The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases

Abstract Background Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive. Results We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state. Conclusions The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

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Structural and Functional Characteristics of ADAMTS-4 and ADAMTS-5: A Review

10.15688/nsr.jvolsu.2020.1.2 ◽

2020 ◽

pp. 12-21

Author(s):

Anastasia Korchagina ◽

Lyudmila Derevshchikova

Keyword(s):

Enzymatic Activity ◽

Binding Sites ◽

Catalytic Domain ◽

Structural Features ◽

Functional Characteristics ◽

Rich Domain ◽

Common Diseases ◽

The Moment ◽

Cysteine Rich Domain

ADAMTS-4 and -5 are aggrecanases that are involved in the development of osteoarthrosis, one of the most common diseases at the moment, due to which a large number of people suffer annually. By some estimates, 9.6% of men and 18% of women over the age of 60 have symptomatic osteoarthrosis. This review discusses the currently known data on the structural features and enzymatic activity of these enzymes. The structures of ADAMTS-4 and -5 are similar, they contain 7 domain sites: the signal section, the N-terminal prodomain, the catalytic domain, the disintegrin-like domain, the thrombospodin motif, the cysteine-rich domain, and the spacer domain. In addition to all these elements, ADAMTS-5 has an additional thrombospodin motif at the end. ADAMTS-4 and -5 cleaves aggrecan in 5 different binding sites. However, the Glu373-Ala374 site probably plays the most important role in the pathogenesis, since when this bond is broken, the whole aggrecan molecule loses its integrity, which leads to the destruction of cartilage and the development of the disease. In the course of the analysis of the information, the authors have found that the participation of ADAMTS-4 and -5 in the development of osteoarthritis varies greatly depending on the type of organism. The researchers have established that in mice ADAMTS-4 plays the largest role in the destruction of aggrecan, while in humans ADAMTS-5 or both aggrecanases influence the development of osteoarthritis. The revealed differences are not fully described; therefore, this review summarizes the already known results in this area, which will facilitate further research in this direction.

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Crystal Structure of a Proteolytic Fragment of ADAMTS13 Reveals Alternative Disulfide Pairings in the Cysteine-Rich Domain

Blood ◽

10.1182/blood.v120.21.3363.3363 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3363-3363

Author(s):

Christopher G. Skipwith ◽

Steven Stayrook ◽

Hanspeter Rottensteiner ◽

Friedrich Scheiflinger ◽

X. Long Zheng

Keyword(s):

Disulfide Bond ◽

Disulfide Bonds ◽

Catalytic Domain ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Full Length ◽

Proteolytic Fragment ◽

Rich Domain ◽

The Difference ◽

Cysteine Rich Domain

Abstract Abstract 3363 Proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 depends on its non-catalytic domain (or exosite) interactions. These exosites, particularly the residues between Tyr659 and Tyr665 and their adjacent residues Arg568 and Phe592 in the ADAMTS13 spacer domain, are the major targets of anti-ADAMTS13 autoantibodies in patients with acquired TTP. In an effort to further determine the potential exosite interactions between ADAMTS13 and VWF, we carried out crystal screening of a recombinant full-length ADAMTS13 purified from stably transfected Chinese hamster ovary cells. After over 900 conditions were tested, we obtained multiple well-formed and diffractable crystals. Upon X-ray diffraction, we discovered that only a proteolytic fragment of full-length ADAMTS13 (Met432-Arg670) had been consistently crystallized. Here, we report the structure of this non-catalytic ADAMTS13 fragment at 2.2Å. The fragment encompasses 8 residues from the first TSP-1 repeat, an entire cysteine-rich domain (CA and CB), and a majority of the spacer domain. While recombinant ADAMTS13 in the present study is fully glycosylated, the overall structure agrees very well with the analogous portion of the ADAMTS13-DTCS structure previously reported (RMSD = 0.02Å, 1817 atoms, 232 residues). A region within the CA domain does vary slightly from that in the ADAMTS13-DTCS (RMSD = 0.08Å, 379 atoms, 51 residues). As with the ADAMTS13-DTCS structure, our structure shows 3 intermolecular disulfide bonds in CA, in addition to 2 free cysteine residues. In this region, our structure shows differences in the disulfide bonding pairing, and a slight shift in the α4 helix from Gly479-Cys483. Specifically, our structure reveals an intermolecular disulfide bond between the residues Cys483 and Cys527 and Cys508 and Cys522. These disulfide pair patterns are different from those reported in the ADAMTS13-DTCS structure, in which disulfide bonds are formed between the residues Cys483 and Cys522 (4.6Å apart in our structure) and the residues Cys508 and Cys527 (3.8Å apart in our structure). Our results indicate that, despite the difference in conditions under which crystals of ADAMTS13 fragments are formed, the overall structure of the Cys-rich and spacer domains are quite similar. The difference in the disulfide bond pattering may provide a novel insight into the mechanism regarding the ADAMTS13 exosite interaction with VWF. Our ongoing work is aimed to determine the complex formation between ADAMTS13 and its substrate or autoantibodies. Disclosures: Rottensteiner: Baxter Innovations GmbH: Employment.

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The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors

Frontiers in Immunology ◽

10.3389/fimmu.2015.00342 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 10

Author(s):

Nicholas V. L. Yap ◽

Fiona J. Whelan ◽

Dawn M. E. Bowdish ◽

G. Brian Golding

Keyword(s):

Scavenger Receptor ◽

Scavenger Receptors ◽

Class A ◽

Rich Domain ◽

Cysteine Rich Domain

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Studies on the structure and binding properties of the cysteine-rich domain of rat low affinity nerve growth factor receptor (p75NGFR).

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)42451-9 ◽

1992 ◽

Vol 267 (12) ◽

pp. 8352-8359

Author(s):

A.N. Baldwin ◽

C.M. Bitler ◽

A.A. Welcher ◽

E.M. Shooter

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Growth Factor Receptor ◽

Nerve Growth Factor Receptor ◽

Binding Properties ◽

Nerve Growth ◽

Rich Domain ◽

Cysteine Rich Domain

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A duplication of 12 bp in the critical cysteine rich domain of the RET proto-oncogene results in a distinct phenotype of multiple endocrine neoplasia type 2A

Human Molecular Genetics ◽

10.1093/hmg/6.4.587 ◽

1997 ◽

Vol 6 (4) ◽

pp. 587-590 ◽

Cited By ~ 31

Author(s):

W Hoppner

Keyword(s):

Multiple Endocrine Neoplasia ◽

Multiple Endocrine Neoplasia Type ◽

Distinct Phenotype ◽

Rich Domain ◽

Endocrine Neoplasia ◽

Endocrine Neoplasia Type ◽

Cysteine Rich Domain

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Elucidation of Binding Determinants and Functional Consequences of Ras/Raf-Cysteine-rich Domain Interactions

Journal of Biological Chemistry ◽

10.1074/jbc.m000397200 ◽

2000 ◽

Vol 275 (29) ◽

pp. 22172-22179 ◽

Cited By ~ 65

Author(s):

Jason G. Williams ◽

Jonelle K. Drugan ◽

Gwan-Su Yi ◽

Geoffrey J. Clark ◽

Channing J. Der ◽

...

Keyword(s):

Rich Domain ◽

Domain Interactions ◽

Functional Consequences ◽

Binding Determinants ◽

Cysteine Rich Domain

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Engineering the substrate binding site of the hyperthermostable archaeal endo-β-1,4-galactanase from Ignisphaera aggregans

Biotechnology for Biofuels ◽

10.1186/s13068-021-02025-6 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Sebastian J. Muderspach ◽

Folmer Fredslund ◽

Verena Volf ◽

Jens-Christian Navarro Poulsen ◽

Thomas H. Blicher ◽

...

Keyword(s):

Binding Site ◽

Plant Cell Wall ◽

Biological Diversity ◽

Catalytic Domain ◽

Substrate Binding ◽

Plant Defence ◽

Structural Diversity ◽

Industrial Applications ◽

Glycoside Hydrolases ◽

Substrate Binding Site

Abstract Background Endo-β-1,4-galactanases are glycoside hydrolases (GH) from the GH53 family belonging to the largest clan of GHs, clan GH-A. GHs are ubiquitous and involved in a myriad of biological functions as well as being widely used industrially. Endo-β-1,4-galactanases, in particular hydrolyse galactan and arabinogalactan in pectin, a major component of the primary plant cell wall, with important functions in plant defence and application in the food and other industries. Here, we explore the family’s biological diversity by characterizing the first archaeal and hyperthermophilic GH53 galactanase, and utilize it as a scaffold for engineering enzymes with different product lengths. Results A galactanase gene was identified in the genome of the anaerobic hyperthermophilic archaeon Ignisphaera aggregans, and the isolated catalytic domain expressed and characterized (IaGal). IaGal presents the typical (βα)8 barrel structure of clan GH-A enzymes, with catalytic carboxylates at the end of the 4th and 7th barrel strands. Its activity optimum of at least 95 °C and melting point over 100 °C indicate extreme thermostability, a very advantageous property for industrial applications. If enzyme depletion is reduced, so is the need for re-addition, and thus costs. The main stabilizing features of IaGal compared to other structurally characterized members are π–π and cation–π interactions. The length of the substrate binding site—and thus produced oligosaccharide products—is intermediate compared to previously characterized galactanases. Variants inspired by the structural diversity in the GH53 family were rationally designed to shorten or extend the substrate binding groove, in order to modulate product length. Subsite-deleted variants produced shorter products than IaGal, as do the fungal galactanases inspiring the design. IaGal variants engineered with a longer binding site produced a less expected degradation pattern, though still different from that of wild-type IaGal. All variants remained extremely stable. Conclusions We have characterized in detail the most thermophilic endo-β-1,4-galactanase known to date and successfully engineered it to modify the degradation profile, while maintaining much of its desirable thermostability. This is an important achievement as oligosaccharide products length is an important property for industrial and natural GHs alike.

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Recovery of Paramyxovirus Simian Virus 5 with a V Protein Lacking the Conserved Cysteine-rich Domain: The Multifunctional V Protein Blocks both Interferon-β Induction and Interferon Signaling

Virology ◽

10.1006/viro.2002.1738 ◽

2002 ◽

Vol 303 (1) ◽

pp. 15-32 ◽

Cited By ~ 152

Author(s):

Biao He ◽

Reay G. Paterson ◽

Nicola Stock ◽

Joan E. Durbin ◽

Russell K. Durbin ◽

...

Keyword(s):

Simian Virus ◽

Interferon Signaling ◽

Interferon Β ◽

V Protein ◽

Rich Domain ◽

Simian Virus 5 ◽

Protein Blocks ◽

Cysteine Rich Domain

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Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity

Biochemical Journal ◽

10.1042/bj20031377 ◽

2004 ◽

Vol 378 (3) ◽

pp. 727-734 ◽

Cited By ~ 161

Author(s):

Paul C. EVANS ◽

Huib OVAA ◽

Maureen HAMON ◽

Peter J. KILSHAW ◽

Svetlana HAMM ◽

...

Keyword(s):

Zinc Finger ◽

Zinc Finger Protein ◽

Catalytic Domain ◽

Cysteine Proteases ◽

Cellular Proteins ◽

Tumour Necrosis Factor Receptor ◽

Specific Substrate ◽

Polyubiquitin Chains ◽

Finger Protein ◽

The Stability

Ubiquitination regulates the stability and/or activity of numerous cellular proteins. The corollary is that de-ubiquitinating enzymes, which ‘trim’ polyubiquitin chains from specific substrate proteins, play key roles in controlling fundamental cellular activities. Ubiquitin is essential at several stages during the activation of NF-κB (nuclear factor κB), a central co-ordinator of inflammation and other immune processes. Ubiquitination is known to cause degradation of the inhibitory molecule IκBα (inhibitor of κB). In addition, activation of TRAF (tumour-necrosis-factor-receptor-associated factor) and IKKγ (IκB kinase γ)/NEMO (NF-κB essential modifier) signal adaptors relies on their modification with ‘nonclassical’ forms of polyubiquitin chains. Ubiquitin also plays a key role in determining cell fate by modulating the stability of numerous pro-apoptotic or anti-apoptotic proteins. The zinc-finger protein A20 has dual functions in inhibiting NF-κB activation and suppressing apoptosis. The molecular mechanisms of these anti-inflammatory and cytoprotective effects are unknown. Here we demonstrate that A20 is a de-ubiquitinating enzyme. It contains an N-terminal catalytic domain that belongs to the ovarian-tumour superfamily of cysteine proteases. A20 cleaved ubiquitin monomers from branched polyubiquitin chains linked through Lys48 or Lys63 and bound covalently to a thiol-group-reactive, ubiquitin-derived probe. Mutation of a conserved cysteine residue in the catalytic site (Cys103) abolished these activities. A20 did not have a global effect on ubiquitinated cellular proteins, which indicates that its activity is target-specific. The biological significance of the catalytic domain is unknown.

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