Leishmania major biotin protein ligase forms a unique cross-handshake dimer

2021 ◽  
Vol 77 (4) ◽  
pp. 510-521
Author(s):  
Manoj Kumar Rajak ◽  
Sonika Bhatnagar ◽  
Shubhant Pandey ◽  
Sunil Kumar ◽  
Shalini Verma ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Size Exclusion ◽  
Covalent Attachment ◽  
Mitochondrial Enzymes ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Terminal Domain ◽  
Exclusion Chromatography ◽  
Partial Unfolding ◽  
Biotin Protein Ligase

Biotin protein ligase catalyses the post-translational modification of biotin carboxyl carrier protein (BCCP) domains, a modification that is crucial for the function of several carboxylases. It is a two-step process that results in the covalent attachment of biotin to the ɛ-amino group of a conserved lysine of the BCCP domain of a carboxylase in an ATP-dependent manner. In Leishmania, three mitochondrial enzymes, acetyl-CoA carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, depend on biotinylation for activity. In view of the indispensable role of the biotinylating enzyme in the activation of these carboxylases, crystal structures of L. major biotin protein ligase complexed with biotin and with biotinyl-5′-AMP have been solved. L. major biotin protein ligase crystallizes as a unique dimer formed by cross-handshake interactions of the hinge region of the two monomers formed by partial unfolding of the C-terminal domain. Interestingly, the substrate (BCCP domain)-binding site of each monomer is occupied by its own C-terminal domain in the dimer structure. This was observed in all of the crystals that were obtained, suggesting a closed/inactive conformation of the enzyme. Size-exclusion chromatography studies carried out using high protein concentrations (0.5 mM) suggest the formation of a concentration-dependent dimer that exists in equilibrium with the monomer.

scholarly journals Effect of heparin administration to sheep on the release profiles of circulating activin A and follistatin

2004 ◽  
Vol 181 (2) ◽  
pp. 307-314 ◽  
Author(s):  
KL Jones ◽  
DM De Kretser ◽  
DJ Phillips
Keyword(s):  
Area Under The Curve ◽  
Activin A ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Large Pool ◽  
Heparin Administration ◽  
Repeat Administration ◽  
Clearance Mechanism ◽  
Exclusion Chromatography ◽  
Dose Dependent

Activin A and follistatin are normally present in relatively low amounts in the circulation. Heparin administration elicits a rapid and robust release of these proteins, although this phenomenon is poorly defined. In the present studies, the response to heparin administration was evaluated in the plasma of adult ewes in terms of whether it was dose-dependent, could be neutralized, was responsive to multiple stimulation, and the nature of the activin A and follistatin released. Activin A and follistatin were rapidly released by heparin in a dose-dependent manner (25, 100 or 250 IU/kg), with differences in the response as adjudged by peak concentration, timing of the peak and area under the curve. The heparin response could be blocked by pretreatment with protamine; conversely protamine injection alone (2 mg/kg) elicited release of follistatin but not activin A. Repeat administration of heparin at three-hourly intervals resulted in activin and follistatin responses to each injection, but each subsequent stimulation increased and extended the responses, consistent with saturation of the heparin clearance mechanism. Size exclusion chromatography of plasma samples confirmed that the majority of activin and follistatin released by heparin was a complex, whereas follistatin released by protamine was unbound. These data are consistent with a large pool of activin A and follistatin resident on extracellular matrices, with the rapid response implicating the vascular endothelium as the prime site of release following administration of these commonly used anticoagulant therapies.

scholarly journals The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

2020 ◽  
Author(s):  
Jaehyoun Lee ◽  
Seunghee Oh ◽  
Saikat Bhattacharya ◽  
Ying Zhang ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Pyruvate Dehydrogenase ◽  
Biochemical Analysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Acetyl Coa ◽  
Cell Extracts ◽  
Exclusion Chromatography ◽  
Dehydrogenase Complex

ABSTRACTThe pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

scholarly journals The N-Terminal Domain That Distinguishes Yeast from Bacterial RNase III Contains a Dimerization Signal Required for Efficient Double-Stranded RNA Cleavage

2000 ◽  
Vol 20 (4) ◽  
pp. 1104-1115 ◽  
Author(s):  
Bruno Lamontagne ◽  
Annie Tremblay ◽  
Sherif Abou Elela
Keyword(s):  
Size Exclusion ◽  
Rna Cleavage ◽  
Rnase Iii ◽  
Double Stranded Rna ◽  
Terminal Domain ◽  
Dsrna Binding ◽  
Exclusion Chromatography ◽  
Dsrna Cleavage ◽  
Two Hybrid

ABSTRACT Yeast Rnt1 is a member of the double-stranded RNA (dsRNA)-specific RNase III family identified by conserved dsRNA binding (dsRBD) and nuclease domains. Comparative sequence analyses have revealed an additional N-terminal domain unique to the eukaryotic homologues of RNase III. The deletion of this domain from Rnt1 slowed growth and led to mild accumulation of unprocessed 25S pre-rRNA. In vitro, deletion of the N-terminal domain reduced the rate of RNA cleavage under physiological salt concentration. Size exclusion chromatography and cross-linking assays indicated that the N-terminal domain and the dsRBD self-interact to stabilize the Rnt1 homodimer. In addition, an interaction between the N-terminal domain and the dsRBD was identified by a two-hybrid assay. The results suggest that the eukaryotic N-terminal domain of Rnt1 ensures efficient dsRNA cleavage by mediating the assembly of optimum Rnt1-RNA ribonucleoprotein complex.

Domain unfolding and the stability of thermolysin in guanidine hydrochloride

1986 ◽  
Vol 64 (10) ◽  
pp. 953-961 ◽  
Author(s):  
Ronald J. T. Corbett ◽  
Faizan Ahmad ◽  
Rodney S. Roche
Keyword(s):  
Guanidine Hydrochloride ◽  
Kinetic Studies ◽  
Conformational Transitions ◽  
Size Exclusion ◽  
Structural Domains ◽  
Terminal Domain ◽  
Exclusion Chromatography ◽  
Terbium Ion ◽  
The Stability ◽  
Denatured States

Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)–S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.

scholarly journals The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243489
Author(s):  
Jaehyoun Lee ◽  
Seunghee Oh ◽  
Saikat Bhattacharya ◽  
Ying Zhang ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Pyruvate Dehydrogenase ◽  
Biochemical Analysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Acetyl Coa ◽  
Cell Extracts ◽  
Exclusion Chromatography ◽  
Dehydrogenase Complex

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

Chromatographic Separation of Single Wall Carbon Nanotubes

2006 ◽  
Vol 922 ◽  
Author(s):  
Barry J. Bauer ◽  
Vardhan Bajpai ◽  
Jeffrey A. Fagan ◽  
Matthew L. Becker ◽  
Erik K. Hobbie
Keyword(s):  
Carbon Nanotubes ◽  
Chemical Modification ◽  
Size Exclusion ◽  
Covalent Attachment ◽  
Single Wall Carbon Nanotubes ◽  
Single Wall Carbon ◽  
Force Microscopy ◽  
Atomic Force ◽  
Exclusion Chromatography ◽  
Chromatographic Detection

AbstractSize exclusion chromatography (SEC) has been used to separate single wall carbon nanotubes (SWNT) dispersed by chemical modification in organic solvents and by DNA in aqueous solution. The chromatographic detection includes size sensitive detectors, multi-angle light scattering (MALS) and intrinsic viscosity (IV), which can provide information on the size and shape of the SEC fractions. The dispersions were also characterized by small angle neutron scattering (SANS) and atomic force microscopy (AFM). Chemical modification was accomplished by covalent attachment of octadecyl amine to acid treated SWNT and by covalent attachment of butyl groups through free radical grafting. Both covalent attachment methods produced dispersions that contained impurities or clusters of SWNT. The DNA dispersions produced the best dispersions, being predominately single nanotubes.

scholarly journals Impact of crystal packing on coiled-coil flexibility.

2014 ◽  
Vol 70 (a1) ◽  
pp. C1599-C1599
Author(s):  
François Ferron ◽  
David Blocquel ◽  
Johnny Habchi ◽  
Eric Durand ◽  
Marion Sevajol ◽  
...  
Keyword(s):  
Measles Virus ◽  
Quaternary Structure ◽  
Crystal Packing ◽  
Coiled Coil ◽  
Size Exclusion ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Partial Unfolding

The structural characterization of various constructs of the Measles virus (MeV) Phosphoprotein (P) multimerization domain (PMD) has brought to light significant discrepancies in the quaternary structure due to both crystal constraints and the flexible nature of this coiled-coil. Indeed, despite a conserved tetrameric parallel coiled-coil core, structural comparison unveiled significant deformations in the C-terminal extremities that even led to the partial unfolding of the coiled-coil. These deformations were induced by intermolecular interactions within the crystal, as well as by the crystallization condition. These deformations also suggest that PMD has the ability to adapt to external mechanical constrains. Using a combination of biophysical methods (size-exclusion chromatography, circular dichroism and small angle X-ray scattering), we assessed the differential flexibility of the C-terminal region of the MeV PMD in solution. Taken together, these results show that crystal packing can be used to "freeze" in a certain state, parts of proteins known to be in a dynamic folding-unfolding equilibrium. They also bring awareness that conclusions about function and mechanism based on analysis of a single crystal structure of a known dynamic protein can be easily biased, and they challenge to some extent the assumption that coiled-coil structures can be reliably predicted from the amino acid sequence.

scholarly journals Palmitoylation of Hedgehog proteins by Hedgehog acyltransferase: roles in signalling and disease

Open Biology ◽  
2021 ◽  
Vol 11 (3) ◽  
Author(s):  
Marilyn D. Resh
Keyword(s):  
Covalent Attachment ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Hedgehog Signalling ◽  
Disease States ◽  
Efficient Delivery ◽  
Hedgehog Proteins ◽  
Hedgehog Acyltransferase

Hedgehog acyltransferase (Hhat), a member of the membrane-boundO-acyltransferase (MBOAT) family, catalyses the covalent attachment of palmitate to the N-terminus of Hedgehog proteins. Palmitoylation is a post-translational modification essential for Hedgehog signalling. This review explores the mechanisms involved in Hhat acyltransferase enzymatic activity, similarities and differences between Hhat and other MBOAT enzymes, and the role of palmitoylation in Hedgehog signalling.In vitroand cell-based assays for Hhat activity have been developed, and residues within Hhat and Hedgehog essential for palmitoylation have been identified. In cells, Hhat promotes the transfer of palmitoyl-CoA from the cytoplasmic to the luminal side of the endoplasmic reticulum membrane, where Shh palmitoylation occurs. Palmitoylation is required for efficient delivery of secreted Hedgehog to its receptor Patched1, as well as for the deactivation of Patched1, which initiates the downstream Hedgehog signalling pathway. While Hhat loss is lethal during embryogenesis, mutations in Hhat have been linked to disease states or abnormalities in mice and humans. In adults, aberrant re-expression of Hedgehog ligands promotes tumorigenesis in an Hhat-dependent manner in a variety of different cancers, including pancreatic, breast and lung. Targeting hedgehog palmitoylation by inhibition of Hhat is thus a promising, potential intervention in human disease.

scholarly journals The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP

2020 ◽  
Vol 295 (50) ◽  
pp. 16960-16974
Author(s):  
Shrestha Chakraborty ◽  
Maitree Biswas ◽  
Sanjay Dey ◽  
Shubhangi Agarwal ◽  
Tulika Chakrabortty ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Regulatory Protein ◽  
Size Exclusion ◽  
Atp Binding ◽  
Dependent Manner ◽  
Class Iii ◽  
Independent Manner ◽  
Atp Binding Site ◽  
Enhancer Binding Protein ◽  
Exclusion Chromatography

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in “cis-mediated” style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC. Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

Structure-function studies of the asparaginyl-tRNA synthetase from Fasciola gigantica: understanding the role of catalytic and non-catalytic domains

2018 ◽  
Vol 475 (21) ◽  
pp. 3377-3391 ◽  
Author(s):  
Vijayakumar Rajendran ◽  
Rohit Shukla ◽  
Harish Shukla ◽  
Timir Tripathi
Keyword(s):  
Trna Synthetase ◽  
Fasciola Gigantica ◽  
Dynamics Simulation ◽  
Size Exclusion ◽  
Intrinsically Disordered ◽  
Catalytic Domains ◽  
Terminal Domain ◽  
Exclusion Chromatography ◽  
Cognate Trna

The asparaginyl-tRNA synthetase (NRS) catalyzes the attachment of asparagine to its cognate tRNA during translation. NRS first catalyzes the binding of Asn and ATP to form the NRS-asparaginyl adenylate complex, followed by the esterification of Asn to its tRNA. We investigated the role of constituent domains in regulating the structure and activity of Fasciola gigantica NRS (FgNRS). We cloned the full-length FgNRS, along with its various truncated forms, expressed, and purified the corresponding proteins. Size exclusion chromatography indicated a role of the anticodon-binding domain (ABD) of FgNRS in protein dimerization. The N-terminal domain (NTD) was not essential for cognate tRNA binding, and the hinge region between the ABD and the C-terminal domain (CTD) was crucial for regulating the enzymatic activity. Molecular docking and fluorescence quenching experiments elucidated the binding affinities of the substrates to various domains. The molecular dynamics simulation of the modeled protein showed the presence of an unstructured region between the NTD and ABD that exhibited a large number of conformations over time, and further analysis indicated this region to be intrinsically disordered. The present study provides information on the structural and functional regulation, protein-substrate(s) interactions and dynamics, and the role of non-catalytic domains in regulating the activity of FgNRS.

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