Crystal structure of a plant ribonuclease, RNase LE

Tanaka, Nobutada; Arai, Jun; Inokuchi, Norio; Koyama, Takashi; Ohgi, Kazuko; Irie, Masachika; Nakamura, Kazuo

doi:10.1006/jmbi.2000.3707

Cited by 51 publications

(56 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2, boxed aa), further solidifying the identity of these Entamoeba enzymes as T2 ribonucleases. Additionally, all members of the T2 ribonuclease family possess at least four structurally conserved cysteine residues involved in intra-chain disulfide bridges, and such bridges are essential for the enzymatic functions of these nucleases (43). In that context, our analyses showed that the mature EhNucI-and EhNucIIdeduced proteins also possess four similarly conserved cysteine residues that could be involved in forming disulfide bridges (Fig.…”

Section: Identification Of E Histolytica Released/secreted Nucleasementioning

confidence: 73%

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

McGugan

Joshi

Dwyer

2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.The ancient eukaryotic human parasite, Entamoeba histolytica, is the causative agent of invasive amebiasis in humans infecting 10% of the world population and resulting in over 100,000 deaths annually, chiefly from amebic liver abscess (1). Infection is acquired through the ingestion of the quadranucleate cyst form of the organism from fecally contaminated food or water supplies. Excystation occurs in the small intestine, releasing immature trophozoites, which move into and colonize the nutrient-poor large intestine.As ancient eukaryotes, Entamoeba parasites contain only rudimentary endoplasmic reticulum and Golgi apparatus (2, 3). Despite this, vesicle trafficking pathways appear to be essential to the pathogenesis of these parasites as (i) uptake and digestions of nutrients by mature trophozoites in the large intestine, (ii) invasion of the intestinal epithelium, (iii) delivery of cyst wall components during encystation, and (iv) dissemination and establishment of extra-intestinal infections all rely on endocytosis and secretion. In addition, these parasites lack many traditional eukaryotic biochemical pathways, including the de novo biosynthesis of purine and pyrimidine nucleobases. As a result, these parasites are completely dependent upon their host for the supply of these essential preformed nutrients. In that regard, it has been reported previously that Entamoeba are capable of salvaging both purines and pyrimidines from the extracellular milieu during in vit...

show abstract

Section: Identification Of E Histolytica Released/secreted Nucleasementioning

confidence: 73%

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

McGugan

Joshi

Dwyer

2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The present structure of CalsepRRP at 2.05 A Ê resolution demonstrates that in spite of its complete lack of activity CalsepRRP adopts the same overall + structure as the active non-base-speci®c RNases from L. esculentum (Tanaka et al, 2000) and M. charantia (Nakagawa et al, 1999). Apart from a few differences, which are mainly restricted to the insertion or deletion of a few residues in loops and the number and location of disul®de bridges, all these proteins share a similar organization of the -sheet strands and -helices.…”

Section: Discussionmentioning

confidence: 73%

“…4), the protein adopts an + structure similar to that of RNases from plants and fungi, with approximate dimensions of 50 Â 40 Â 25 A Ê . The threedimensional fold closely resembles that of RNase Rh from the fungus R. niveus (Kurihara et al, 1996) and the plant RNases from L. esculentum RNase LE (Tanaka et al, 2000) and M. charantia RNase MC1 (Nakagawa et al, 1999) (Fig. 5), with C r.m.s.…”

Section: Structure Descriptionmentioning

confidence: 68%

“…The phase problem was solved with the molecularreplacement technique in AMoRe (Navaza, 1994) using the coordinates of the extracellular ribonuclease from L. esculentum RNase LE (PDB code 1dix; Tanaka et al, 2000) as a search model. Using space group P6 2 , two peaks were found in the translation search corresponding to the two molecules in the asymmetric unit.…”

Section: Determination and Refinement Of The Structurementioning

confidence: 99%

“…The so-called B1 site (base-recognition site for the base at the 5 H side of the scissile phosphodiester bond) of RNases (Richards & Wyckoff, 1971), which is responsible for the accommodation of a purine base (adenine or guanine) of the substrate RNA, consists of a hydrophobic pocket located in the vicinity of the P1 site and is comprised of aromatic residues (Tyr8, Trp42, Tyr50 and Tyr175 of RNase LE; Tanaka et al, 2000). The corresponding pocket of CalsepRRP comprises residues Thr9, Trp46, Trp54 and Gln179.…”

Section: The Putative B1 Site and B2 Sitementioning

confidence: 99%

See 2 more Smart Citations

Structure of an RNase-related protein fromCalystegia sepium

Rabijns

Verboven

Rougé

et al. 2002

Acta Crystallogr D Biol Cryst

View full text Add to dashboard Cite

The structure of a catalytically inactive RNase-related protein from Calystegia sepium (CalsepRRP) has been resolved by protein crystallography at a resolution of 2.05 A Ê and an R factor of 20.74%. Although the protein is completely devoid of ribonuclease activity, it adopts the typical + structure of non-base-speci®c RNases. Analysis of the structure revealed that two amino-acid substitutions in the`active' P1 site, in combination with the less hydrophobic/aromatic character of the B1 base-recognition site and a completely disrupted B2 base-recognition site, might account for this complete lack of activity.

show abstract

Prediction of the Repeat Domain Structures and Impact of Parkinsonism-Associated Variations on Structure and Function of all Functional Domains of Leucine-Rich Repeat Kinase 2 (LRRK2)

et al. 2014

View full text Add to dashboard Cite

Genetic variations of leucine-rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras-like GTPase (ROC) domain and C-terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD-causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three-dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three-dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three-dimensional structures of repeat domains of other proteins, the tandem Roc-COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein-protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc-COR and kinase domains, and (3) the effects of some PD-associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.

show abstract

Crystal structure of a plant ribonuclease, RNase LE

Cited by 51 publications

References 41 publications

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

Structure of an RNase-related protein fromCalystegia sepium

Prediction of the Repeat Domain Structures and Impact of Parkinsonism-Associated Variations on Structure and Function of all Functional Domains of Leucine-Rich Repeat Kinase 2 (LRRK2)

Contact Info

Product

Resources

About