Lyudmila G. Doudeva

Structural insights into TDP-43 in nucleic-acid binding and domain interactions

TDP-43 is a pathogenic protein: its normal function in binding to UG-rich RNA is related to cystic fibrosis, and inclusion of its C-terminal fragments in brain cells is directly linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Here we report the 1.65 Å crystal structure of the C-terminal RRM2 domain of TDP-43 in complex with a single-stranded DNA. We show that TDP-43 is a dimeric protein with two RRM domains, both involved in DNA and RNA binding. The crystal structure reveals the basis of TDP-43s TG/UG preference in nucleic acids binding. It also reveals that RRM2 domain has an atypical RRM-fold with an additional β-strand involved in making protein–protein interactions. This self association of RRM2 domains produced thermal-stable RRM2 assemblies with a melting point greater than 85°C as monitored by circular dichroism at physiological conditions. These studies thus characterize the recognition between TDP-43 and nucleic acids and the mode of RRM2 self association, and provide molecular models for understanding the role of TDP-43 in cystic fibrosis and the neurodegenerative diseases related to TDP-43 proteinopathy.

Metal ions and phosphate binding in the H-N-H motif: Crystal structures of the nuclease domain of ColE7/Im7 in complex with a phosphate ion and different divalent metal ions

H‐N‐H is a motif found in the nuclease domain of a subfamily of bacteria toxins, including colicin E7, that are capable of cleaving DNA nonspecifically. This H‐N‐H motif has also been identified in a subfamily of homing endonucleases, which cleave DNA site specifically. To better understand the role of metal ions in the H‐N‐H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 (nuclease‐ColE7) in complex with its inhibitor Im7 in two different crystal forms, and we resolved the structures of EDTA‐treated, Zn2+‐bound and Mn2+‐bound complexes in the presence of phosphate ions at resolutions of 2.6 Å to 2.0 Å. This study offers the first determination of the structure of a metal‐free and substrate‐free enzyme in the H‐N‐H family. The H‐N‐H motif contains two antiparallel β‐strands linked to a C‐terminal α‐helix, with a divalent metal ion located in the center. Here we show that the metal‐binding sites in the center of the H‐N‐H motif, for the EDTA‐treated and Mg2+‐soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H‐N‐H motif. However, a Zn2+ or Mn2+ ions were observed in the center of the H‐N‐H motif in cases of Zn2+ or Mn2+‐soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H‐N‐H motif in stabilizing the phosphoanion in the transition state during hydrolysis.

The crystal structure of the nuclease domain of colicin E7 suggests a mechanism for binding to double-stranded DNA by the H-N-H endonucleases.

The bacterial toxin ColE7 contains an H-N-H endonuclease domain (nuclease ColE7) that digests cellular DNA or RNA non-specifically in target cells, leading to cell death. In the host cell, protein Im7 forms a complex with ColE7 to inhibit its nuclease activity. Here, we present the crystal structure of the unbound nuclease ColE7 at a resolution of 2.1A. Structural comparison between the unbound and bound nuclease ColE7 in complex with Im7, suggests that Im7 is not an allosteric inhibitor that induces backbone conformational changes in nuclease ColE7, but rather one that inhibits by blocking the substrate-binding site. There were two nuclease ColE7 molecules in the P1 unit cell in crystals and they appeared as a dimer related to each other by a non-crystallographic dyad symmetry. Gel-filtration and cross-linking experiments confirmed that nuclease ColE7 indeed formed dimers in solution and that the dimeric conformation was more favored in the presence of double-stranded DNA. Structural comparison of nuclease ColE7 with the His-Cys box homing endonuclease I-PpoI further demonstrated that H-N-H motifs in dimeric nuclease ColE7 were oriented in a manner very similar to that of the betabetaalpha-fold of the active sites found in dimeric I-PpoI. A mechanism for the binding of double-stranded DNA by dimeric H-N-H nuclease ColE7 is suggested.

Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+

The nuclease domain of ColE7 (N‐ColE7) contains an H‐N‐H motif that folds in a ββα‐metal topology. Here we report the crystal structures of a Zn2+‐bound N‐ColE7 (H545E mutant) in complex with a 12‐bp duplex DNA and a Ni2+‐bound N‐ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 Å and 2.0 Å, respectively. Metal‐dependent cleavage assays showed that N‐ColE7 cleaves double‐stranded DNA with a single metal ion cofactor, Ni2+, Mg2+, Mn2+, and Zn2+. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg2+ atconcentrations of <25mM, indicating thatzincisthe physiologically relevant metal ion in N‐ColE7 in host E. coli. In the crystal structure of N‐ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni2+ is bound in N‐ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His‐metal finger motif can be coordinated to six ligands, such as Mg2+ in I‐PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni2+ or Zn2+ in ColE7. Universally, the metal ion in the His‐metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P–O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.

Structural basis for sequence-dependent DNA cleavage by nonspecific endonucleases

Nonspecific endonucleases hydrolyze DNA without sequence specificity but with sequence preference, however the structural basis for cleavage preference remains elusive. We show here that the nonspecific endonuclease ColE7 cleaves DNA with a preference for making nicks after (at 3′O-side) thymine bases but the periplasmic nuclease Vvn cleaves DNA more evenly with little sequence preference. The crystal structure of the ‘preferred complex’ of the nuclease domain of ColE7 bound to an 18 bp DNA with a thymine before the scissile phosphate had a more distorted DNA phosphate backbone than the backbones in the non-preferred complexes, so that the scissile phosphate was compositionally closer to the endonuclease active site resulting in more efficient DNA cleavage. On the other hand, in the crystal structure of Vvn in complex with a 16 bp DNA, the DNA phosphate backbone was similar and not distorted in comparison with that of a previously reported complex of Vvn with a different DNA sequence. Taken together these results suggest a general structural basis for the sequence-dependent DNA cleavage catalyzed by nonspecific endonucleases, indicating that nonspecific nucleases could induce DNA to deform to distinctive levels depending on the local sequence leading to different cleavage rates along the DNA chain.

The crystal structure of TDP-43 RRM1-DNA complex reveals the specific recognition for UG- and TG-rich nucleic acids

TDP-43 is an important pathological protein that aggregates in the diseased neuronal cells and is linked to various neurodegenerative disorders. In normal cells, TDP-43 is primarily an RNA-binding protein; however, how the dimeric TDP-43 binds RNA via its two RNA recognition motifs, RRM1 and RRM2, is not clear. Here we report the crystal structure of human TDP-43 RRM1 in complex with a single-stranded DNA showing that RRM1 binds the nucleic acid extensively not only by the conserved β-sheet residues but also by the loop residues. Mutational and biochemical assays further reveal that both RRMs in TDP-43 dimers participate in binding of UG-rich RNA or TG-rich DNA with RRM1 playing a dominant role and RRM2 playing a supporting role. Moreover, RRM1 of the amyotrophic lateral sclerosis-linked mutant D169G binds DNA as efficiently as the wild type; nevertheless, it is more resistant to thermal denaturation, suggesting that the resistance to degradation is likely linked to TDP-43 proteinopathies. Taken together all the data, we suggest a model showing that the two RRMs in each protomer of TDP-43 homodimer work together in RNA binding and thus the dimeric TDP-43 recognizes long clusters of UG-rich RNA to achieve high affinity and specificity.

Structural analysis of disease-related TDP-43 D169G mutation: linking enhanced stability and caspase cleavage efficiency to protein accumulation

The RNA-binding protein TDP-43 forms intracellular inclusions in amyotrophic lateral sclerosis (ALS). While TDP-43 mutations have been identified in ALS patients, how these mutations are linked to ALS remains unclear. Here we examined the biophysical properties of six ALS-linked TDP-43 mutants and found that one of the mutants, D169G, had higher thermal stability than wild-type TDP-43 and that it was cleaved by caspase 3 more efficiently, producing increased levels of the C-terminal 35 kD fragments (TDP-35) in vitro and in neuroblastoma cells. The crystal structure of the TDP-43 RRM1 domain containing the D169G mutation in complex with DNA along with molecular dynamics simulations reveal that the D169G mutation induces a local conformational change in a β turn and increases the hydrophobic interactions in the RRM1 core, thus enhancing the thermal stability of the RRM1 domain. Our results provide the first crystal structure of TDP-43 containing a disease-linked D169G mutation and a disease-related mechanism showing that D169G mutant is more susceptible to proteolytic cleavage by caspase 3 into the pathogenic C-terminal 35-kD fragments due to its increased stability in the RRM1 domain. Modulation of TDP-43 stability and caspase cleavage efficiency could present an avenue for prevention and treatment of TDP-43-linked neurodegeneration.

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.

Structural and functional insight into cell-defending non-specific nucleases

C85 MS65.28.5 Acta Cryst. (2005). A61, C85 Structural and Functional Insight into Celldefending Non-specific Nucleases Hanna S. Yuan, Kuo-Chiang Hsia, Lyudmila G. Doudeva, Hsinchin Huang, Wei-Zen Yang, Woei-Chyn Chu, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC. Institute of Biomedical Engineering, National Yang Ming University, Taipei, Taiwan, ROC. E-mail: hanna@sinica.edu.tw