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

Blood ◽  
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.

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

Mobile DNA ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marc Guérineau ◽  
Luiza Bessa ◽  
Séverine Moriau ◽  
Ewen Lescop ◽  
François Bontems ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Dna Cleavage ◽  
Trichoplusia Ni ◽  
Catalytic Domain ◽  
Structural Diversity ◽  
Paramecium Tetraurelia ◽  
Lysine Methylation ◽  
Unusual Structure ◽  
Rich Domain ◽  
Cysteine Rich Domain

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.

scholarly journals Glycoprotein Ibα forms disulfide bonds with 2 glycoprotein Ibβ subunits in the resting platelet

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 603-609 ◽  
Author(s):  
Shi-Zhong Luo ◽  
Xi Mo ◽  
Vahid Afshar-Kharghan ◽  
Sankaranarayanan Srinivasan ◽  
José A. López ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Cho Cells ◽  
Disulfide Bonds ◽  
Transmembrane Domain ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Sequence Polymorphism ◽  
Size Difference ◽  
Receptor Complex ◽  
Subunit Stoichiometry

Abstract It is widely accepted that glycoprotein (GP) Ib contains one Ibα and one Ibβ subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ibα, C484 or C485, forms the disulfide bond with Ibβ. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibβ. In the context of isolated peptides containing the Ibα or Ibβ transmembrane domain and nearby Cys residue, C484 and C485 in the Ibα peptide were both capable of forming a disulfide bond with the Ibβ peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibβ subunits but only 1 Ibα and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ibα, not the number of Ibβ subunits therein. Overall, these results demonstrate that Ibα is covalently connected to 2 Ibβ subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The αβ2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.

scholarly journals Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1

1999 ◽  
Vol 340 (3) ◽  
pp. 687-692 ◽  
Author(s):  
Jun-ichi MIYAZAKI ◽  
Shigetada NAKANISHI ◽  
Hisato JINGAMI
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Full Length ◽  
Soluble Form ◽  
Soluble Receptor ◽  
Glycine Site ◽  
Binding Characteristics ◽  
Aspartate Receptor ◽  
Extracellular Region ◽  
Membrane Bound

N-Methyl-D-aspartate receptor channels are composed of an NR1 subunit and at least one of the NR2 subunits (NR2A-D). Activation of the N-methyl-D-aspartate receptor requires the co-agonists glycine and glutamate. It has been proposed that the NR1 subunit possesses a glycine-binding site. We have expressed a soluble form of the NR1 subunit, which was produced by connecting the N-terminal extracellular region with the extracellular loop between the third and fourth membrane segments, by a baculovirus system along with full-length and truncated membrane-bound forms. The soluble NR1 receptor was efficiently secreted into the culture medium and showed a high affinity for ligands. The Kd of a glycine-site antagonist, [3H]MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-carboxylic acid], for the soluble receptor was 3.89±0.97 nM, which was comparable to the Kd of 4.47±1.39 nM for the membrane-bound full-length form. These values were close to the values reported previously with the use of rat brain membranes and Chinese hamster ovary cells expressing the full-length form of the NR1 subunit. The Ki values of other glycine-site antagonists, L-689,560 (trans-2-carboxy-5,7-dichloro - 4 - phenylaminocarbonylamino - 1,2,3,4 - tetrahydroquinoline), 5,7-dichlorokynurenate and 5,7-dinitroquinoxaline-2,3-dione, for the soluble receptor were also similar to those for the full-length form of NR1. [3H]MDL 105,519 binding was also inhibited by the agonists glycine and D-serine. Thus the affinity and selectivity of ligand-binding characteristics of the NR1 subunit is conferred on the soluble form of the NR1 subunit. This soluble receptor provides a good experimental tool for initiating a biophysical analysis of the N-methyl-D-aspartate receptor channel protein.

α1C (CaV1.2) L-type calcium channel mediates mechanosensitive calcium regulation

2002 ◽  
Vol 283 (3) ◽  
pp. C1001-C1008 ◽  
Author(s):  
Greg L. Lyford ◽  
Peter R. Strege ◽  
Allan Shepard ◽  
Yijun Ou ◽  
Leonid Ermilov ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Channel ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Calcium Entry ◽  
Intestinal Smooth Muscle ◽  
Ovary Cells ◽  
Hek 293 ◽  
Rich Domain

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the α1C L-type calcium channel subunit (CaV1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a β2 calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac α1C splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the α1C COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming α1C-subunit.

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

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.

scholarly journals The Entire Nucleotide Sequence of Friend-Related and Paralysis-Inducing PVC-441 Murine Leukemia Virus (MuLV) and Its Comparison with Those of PVC-211 MuLV and Friend MuLV

1998 ◽  
Vol 72 (4) ◽  
pp. 3423-3426 ◽  
Author(s):  
Atsushi Tanaka ◽  
Kiyomasa Oka ◽  
Keiji Tanaka ◽  
Atsushi Jinno ◽  
Sandra K. Ruscetti ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Murine Leukemia Virus ◽  
Chinese Hamster Ovary Cells ◽  
Leukemia Virus ◽  
Chinese Hamster ◽  
Brain Capillary Endothelial Cell ◽  
Ovary Cells ◽  
The Difference ◽  
Entire Nucleotide Sequence ◽  
Murine Leukemia

ABSTRACT PVC-441 murine leukemia virus (MuLV) is a member of the PVC group of Friend MuLV (F-MuLV)-derived neuropathogenic retroviruses. In order to determine the molecular basis for the difference in neuropathogenicity between PVC-441 and the previously characterized PVC-211 MuLVs, the entire nucleotide sequence of PVC-441 MuLV was determined and compared with those of PVC-211 and F-MuLV. The results suggest that PVC-441 and PVC-211 MuLVs were formed as a result of random mutations of F-MuLV and developed differently. The distinct pathogenicities of PVC-441 and PVC-211 MuLVs were maintained in the viruses regenerated from their molecular clones, and the sequences responsible for the pathological differences observed can be localized to the env gene. The amino acid sequence of PVC-441 deduced from its nucleotide sequence revealed a number of differences from PVC-211, the most striking of which was a difference at position 129 of the SU proteins in the two viruses. Host range studies with a brain capillary endothelial cell line (RTEC-6) and Chinese hamster ovary cells (CHO-K1) revealed that PVC-441, like PVC-211, could infect these cells but its efficiency of infection was lower than that of PVC-211. These results may account for the difference in neuropathogenicity between PVC-441 and PVC-211.

scholarly journals Redox Potentials of Disulfide Bonds in LOXL2 Studied by Nonequilibrium Alchemical Simulation

2021 ◽  
Vol 9 ◽  
Author(s):  
Lirui Lin ◽  
Haiying Zou ◽  
Wenjin Li ◽  
Li-Yan Xu ◽  
En-Min Li ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Functional Role ◽  
Catalytic Domain ◽  
Lysyl Oxidase ◽  
Scavenger Receptor ◽  
Redox Potentials ◽  
Catalytic Role

Lysyl oxidase-like 2 (LOXL2) is a metalloenzyme that catalyzes the oxidative deamination ε-amino group of lysine. It is found that LOXL2 is a promotor for the metastasis and invasion of cancer cells. Disulfide bonds are important components in LOXL2, and they play a stabilizing role for protein structure or a functional role for regulating protein bioactivity. The redox potential of disulfide bond is one important property to determine the functional role of disulfide bond. In this study, we have calculated the reduction potential of all the disulfide bonds in LOXL2 by non-equilibrium alchemical simulations. Our results show that seven of seventeen disulfide bonds have high redox potentials between −182 and −298 mV and could have a functional role, viz., Cys573–Cys625, Cys579–Cys695, Cys657–Cys673, and Cys663–Cys685 in the catalytic domain, Cys351–Cys414, Cys464–Cys530, and Cys477–Cys543 in the scavenger receptor cysteine-rich (SRCR) domains. The disulfide bond of Cys351–Cys414 is predicted to play an allosteric function role, which could affect the metastasis and invasion of cancer cells. Other functional bonds have a catalytic role related to enzyme activity. The rest of disulfide bonds are predicted to play a structural role. Our study provides an important insight for the classification of disulfide bonds in LOXL2 and can be utilized for the drug design that targets the cysteine residues in LOXL2.

Removing the Covalent Links between GP Ibα and GP Ibβ Hampers Binding of von Willebrand Factor to GP Ib-IX Complex in CHO Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3641-3641
Author(s):  
Xi Mo ◽  
Shi-Zhong Luo ◽  
Yuandong Peng ◽  
Adam D. Munday ◽  
Carol Sun ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Cell Model ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Binding Activity ◽  
Ovary Cells ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In the glycoprotein (GP) Ib-IX complex, GP Ibα contains the binding region for its ligand von Willebrand factor (vWF). How the other subunits in the complex, GP Ibβ and GP IX, regulate the GP Ibα-vWF interaction is not clear. Since GP Ibα connects with two GP Ibβ subunits via disulfide bonds, these inter-subunit covalent links may be important to the proper vWF-binding activity of the GP Ib-IX complex. To test this idea, we have obtained Chinese hamster ovary cells stably expressing the mutant GP Ib-IX complex (CHOαSSβIX) in which both residues C484 and C485 of GP Ibα were changed to Ser. As expected, the mutant GP Ibα did not form any disulfide bonds with GP Ibβ in these cells. Nonetheless, co-immunoprecipitation experiments in the digitonin-containing buffer demonstrated that association of GP Ibα with GP Ibβ and GP IX was retained in CHOαSSβIX cells. The expression level of GP Ibα in CHOαSSβIX cells, detected by its binding to WM23, a conformation-insensitive antibody, was comparable to that in the transfected cells stably expressing the wild type GP Ib-IX complex (CHOαβIX). In contrast, binding of CHOαSSβIX cells to AK2, SZ2 or AN51, all of which are GP Ibα-specific conformation-sensitive antibodies, was significantly different from that of CHOαβIX cells. Compared to CHOαβIX cells, ristocetin-induced binding of CHOαSSβIX cells to vWF under static conditions was reduced by about 50% as determined by flow cytometry. Consistently, rolling of CHOαSSβIX cells on the vWF-coated glass slide was significantly faster than CHOαβIX cells under various flow conditions. Thus, in a transfected cell model, removing the disulfide links between GP Ibα and GP Ibβ in the GP Ib-IX complex leads to an alteration in the conformation of the ligand-binding domain in GP Ibα and as a consequence hampers the complex binding to vWF. Our finding suggests that the relative position of GP Ibα and GP Ibβ in the receptor complex is crucial to its vWF-binding activity, which may be exploited to modulate the GP Ibα-vWF interaction.

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