1,720,986 research outputs found
Effects of microwaves (900 MHz) on peroxidase systems: a comparison between lactoperoxidase and horseradish peroxidase
No full textSubstrate-induced conformational change in cytochrome P450 OleP
No full textThe regulation of cytochrome P450 activity is often achieved by structural transitions induced by substrate binding. We describe the conformational transition experienced upon binding by the P450 OleP, an epoxygenase involved in oleandomycin biosynthesis. OleP bound to the substrate analog 6DEB crystallized in 2 forms: one with an ensemble of open and closed conformations in the asymmetric unit and another with only the closed conformation. Characterization of OleP-6DEB binding kinetics, also using the P450 inhibitor clotrimazole, unveiled a complex binding mechanism that involves slow conformational rearrangement with the accumulation of a spectroscopically detectable intermediate where 6DEB is bound to open OleP. Data reported herein provide structural snapshots of key precatalytic steps in the OleP reaction and explain how structural rearrangements induced by substrate binding regulate activity
Side-chain interactions form late and cooperatively in the binding reaction between disordered peptides and PDZ domains
No full textLack of orientation selectivity of the heme insertion in murine neuroglobin revealed by resonance Raman spectroscopy
No full textDifferent murine neuroglobin variants showing structural and dynamic alterations which are associated with perturbation of ligand binding have been studied. The CD loop mutants characterized by an enhanced flexibility (Gly-loop40-48 and Gly-loop44-47 ), the F106A mutant, and the double Gly-loop44-47 /F106A mutant. Their ferric resonance Raman spectra in solution and in crystals are almost identical. In the high frequency region, the identification of a double set of core size marker bands indicates the presence of two 6-coordinate low spin species. The resonance Raman data, together with the corresponding crystal structures, indicate the presence of two neuroglobin conformers with a reversed (A conformer) or a canonical (B conformer) heme insertion orientation. With the identification of the marker bands corresponding to each conformer, the data indicates that the B conformer increases at the expense of the A form, predominantly in the Gly-loop44-47 /F106A double mutant, as confirmed by X-ray crystallography. This is the first time that a reversed heme insertion has been identified by resonance Raman in a native 6-coordinate low spin heme protein. This diagnostic tool could be extended to other heme proteins in order to detect heme orientational disorder, which are likely to be correlated to functionally relevant heme dynamics
Proximal and distal control for ligand binding in neuroglobin: role of the CD loop and evidence for His64 gating
Get PDFNeuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant "Gly-loop", the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs
Cryo-EM structure of the human ferritin–transferrin receptor 1 complex
Get PDFHuman transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate
Structure of the adenylation domain thr1 involved in the biosynthesis of 4-chlorothreonine in Streptomyces sp. OH-5093 - protein flexibility and molecular bases of substrate specificity
No full textWe determined the crystal structure of Thr1, the self-standing adenylation domain involved in the nonribosomal-like biosynthesis of free 4-chlorothreonine in Streptomyces sp. OH-5093. Thr1 shows two monomers in the crystallographic asymmetric unit with different relative orientations of the C- and N-terminal subdomains both in the presence of substrates and in the unliganded form. Cocrystallization with substrates, adenosine 5'-triphosphate and l-threonine, yielded one monomer containing the two substrates and the other in complex with l-threonine adenylate, locked in a postadenylation state. Steady-state kinetics showed that Thr1 activates l-Thr and its stereoisomers, as well as d-Ala, l- and d-Ser, albeit with lower efficiency. Modeling of these substrates in the active site highlighted the molecular bases of substrate discrimination. This work provides the first crystal structure of a threonine-activating adenylation enzyme, a contribution to the studies on conformational rearrangement in adenylation domains and on substrate recognition in nonribosomal biosynthesis
The Nuts and Bolts of SARS-CoV-2 Spike Receptor-Binding Domain Heterologous Expression
Get PDFCOVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful “tool” to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry
Effect of Salts on the Conformational Dynamics of the Cytochrome P450 OleP
Get PDFCytochrome P450 OleP catalytic activity is strongly influenced by its structural dynamic conformational behavior. Here, we combine equilibrium-binding experiments with all-atom molecular dynamics simulations to clarify how different environments affect OleP conformational equilibrium between the open and the closed—catalytic competent—forms. Our data clearly show that at high-ionic strength conditions, the closed form is favored, and, very interestingly, different mechanisms, depending on the chemistry of the cations, can be used to rationalize such an effect
Subcellular localization of the five members of the human steroid 5α-reductase family
Get PDFIn humans the steroid 5a-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in
lipidic substrates: D4-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the
more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum
localization.
We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5a-reductase family as both N- and Cterminus
green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the
subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of
the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates
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