Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes

Enzymes known as bifunctional and bimodular prokaryotic type-I FAD synthetase (FADS) exhibit ATP:riboflavin kinase (RFK) and FMN:ATP adenylyltransferase (FMNAT) activities in their C-Terminal and N-Terminal modules, respectively, and produce flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These act as cofactors of a plethora of flavoproteins in all organisms. Therefore, regulation of their production maintains the cellular flavoproteome homeostasis. Here, we focus on regulation of the FMN synthesis in Corynebacterium ammoniagenes (Ca) by the inhibition of its RFK activity by substrates and products of the reaction. We use a truncated CaFADS variant consisting in the isolated C-Terminal RFK module, whose RFK activity is similar to that of the full-length enzyme. Inhibition of the RFK activity by the RF substrate is independent of the FMNAT module, and FMN production, in addition to being inhibited by an excess of RF, is also inhibited by both of the reaction products. Pre-steady-state kinetic and thermodynamic studies reveal key aspects to the substrates induced fit to produce the catalytically competent complex. Among them, the role of Mg2+ in the concerted allocation of substrates for catalysis and the ensemble of non-competent complexes that contribute to the regulated inhibition of the RFK activity are particularly relevant

A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility.

Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A-H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A-H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A-H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription

The paralogue of the intrinsically disordered nuclear protein 1 has a nuclear localization sequence that binds to human importin a3

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin a, with several human isoforms; among them, importin a3 (Impa3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Impa3 and its depleted species, ¿Impa3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp a3 with a low micromolar affinity (~5 µM). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to ¿Impa3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Impa3, confirming that the IBB could act as an auto-inhibition region of Impa3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation

Residual helicity at the active site of the histidine phosphocarrier, HPr, modulates binding affinity to its natural partners

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first a-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first a-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. The refore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Streptococcus pneumoniae TIGR4 flavodoxin: Structural and biophysical characterization of a novel drug target

Streptococcus pneumoniae (Sp) strain TIGR4 is a virulent, encapsulated serotype that causes bacteremia, otitis media, meningitis and pneumonia. Increased bacterial resistance and limited efficacy of the available vaccine to some serotypes complicate the treatment of diseases associated to this microorganism. Flavodoxins are bacterial proteins involved in several important metabolic pathways. The Sp flavodoxin (Spfld) gene was recently reported to be essential for the establishment of meningitis in a rat model, which makes SpFld a potential drug target. To facilitate future pharmacological studies, we have cloned and expressed SpFld in E. coli and we have performed an extensive structural and biochemical characterization of both the apo form and its active complex with the FMN cofactor. SpFld is a short-chain flavodoxin containing 146 residues. Unlike the well-characterized long-chain apoflavodoxins, the Sp apoprotein displays a simple two-state thermal unfolding equilibrium and binds FMN with moderate affinity. The X-ray structures of the apo and holo forms of SpFld differ at the FMN binding site, where substantial rearrangement of residues at the 91-100 loop occurs to permit cofactor binding. This work will set up the basis for future studies aiming at discovering new potential drugs to treat S. pneumoniae diseases through the inhibition of SpFld.We acknowledge financial support from BFU2010-16297 and BFU2010-19504 [Ministerio de Ciencia e Innovación Spain], BFU2013-47064-P, BIO2014-57314-REDT and CTQ2013-44367-C2-2-P [Ministerio de Economía y Competitividad, Spain], and DGA (Protein Targets B89). We also thank synchrotron radiation sources DLS (Oxford), and in particular beamline I04-1 (experiment number MX8035-3 and MX8035-11). The research leading to these results has also received funding from the FP7 (2007–2013) under BIOSTRUCTX-7687. A.R.C. was funded by a Banco Santander Central Hispano/Universidad de Zaragoza predoctoral fellowship. M. C-G was recipient of a predoctoral fellowship from the Government of Aragón.Peer Reviewe

Function of cofactor Akirin2 in the regulation of gene expression in model human Caucasian neutrophil-like HL60 cells

The Akirin family of transcription cofactors are involved throughout the metazoan in the regulation of different biological processes (BPs) such as immunity, interdigital regression, muscle and neural development. Akirin do not have catalytic or DNA-binding capability and exert its regulatory function primarily through interacting proteins such as transcription factors, chromatin remodelers, and RNA-associated proteins. In the present study, we focused on the human Akirin2 regulome and interactome in neutrophil-like model human Caucasian promyelocytic leukemia HL60 cells. Our hypothesis is that metazoan evolved to have Akirin2 functional complements and different Akirin2-mediated mechanisms for the regulation of gene expression. To address this hypothesis, experiments were conducted using transcriptomics, proteomics and systems biology approaches in akirin2 knockdown and wildtype (WT) HL60 cells to characterize Akirin2 gene/protein targets, functional complements and to provide evidence of different mechanisms that may be involved in Akirin2-mediated regulation of gene expression. The results revealed Akirin2 gene/protein targets in multiple BPs with higher representation of immunity and identified immune response genes as candidate Akirin2 functional complements. In addition to linking chromatin remodelers with transcriptional activation, Akirin2 also interacts with histone H3.1 for regulation of gene expression. © 2021 The Author(s)

A phosphorylation-induced switch in the nuclear localization sequence of the intrinsically disordered nupr1 hampers binding to importin

Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin a, of which there are several human isoforms; among them, importin a3 (Impa3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impa3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impa3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impa3 with low micromolar affinity (1.7–27 µM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i, i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impa3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure

Inhibition of the PP2A activity by the histone chaperone ANP32B is long-range allosterically regulated by respiratory cytochrome c

Repair of injured DNA relies on nucleosome dismantling by histone chaperones and de-phosphorylation events carried out by Protein Phosphatase 2A (PP2A). Typical histone chaperones are the Acidic leucine-rich Nuclear Phosphoprotein 32 family (ANP32) members, e.g. ANP32A, which is also a well-known PP2A inhibitor (a.k.a. I1PP2A). Here we report the novel interaction between the endogenous family member B—so-called ANP32B—and endogenous cytochrome c in cells undergoing camptothecin-induced DNA damage. Soon after DNA lesions but prior to caspase cascade activation, the hemeprotein translocates to the nucleus to target the Low Complexity Acidic Region (LCAR) of ANP32B; in a similar way, our group recently reported that the hemeprotein targets the acidic domain of SET/Template Activating Factor-Iß (SET/TAF-Iß), which is another histone chaperone and PP2A inhibitor (a.k.a. I2PP2A). The nucleosome assembly activity of ANP32B is indeed unaffected by cytochrome c binding. Like ANP32A, ANP32B inhibits PP2A activity and is thus herein referred to as I3PP2A. Our data demonstrates that ANP32B-dependent inhibition of PP2A is regulated by respiratory cytochrome c, which induces long-distance allosteric changes in the structured N-terminal domain of ANP32B upon binding to the C-terminal LCAR. In agreement with the reported role of PP2A in the DNA damage response, we propose a model wherein cytochrome c is translocated from the mitochondria into the nucleus upon DNA damage to modulate PP2A activity via its interaction with ANP32B. © 2021 The Author(s

Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47

Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c – namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death – is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.Peer reviewe

Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches