Active site characterization and activity of the human aspartyl (asparaginyl) β-hydroxylase

Metallomics ◽  
2021 ◽  
Vol 13 (10) ◽  
Author(s):  
Jenna M Greve ◽  
Andrew M Pinkham ◽  
Zechariah Thompson ◽  
J A Cowan
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Catalytic Domain ◽  
Negative Impact ◽  
Enzyme Stability ◽  
Titration Calorimetry ◽  
Cellular Biochemistry ◽  
Biophysical Approach

Abstract Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the β-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

scholarly journals A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

2018 ◽  
Vol 293 (21) ◽  
pp. 7993-8008 ◽  
Author(s):  
Subrata Debnath ◽  
Dalibor Kosek ◽  
Harichandra D. Tagad ◽  
Stewart R. Durell ◽  
Daniel H. Appella ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Metal Ions ◽  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Catalytic Domain ◽  
Protein Phosphatases ◽  
Random Order ◽  
Structural Features

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E–c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

scholarly journals A Wolf in Another Wolf’s Clothing: Post-Genomic Regulation Dictates Venom Profiles of Medically-Important Cryptic Kraits in India

Toxins ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 69 ◽  
Author(s):  
Kartik Sunagar ◽  
Suyog Khochare ◽  
R. R. Senji Laxme ◽  
Saurabh Attarde ◽  
Paulomi Dam ◽  
...  
Keyword(s):  
Negative Impact ◽  
Indian Subcontinent ◽  
Clinical Effectiveness ◽  
Venom Gland ◽  
Western India ◽  
The Common ◽  
Genomic Regulation

The Common Krait (Bungarus caeruleus) shares a distribution range with many other ‘phenotypically-similar’ kraits across the Indian subcontinent. Despite several reports of fatal envenomings by other Bungarus species, commercial Indian antivenoms are only manufactured against B. caeruleus. It is, therefore, imperative to understand the distribution of genetically distinct lineages of kraits, the compositional differences in their venoms, and the consequent impact of venom variation on the (pre)clinical effectiveness of antivenom therapy. To address this knowledge gap, we conducted phylogenetic and comparative venomics investigations of kraits in Southern and Western India. Phylogenetic reconstructions using mitochondrial markers revealed a new species of krait, Romulus’ krait (Bungarus romulusi sp. nov.), in Southern India. Additionally, we found that kraits with 17 mid-body dorsal scale rows in Western India do not represent a subspecies of the Sind Krait (B. sindanus walli) as previously believed, but are genetically very similar to B. sindanus in Pakistan. Furthermore, venom proteomics and comparative transcriptomics revealed completely contrasting venom profiles. While the venom gland transcriptomes of all three species were highly similar, venom proteomes and toxicity profiles differed significantly, suggesting the prominent role of post-genomic regulatory mechanisms in shaping the venoms of these cryptic kraits. In vitro venom recognition and in vivo neutralisation experiments revealed a strong negative impact of venom variability on the preclinical performance of commercial antivenoms. While the venom of B. caeruleus was neutralised as per the manufacturer’s claim, performance against the venoms of B. sindanus and B. romulusi was poor, highlighting the need for regionally-effective antivenoms in India.

scholarly journals Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-β-lactamase

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 5797
Author(s):  
Gayathri Selvaraju ◽  
Thean Chor Leow ◽  
Abu Bakar Salleh ◽  
Yahaya M. Normi
Keyword(s):  
Active Site ◽  
Protein Complexes ◽  
Hypothetical Protein ◽  
Titration Calorimetry ◽  
Binding Properties ◽  
Vitro Assay ◽  
In Silico Docking ◽  
Inhibitory Peptides ◽  
Catalytic Function

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide–protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.

scholarly journals IκB kinase 2 is not essential for platelet activation

2020 ◽  
Vol 4 (4) ◽  
pp. 638-643
Author(s):  
Manuel Salzmann ◽  
Sonja Bleichert ◽  
Bernhard Moser ◽  
Marion Mussbacher ◽  
Mildred Haase ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Active Site ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Iκb Kinase ◽  
Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

scholarly journals TET-mediated 5-methylcytosine oxidation in tRNA promotes translation

2020 ◽  
pp. jbc.RA120.014226
Author(s):  
Hui Shen ◽  
Robert Jordan Ontiveros ◽  
Michael C Owens ◽  
Monica Yun Liu ◽  
Uday Ghanty ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Biological Function ◽  
Catalytic Domain ◽  
Embryonic Stem ◽  
Wild Type ◽  
Oxidation Activity ◽  
Tet Enzymes ◽  
Mediated Oxidation

Oxidation of 5-methylcytosine (5mC) in DNA by the Ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNAs). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison to wild type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.

scholarly journals Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

2013 ◽  
Vol 288 (23) ◽  
pp. 17008-17018 ◽  
Author(s):  
D. Fernando Estrada ◽  
Jennifer S. Laurence ◽  
Emily E. Scott
Keyword(s):  
Cytochrome P450 ◽  
Binding Site ◽  
Active Site ◽  
Catalytic Domain ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Reaction Conditions ◽  
Reversible Binding ◽  
Important Interaction

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

scholarly journals RAG Transposase Can Capture and Commit to Target DNA before or after Donor Cleavage

2001 ◽  
Vol 21 (13) ◽  
pp. 4302-4310 ◽  
Author(s):  
Matthew B. Neiditch ◽  
Gregory S. Lee ◽  
Mark A. Landree ◽  
David B. Roth
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Cell Receptor ◽  
Rate Limiting Step ◽  
Target Capture ◽  
Target Dna ◽  
Before And After ◽  
Rag 1 ◽  
Antigen Receptor Loci

ABSTRACT The discovery that the V(D)J recombinase functions as a transposase in vitro suggests that transposition by this system might be a potent source of genomic instability. To gain insight into the mechanisms that regulate transposition, we investigated a phenomenon termed target commitment that reflects a functional association between the RAG transposase and the target DNA. We found that the V(D)J recombinase is quite promiscuous, forming productive complexes with target DNA both before and after donor cleavage, and our data indicate that the rate-limiting step for transposition occurs after target capture. Formation of stable target capture complexes depends upon the presence of active-site metal binding residues (the DDE motif), suggesting that active-site amino acids in RAG-1 are critical for target capture. The ability of the RAG transposase to commit to target prior to cleavage may result in a preference for transposition into nearby targets, such as immunoglobulin and T-cell receptor loci. This could bias transposition toward relatively “safe” regions of the genome. A preference for localized transposition may also have influenced the evolution of the antigen receptor loci.

scholarly journals A novel function of M. tuberculosis chaperonin paralog GroEL1 in copper homeostasis

2019 ◽  
Author(s):  
Mohammed Yousuf Ansari ◽  
Sakshi D. Batra ◽  
Hina Ojha ◽  
Ashish ◽  
Jaya S. Tyagi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Functional Divergence ◽  
Cellular Protein ◽  
Copper Stress ◽  
Titration Calorimetry ◽  
Evolutionary Divergence ◽  
Wild Type ◽  
Knock Out

AbstractMycobacterial GroELs namely GroEL1 and GroEL2 belong to the family of molecular chaperones, chaperonins. Chaperonins in Escherichia coli are termed as GroEL and GroES which are encoded by essential genes and are involved in cellular protein folding. GroEL1 has a characteristic Histidine-rich C-terminus contrary to its essential paralog GroEL2 and E. coli GroEL which have hydrophobic (GGM) repeats. Since Histidine richness is likely to be involved in metal binding, in this study we have attempted to decipher the role of GroEL1 protein in chelating metals and the consequent role on M. tuberculosis physiology. Using isothermal titration calorimetry (ITC), we found that GroEL1 binds copper, nickel and cobalt, with the highest binding affinity to copper. Since copper is known to be toxic at higher concentration, we cultured Wild Type M. tuberculosis H37Rv, groEL1 knock-out and groEL1-complemented strain with increasing concentrations of copper. We found that M. tuberculosis groEL1 knock out strain is more sensitive to copper than the wild type. Further hypothesizing that the probable mode of action of copper is by induction of oxidative stress, we attempted to understand the role of GroEL1 in redox silencing and hydroxyl radical mediated DNA damage. We interestingly found through our in vitro studies that GroEL1 is helpful in protection from copper stress through maintaining redox balance and free radical mediated DNA damage. Thus, these results indicate that the duplication of chaperonin genes in M. tuberculosis might have led to their evolutionary divergence and resulted in a functional divergence of chaperonins.

scholarly journals The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients

2020 ◽  
Vol 295 (37) ◽  
pp. 12900-12909
Author(s):  
Zongwei Guo ◽  
Ting Song ◽  
Ziqian Wang ◽  
Donghai Lin ◽  
Keke Cao ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Cell Fate ◽  
Protein Misfolding ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Allosteric Site ◽  
Transverse Relaxation ◽  
Bh3 Domain

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.

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