Hanna S. Yuan

Stabilization and enhancement of the antiapoptotic activity of mcl-1 by TCTP.

ABSTRACT Mcl-1 is one Bcl-2 family member that plays a pivotal role in animal development. The extremely labile nature of the Mcl-1 protein itself and the fact that the Mcl-1 level is a critical determinant in various cell survival pathways suggest that cellular processes that regulate Mcl-1 stability are as important as those that regulate Mcl-1 synthesis. Although transcriptional stimulation of Mcl-1 synthesis in response to various stimuli has been well documented, regulation of Mcl-1 stability has been hardly explored. In this study, we identified that the translationally controlled tumor protein (TCTP) was one cellular factor that interacted with Mcl-1 and modulated Mcl-1 stability. While overexpression of TCTP augmented the protein stability of Mcl-1, knockdown expression of TCTP by RNA interference destabilized Mcl-1. Furthermore, TCTP stabilized Mcl-1 through interfering with Mcl-1s degradation by the ubiquitin-dependent proteasome degradation pathway, and the TCTP binding-defective mutant of Mcl-1 (K257V) was much more susceptible to degradation and manifested a compromised antiapoptotic activity. Taken together, these results suggest that TCTP modulates Mcl-1s antiapoptotic activity by modulating its protein stability. The possible mechanism(s) involved in TCTPs modulation process is discussed.

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.

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

BACKGROUND Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition. RESULTS The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 A resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel alpha/beta fold and contains a Zn2+ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (alpha1(*)-loop12-alpha2(*); where * represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23-alpha3(*)-loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7. CONCLUSIONS The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.

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.

Crystal Structure of a Natural Circularly Permuted Jellyroll Protein: 1,3-1,4-β-d-Glucanase from Fibrobacter succinogenes

The 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes (Fsbeta-glucanase) is classified as one of the family 16 glycosyl hydrolases. It hydrolyzes the glycosidic bond in the mixed-linked glucans containing beta-1,3- and beta-1,4-glycosidic linkages. We constructed a truncated form of recombinant Fsbeta-glucanase containing the catalytic domain from amino acid residues 1-258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme. The crystal structure of the truncated Fsbeta-glucanase was solved at a resolution of 1.7A by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein. The overall topology of the truncated Fsbeta-glucanase consists mainly of two eight-stranded anti-parallel beta-sheets arranged in a jellyroll beta-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules. Sequence comparison with other bacterial glucanases showed that Fsbeta-glucanase is the only naturally occurring circularly permuted beta-glucanase with reversed sequences. Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share approximately 70% sequence identity, than to the naturally occurring Fsbeta-glucanase of similar topology with 30% identity. This result suggests that protein structure relies more on sequence identity than topology. The high-resolution structure of Fsbeta-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability.

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.

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.

Structural Studies of the Pigeon Cytosolic Nadp+ -Dependent Malic Enzyme

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2 in the presence of divalent cations (Mg2+, Mn2+). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD+ or NADP+, but not both. Structural studies of the human mitochondrial NAD+‐dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP+‐dependent malic enzyme, in a closed form, in a quaternary complex with NADP+, Mn2+, and oxalate. This represents the first structural information on an NADP+‐dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2′‐phosphate group of the NADP+ cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP+ selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.