Yu-Yuan Hsiao

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.

Crystal structure of CRN-4: implications for domain function in apoptotic DNA degradation.

ABSTRACT Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4s biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn2+-bound active form, and Er3+-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn2+-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4s function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis.

How an exonuclease decides where to stop in trimming of nucleic acids: crystal structures of RNase T-product complexes

Exonucleases are key enzymes in the maintenance of genome stability, processing of immature RNA precursors and degradation of unnecessary nucleic acids. However, it remains unclear how exonucleases digest nucleic acids to generate correct end products for next-step processing. Here we show how the exonuclease RNase T stops its trimming precisely. The crystal structures of RNase T in complex with a stem-loop DNA, a GG dinucleotide and single-stranded DNA with different 3′-end sequences demonstrate why a duplex with a short 3′-overhang, a dinucleotide and a ssDNA with a 3′-end C cannot be further digested by RNase T. Several hydrophobic residues in RNase T change their conformation upon substrate binding and induce an active or inactive conformation in the active site that construct a precise machine to determine which substrate should be digested based on its sequence, length and structure. These studies thus provide mechanistic insights into how RNase T prevents over digestion of its various substrates, and the results can be extrapolated to the thousands of members of the DEDDh family of exonucleases.

Structural Insights Into DNA Repair by RNase T—An Exonuclease Processing 3′ End of Structured DNA in Repair Pathways

Structure analysis of the exonuclease RNase T reveals that it also functions in DNA repair pathways where it binds and processes bulge, bubble, and Y-structured DNA to trim the DNA 3′ ends.

The 1.5 A structure of endo-1,3-beta-glucanase from Streptomyces sioyaensis: evolution of the active-site structure for 1,3-beta-glucan-binding specificity and hydrolysis.

The catalytic domain structure of Streptomyces sioyaensis 1,3-beta-glucanase (278 amino acids), a member of glycosyl hydrolase family 16 (GHF16), was determined to 1.5 A resolution in space group P2(1)2(1)2(1). The enzyme specifically hydrolyzes the glycosidic bond of the 1,3-beta-linked glucan substrate. The overall structure contains two antiparallel six-and seven-stranded beta-sheets stacked in a beta-sandwich jelly-roll motif similar to the fold of GHF16 1,3-1,4-beta-glucanases. The active-site cleft of the enzyme is distinct, with the closure of one end primarily caused by two protruding loop insertions and two key residues, Tyr38 and Tyr134. The current known structures of 1,3-1,4-beta-glucanases and 1,3-beta-glucanase from Nocardiopsis sp., on the other hand, have open-channel active-site clefts that can accommodate six beta-D-glucopyranosyl units. The active-site structure of 1,3-beta-glucanase was compared with those of other homologous structures in order to address the binding and enzymatic specificity for 1,3-beta-linked glucans in Streptomyces. This information could be helpful in the development of specific antifungal agents.

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.

Aromatic residues in RNase T stack with nucleobases to guide the sequence‐specific recognition and cleavage of nucleic acids

RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preference in that its 3′‐to‐5′ exonuclease activity is blocked by a 3′‐terminal dinucleotide CC in digesting both single‐stranded RNA and DNA. Our previous crystal structure analysis of RNase T‐DNA complexes show that four phenylalanine residues, F29, F77, F124, and F146, stack with the two 3′‐terminal nucleobases. To elucidate if the π–π stacking interactions between aromatic residues and nucleobases play a critical role in sequence‐specific protein–nucleic acid recognition, here we mutated two to four of the phenylalanine residues in RNase T to tryptophan (W mutants) and tyrosine (Y mutants). The Escherichia coli strains expressing either the W mutants or the Y mutants had slow growth phenotypes, suggesting that all of these mutants could not fully substitute the function of the wild‐type RNase T in vivo. DNA digestion assays revealed W mutants shared similar sequence specificity with wild‐type RNase T. However, the Y mutants exhibited altered sequence‐dependent activity, digesting ssDNA with both 3′‐end CC and GG sequences. Moreover, the W and Y mutants had reduced DNA‐binding activity and lower thermal stability as compared to wild‐type RNase T. Taken together, our results suggest that the four phenylalanine residues in RNase T not only play critical roles in sequence‐specific recognition, but also in overall protein stability. Our results provide the first evidence showing that the π−π stacking interactions between nucleobases and protein aromatic residues may guide the sequence‐specific activity for DNA and RNA enzymes.

Structure and function of TatD exonuclease in DNA repair

TatD is an evolutionarily conserved protein with thousands of homologues in all kingdoms of life. It has been suggested that TatD participates in DNA fragmentation during apoptosis in eukaryotic cells. However, the cellular functions and biochemical properties of TatD in bacterial and non-apoptotic eukaryotic cells remain elusive. Here we show that Escherichia coli TatD is a Mg2+-dependent 3′–5′ exonuclease that prefers to digest single-stranded DNA and RNA. TatD-knockout cells are less resistant to the DNA damaging agent hydrogen peroxide, and TatD can remove damaged deaminated nucleotides from a DNA chain, suggesting that it may play a role in the H2O2-induced DNA repair. The crystal structure of the apo-form TatD and TatD bound to a single-stranded three-nucleotide DNA was determined by X-ray diffraction methods at a resolution of 2.0 and 2.9 Å, respectively. TatD has a TIM-barrel fold and the single-stranded DNA is bound at the loop region on the top of the barrel. Mutational studies further identify important conserved metal ion-binding and catalytic residues in the TatD active site for DNA hydrolysis. We thus conclude that TatD is a new class of TIM-barrel 3′–5′ exonuclease that not only degrades chromosomal DNA during apoptosis but also processes single-stranded DNA during DNA repair.

Structural Insights into Substrate Recognition by Clostridium difficile Sortase

Sortases function as cysteine transpeptidases that catalyze the covalent attachment of virulence-associated surface proteins into the cell wall peptidoglycan in Gram-positive bacteria. The substrate proteins targeted by sortase enzymes have a cell wall sorting signal (CWSS) located at the C-terminus. Up to date, it is still not well understood how sortases with structural resemblance among different classes and diverse species of bacteria achieve substrate specificity. In this study, we focus on elucidating the molecular basis for specific recognition of peptide substrate PPKTG by Clostridium difficile sortase B (Cd-SrtB). Combining structural studies, biochemical assays and molecular dynamics simulations, we have constructed a computational model of Cd-SrtBΔN26–PPKTG complex and have validated the model by site-directed mutagensis studies and fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we have revealed that the fourth amino acid in the N-terminal direction from cleavage site of PPKTG forms specific interaction with Cd-SrtB and plays an essential role in configuring the peptide to allow more efficient substrate-specific cleavage by Cd-SrtB.

Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability

ABSTRACT Structural calcium sites control protein thermostability and activity by stabilizing native folds and changing local conformations. Alicyclobacillus acidocaldarius survives in thermal-acidic conditions and produces an endoglucanase Cel9A (AaCel9A) which contains a calcium-binding site (Ser465 to Val470) near the catalytic cleft. By superimposing the Ca2+-free and Ca2+-bounded conformations of the calcium site, we found that Ca2+ induces hydrophobic interactions between the calcium site and its nearby region by driving a conformational change. The hydrophobic interactions at the high-B-factor region could be enhanced further by replacing the surrounding polar residues with hydrophobic residues to affect enzyme thermostability and activity. Therefore, the calcium-binding residue Asp468 (whose side chain directly ligates Ca2+), Asp469, and Asp471 of AaCel9A were separately replaced by alanine and valine. Mutants D468A and D468V showed increased activity compared with those of the wild type with 0 mM or 10 mM Ca2+ added, whereas the Asp469 or Asp471 substitution resulted in decreased activity. The D468A crystal structure revealed that mutation D468A triggered a conformational change similar to that induced by Ca2+ in the wild type and developed a hydrophobic interaction network between the calcium site and the neighboring hydrophobic region (Ala113 to Ala117). Mutations D468V and D468A increased 4.5°C and 5.9°C, respectively, in melting temperature, and enzyme half-life at 75°C increased approximately 13 times. Structural comparisons between AaCel9A and other endoglucanases of the GH9 family suggested that the stability of the regions corresponding to the AaCel9A calcium site plays an important role in GH9 endoglucanase catalysis at high temperature.