondrial protein that traverses to the nucleus and participates in chromosomal DNA degradation during apoptosis in yeast, worms, flies, and mammals. However, it remains unclear how EndoG binds and digests DNA. Here we show that the Caenorhabditis elegans CPS-6, a homolog of EndoG, is a homodimeric Mg2+-dependent nuclease, binding preferentially to G-tract DNA in the optimum low salt buffer at pH 7. The crystal structure of CPS-6 was determined at 1.8 Å resolution, revealing a mixed αβ topology with the two ββα-metal finger nuclease motifs located distantly at the two sides of the dimeric enzyme. A structural model of the CPS-6-DNA complex suggested a positively charged DNA-binding groove near the Mg2+-bound active site. Mutations of four aromatic and basic residues: Phe122, Arg146, Arg156, and Phe166, in the protein-DNA interface significantly reduced the DNA binding and cleavage activity of CPS-6, confirming that these residues are critical for CPS-6-DNA interactions. In vivo transformation rescue experiments further showed that the reduced DNase activity of CPS-6 mutants was positively correlated with its diminished cell killing activity in C. elegans. Taken together, these biochemical, structural, mutagenesis, and in vivo data reveal a molecular basis of how CPS-6 binds and hydrolyzes DNA to promote cell death.

Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer

Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the ββα-metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS-6 degrades DNA without sequence specificity. Two obligatory monomeric CPS-6 mutants (P207E and K131D/F132N) were constructed, and they degrade DNA with diminished activity due to poorer DNA-binding affinity as compared to wild-type CPS-6. Moreover, the P207E mutant exhibits predominantly 3′-to-5′ exonuclease activity, indicating a possible endonuclease to exonuclease activity change. Thus, the dimer conformation of CPS-6 is essential for maintaining its optimal DNA-binding and endonuclease activity. Compared to other non-specific endonucleases, which are usually monomeric enzymes, EndoG is a unique dimeric endonuclease, whose activity hence can be modulated by oxidation to induce conformational changes.

Crystal structure of dimeric human PNPase reveals why disease-linked mutants suffer from low RNA import and degradation activities

Abstract Human polynucleotide phosphorylase (PNPase) is an evolutionarily conserved 3′-to-5′ exoribonuclease principally located in mitochondria where it is responsible for RNA turnover and import. Mutations in PNPase impair structured RNA transport into mitochondria, resulting in mitochondrial dysfunction and disease. PNPase is a trimeric protein with a doughnut-shaped structure hosting a central channel for single-stranded RNA binding and degradation. Here, we show that the disease-linked human PNPase mutants, Q387R and E475G, form dimers, not trimers, and have significantly lower RNA binding and degradation activities compared to wild-type trimeric PNPase. Moreover, S1 domain-truncated PNPase binds single-stranded RNA but not the stem–loop signature motif of imported structured RNA, suggesting that the S1 domain is responsible for binding structured RNAs. We further determined the crystal structure of dimeric PNPase at a resolution of 2.8 Å and, combined with small-angle X-ray scattering, show that the RNA-binding K homology and S1 domains are relatively inaccessible in the dimeric assembly. Taken together, these results show that mutations at the interface of the trimeric PNPase tend to produce a dimeric protein with destructive RNA-binding surfaces, thus impairing both of its RNA import and degradation activities and leading to mitochondria disorders.