Molecular basis for fibroblast growth factor 23 O-glycosylation by GalNAc-T3.

Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3's structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.

Protein O-Fucosyltransferase 1 Undergoes Interdomain Flexibility in Solution.

Protein O-fucosyltransferase 1 (PoFUT1) is a GT-B fold enzyme that fucosylates proteins containing EGF-like repeats. GT-B glycosyltransferases have shown a remarkable grade of plasticity adopting closed and open conformations as a way of tuning their catalytic cycle, a feature that has not been observed for PoFUT1. Here, we analyzed Caenorhabditis elegans PoFUT1 (CePoFUT1) conformational behavior in solution by atomic force microscopy (AFM) and single-molecule fluorescence resonance energy transfer (SMF-FRET). Our results show that this enzyme is very flexible and adopts mainly compact conformations and to a lesser extend a highly dynamic population that oscillates between compact and highly extended conformations. Overall, our experiments illustrate the inherent complexity of CePoFUT1 dynamics, which might play a role during its catalytic cycle.

Molecular basis for the integration of environmental signals by FurB from Anabaena sp. PCC 7120.

FUR (Ferric uptake regulator) proteins are among the most important families of transcriptional regulators in prokaryotes, often behaving as global regulators. In the cyanobacterium Anabaena PCC 7120, FurB (Zur, Zinc uptake regulator) controls zinc and redox homeostasis through the repression of target genes in a zinc-dependent manner. In vitro, non-specific binding of FurB to DNA elicits protection against oxidative damage and avoids cleavage by deoxyribonuclease I. The present study provides, for the first time, evidence of the influence of redox environment in the interaction of FurB with regulatory zinc and its consequences in FurB-DNA-binding affinity. Calorimetry studies showed that, in addition to one structural Zn(II), FurB is able to bind two additional Zn(II) per monomer and demonstrated the implication of cysteine C93 in regulatory Zn(II) coordination. The interaction of FurB with the second regulatory zinc occurred only under reducing conditions. While non-specific FurB-DNA interaction is Zn(II)-independent, the optimal binding of FurB to target promoters required loading of two regulatory zinc ions. Those results combined with site-directed mutagenesis and gel-shift assays evidenced that the redox state of cysteine C93 conditions the binding of the second regulatory Zn(II) and, in turn, modulates the affinity for a specific DNA target. Furthermore, differential spectroscopy studies showed that cysteine C93 could also be involved in heme coordination by FurB, either as a direct ligand or being located near the binding site. The results indicate that besides controlling zinc homeostasis, FurB could work as a redox-sensing protein probably modifying its zinc and DNA-binding abilities depending upon environmental conditions.

A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.

Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions.

Microcystin-LR Binds Iron, and Iron Promotes Self-Assembly.

The microcystin-producing Microcystis aeruginosa PCC 7806 and its close strain, the nonproducing Microcystis aeruginosa PCC 7005, grow similarly in the presence of 17 µM iron. Under severe iron deficient conditions (0.05 µM), the toxigenic strain grows slightly less than in iron-replete conditions, while the nonproducing microcystin strain is not able to grow. Isothermal titration calorimetry performed at cyanobacterial cytosol or meaningful environmental pHs values shows a microcystin-LR dissociaton constant for Fe2+ and Fe3+ of 2.4 µM. Using atomic force microscopy, 40% of microcystin-LR dimers were observed, and the presence of iron promoted its oligomerization up to six units. Microcystin-LR binds also Mo6+, Cu2+, and Mn2+. Polymeric microcystin binding iron may be related with a toxic cell colony advantage, providing enhanced iron bioavailability and perhaps affecting the structure of the gelatinous sheath. Inside cells, with microcystin implicated in the fitness of the photosynthetic machinery under stress conditions, the toxin would be involved in avoiding metal-dependent Fenton reactions when photooxidation causes disassembly of the iron-rich photosystems. Additionally, it could be hypothesized that polymerization-depolymerization dynamics may be an additional signal that could trigger changes (for example, in the binding of microcystin to proteins).

A physical picture for mechanical dissociation of biological complexes: from forces to free energies.

Single-molecule force spectroscopy is a powerful technique based on the application of controlled forces to macromolecules. In order to relate the measured response of the molecule to its equilibrium and dynamic properties, a suitable physical picture of the involved process is necessary. In this work, we introduce a plausible model for mechanical unbinding of some molecular complexes, based on a novel free energy profile. We combine two standard theoretical frameworks for analyzing force spectroscopy experiments on two protein:protein complexes, obtaining key magnitudes of the underlying free energy profile, which are only understood within the mentioned model. Additionally, we carry out detailed stochastic dynamics simulations to prove the validity of the analysis protocol and the reliability of the free energy profile. Remarkably, we can compare directly the obtained unbinding free energies with the previously known bulk binding free energies, bridging the gap between bulk and single molecule techniques.

Quaternary organization in a bifunctional prokaryotic FAD synthetase: Involvement of an arginine at its adenylyltransferase module on the riboflavin kinase activity.

Prokaryotic FAD synthetases (FADSs) are bifunctional enzymes composed of two modules, the C-terminal module with ATP:riboflavin kinase (RFK) activity, and the N-terminus with ATP:FMN adenylyltransferase (FMNAT) activity. The FADS from Corynebacterium ammoniagenes, CaFADS, forms transient oligomers during catalysis. These oligomers are stabilized by several interactions between the RFK and FMNAT sites from neighboring protomers, which otherwise are separated in the monomeric enzyme. Among these inter-protomer interactions, the salt bridge between E268 at the RFK site and R66 at the FMNAT-module is particularly relevant, as E268 is the catalytic base of the kinase reaction. Here we have introduced point mutations at R66 to analyze the impact of the salt-bridge on ligand binding and catalysis. Interestingly, these mutations have only mild effects on ligand binding and kinetic properties of the FMNAT-module (where R66 is located), but considerably impair the RFK activity turnover. Substitutions of R66 also modulate the ratio between monomeric and oligomeric species and modify the quaternary arrangement observed by single-molecule methods. Therefore, our data further support the cross-talk between the RFK- and FMNAT-modules of neighboring protomers in the CaFADS enzyme, and establish the participation of R66 in the modulation of the geometry of the RFK active site during catalysis.

Key Residues Regulating the Reductase Activity of the Human Mitochondrial Apoptosis Inducing Factor.

The human Apoptosis Inducing Factor (hAIF) is a bifunctional NAD(P)H-dependent flavoreductase involved in both mitochondrial energy metabolism and caspase-independent cell death. Even though several studies indicate that both functions are redox controlled by NADH binding, the exact role of hAIF as a reductase in healthy mitochondria remains unknown. Upon reduction by NADH, hAIF dimerizes and produces very stable flavin/nicotinamide charge transfer complexes (CTC), by stacking of the oxidized nicotinamide moiety of the NAD(+) coenzyme against the re-face of the reduced flavin ring of its FAD cofactor. Such complexes are critical to restrict the hAIF efficiency as a reductase. The molecular basis of the hAIF reductase activity is here investigated by analyzing the role played by residues contributing to the interaction of the FAD isoalloxazine ring and of the nicotinamide moiety of NADH at the active site. Mutations at K177 and E314 produced drastic effects on the hAIF ability to retain the FAD cofactor, indicating that these residues are important to set up the holo-enzyme active site conformation. Characterization of P173G hAIF indicates that the stacking of P173 against the isoalloxazine ring is relevant to determine the flavin environment and to modulate the enzyme affinity for NADH. Finally, the properties of the F310G and H454S hAIF mutants indicate that these two positions contribute to form a compact active site essential for NADH binding, CTC stabilization, and NAD(+) affinity for the reduced state of hAIF. These features are key determinants of the particular behavior of hAIF as a NADH-dependent oxidoreductase.

Sequential binding of FurA from Anabaena sp. PCC 7120 to iron boxes: exploring regulation at the nanoscale.

Fur (ferric uptake regulator) proteins are involved in the control of a variety of processes in most prokaryotes. Although it is assumed that this regulator binds its DNA targets as a dimer, the way in which this interaction occurs remains unknown. We have focused on FurA from the cyanobacterium Anabaena sp. PCC 7120. To assess the molecular mechanism by which FurA specifically binds to "iron boxes" in PfurA, we examined the topology arrangement of FurA-DNA complexes by atomic force microscopy. Interestingly, FurA-PfurA complexes exhibit several populations, in which one is the predominant and depends clearly on the regulator/promoter ratio on the environment. Those results together with EMSA and other techniques suggest that FurA binds PfurA using a sequential mechanism: (i) a monomer specifically binds to an "iron box" and bends PfurA; (ii) two situations may occur, that a second FurA monomer covers the free "iron box" or that joins to the previously used forming a dimer which would maintain the DNA kinked; (iii) trimerization in which the DNA is unbent; and (iv) finally undergoes a tetramerization; the next coming molecules cover the DNA strands unspecifically. In summary, the bending appears when an "iron box" is bound to one or two molecules and decreases when both "iron boxes" are covered. These results suggest that DNA bending contributes at the first steps of FurA repression promoting the recruitment of new molecules resulting in a fine regulation in the Fur-dependent cluster associated genes.

Mechanostability of the Single-Electron-Transfer Complexes of Anabaena Ferredoxin-NADP(+) Reductase.

The complexes formed between the flavoenzyme ferredoxin-NADP(+) reductase (FNR; NADP(+) =nicotinamide adenine dinucleotide phosphate) and its redox protein partners, ferredoxin (Fd) and flavodoxin (Fld), have been analysed by using dynamic force spectroscopy through AFM. A strategy is developed to immobilise proteins on a substrate and AFM tip to optimise the recognition ability. The differences in the recognition efficiency regarding a random attachment procedure, together with nanomechanical results, show two binding models for these systems. The interaction of the reductase with the natural electron donor, Fd, is threefold stronger and its lifetime is longer and more specific than that with the substitute under iron-deficient conditions, Fld. The higher bond probability and two possible dissociation pathways in Fld binding to FNR are probably due to the nature of this complex, which is closer to a dynamic ensemble model. This is in contrast with the one-step dissociation kinetics that has been observed and a specific interaction described for the FNR:Fd complex.

Structural insights into the coenzyme mediated monomer-dimer transition of the pro-apoptotic apoptosis inducing factor.

The apoptosis-inducing factor (AIF) is a mitochondrial-flavoprotein that, after cell death induction, is distributed to the nucleus to mediate chromatinolysis. In mitochondria, AIF is present in a monomer-dimer equilibrium that after reduction by NADH gets displaced toward the dimer. The crystal structure of the human AIF (hAIF):NAD(H)-bound dimer revealed one FAD and, unexpectedly, two NAD(H) molecules per protomer. A 1:2 hAIF:NAD(H) binding stoichiometry was additionally confirmed in solution by using surface plasmon resonance. The here newly discovered NAD(H)-binding site includes residues mutated in human disorders, and accommodation of the coenzyme in it requires restructuring of a hAIF portion within the 509-560 apoptogenic segment. Disruption of interactions at the dimerization surface by production of the hAIF E413A/R422A/R430A mutant resulted in a nondimerizable variant considerably less efficiently stabilizing charge-transfer complexes upon coenzyme reduction than WT hAIF. These data reveal that the coenzyme-mediated monomer-dimer transition of hAIF modulates the conformation of its C-terminal proapoptotic domain, as well as its mechanism as reductase. These observations suggest that both the mitochondrial and apoptotic functions of hAIF are interconnected and coenzyme controlled: a key information in the understanding of the physiological role of AIF in the cellular life and death cycle.

Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level.

Biochemical characterization of Corynebacterium ammoniagenes FADS (CaFADS) pointed to certain confusion about the stoichiometry of this bifunctional enzyme involved in the production of FMN and FAD in prokaryotes. Resolution of its crystal structure suggested that it might produce a hexameric ensemble formed by a dimer of trimers. We used atomic force microscopy (AFM) to direct imaging single CaFADS molecules bound to mica surfaces, while preserving their catalytic properties. AFM allowed solving individual CaFADS monomers, for which it was even possible to distinguish their sub-molecular individual N- and C-terminal modules in the elongated enzyme. Differences between monomers and higher stoichiometries were easily imaged, enabling us to detect formation of oligomeric species induced by ligand binding. The presence of ATP:Mg(2+) particularly induced the appearance of the hexameric assembly whose mean molecular volume resembles the crystallographic dimer of trimers. Finally, the AFM results are confirmed in cross-linking solution, and the presence of such oligomeric CaFADS species detected in cell extracts. All these results are consistent with the formation of a dimer of trimers during the enzyme catalytic cycle that might bear biological relevance.

NS3 protease from hepatitis C virus: biophysical studies on an intrinsically disordered protein domain.

The nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) is responsible for processing the non-structural region of the viral precursor polyprotein in infected hepatic cells. NS3 protease activity, located at the N-terminal domain, is a zinc-dependent serine protease. A zinc ion, required for the hydrolytic activity, has been considered as a structural metal ion essential for the structural integrity of the protein. In addition, NS3 interacts with another cofactor, NS4A, an accessory viral protein that induces a conformational change enhancing the hydrolytic activity. Biophysical studies on the isolated protease domain, whose behavior is similar to that of the full-length protein (e.g., catalytic activity, allosteric mechanism and susceptibility to inhibitors), suggest that a considerable global conformational change in the protein is coupled to zinc binding. Zinc binding to NS3 protease can be considered as a folding event, an extreme case of induced-fit binding. Therefore, NS3 protease is an intrinsically (partially) disordered protein with a complex conformational landscape due to its inherent plasticity and to the interaction with its different effectors. Here we summarize the results from a detailed biophysical characterization of this enzyme and present new experimental data.

Recent advances in sensing the inter-biomolecular interactions at the nanoscale - A comprehensive review of AFM-based force spectroscopy.

Biomolecular interactions underpin most processes inside the cell. Hence, a precise and quantitative understanding of molecular association and dissociation events is crucial, not only from a fundamental perspective, but also for the rational design of biomolecular platforms for state-of-the-art biomedical and industrial applications. In this context, atomic force microscopy (AFM) appears as an invaluable experimental technique, allowing the measurement of the mechanical strength of biomolecular complexes to provide a quantitative characterization of their interaction properties from a single molecule perspective. In the present review, the most recent methodological advances in this field are presented with special focus on bioconjugation, immobilization and AFM tip functionalization, dynamic force spectroscopy measurements, molecular recognition imaging and theoretical modeling. We expect this work to significantly aid in grasping the principles of AFM-based force spectroscopy (AFM-FS) technique and provide the necessary tools to acquaint the type of data that can be achieved from this type of experiments. Furthermore, a critical assessment is done with other nanotechnology techniques to better visualize the future prospects of AFM-FS.

Beyond a platform protein for the degradosome assembly: The Apoptosis-Inducing Factor as an efficient nuclease involved in chromatinolysis.

The Apoptosis-Inducing Factor (AIF) is a moonlighting flavoenzyme involved in the assembly of mitochondrial respiratory complexes in healthy cells, but also able to trigger DNA cleavage and parthanatos. Upon apoptotic-stimuli, AIF redistributes from the mitochondria to the nucleus, where upon association with other proteins such as endonuclease CypA and histone H2AX, it is proposed to organize a DNA-degradosome complex. In this work, we provide evidence for the molecular assembly of this complex as well as for the cooperative effects among its protein components to degrade genomic DNA into large fragments. We have also uncovered that AIF has nuclease activity that is stimulated in the presence of either Mg2+ or Ca2+. Such activity allows AIF by itself and in cooperation with CypA to efficiently degrade genomic DNA. Finally, we have identified TopIB and DEK motifs in AIF as responsible for its nuclease activity. These new findings point, for the first time, to AIF as a nuclease able to digest nuclear dsDNA in dying cells, improving our understanding of its role in promoting apoptosis and opening paths for the development of new therapeutic strategies.

Hydrocolloids of Egg White and Gelatin as a Platform for Hydrogel-Based Tissue Engineering.

Innovative materials are needed to produce scaffolds for various tissue engineering and regenerative medicine (TERM) applications, including tissue models. Materials derived from natural sources that offer low production costs, easy availability, and high bioactivity are highly preferred. Chicken egg white (EW) is an overlooked protein-based material. Whilst its combination with the biopolymer gelatin has been investigated in the food technology industry, mixed hydrocolloids of EW and gelatin have not been reported in TERM. This paper investigates these hydrocolloids as a suitable platform for hydrogel-based tissue engineering, including 2D coating films, miniaturized 3D hydrogels in microfluidic devices, and 3D hydrogel scaffolds. Rheological assessment of the hydrocolloid solutions suggested that temperature and EW concentration can be used to fine-tune the viscosity of the ensuing gels. Fabricated thin 2D hydrocolloid films presented globular nano-topography and in vitro cell work showed that the mixed hydrocolloids had increased cell growth compared with EW films. Results showed that hydrocolloids of EW and gelatin can be used for creating a 3D hydrogel environment for cell studies inside microfluidic devices. Finally, 3D hydrogel scaffolds were fabricated by sequential temperature-dependent gelation followed by chemical cross-linking of the polymeric network of the hydrogel for added mechanical strength and stability. These 3D hydrogel scaffolds displayed pores, lamellae, globular nano-topography, tunable mechanical properties, high affinity for water, and cell proliferation and penetration properties. In conclusion, the large range of properties and characteristics of these materials provide a strong potential for a large variety of TERM applications, including cancer models, organoid growth, compatibility with bioprinting, or implantable devices.

Molecular Recognition of Proteins through Quantitative Force Maps at Single Molecule Level.

Intermittent jumping force is an operational atomic-force microscopy mode that produces simultaneous topography and tip-sample maximum-adhesion images based on force spectroscopy. In this work, the operation conditions have been implemented scanning in a repulsive regime and applying very low forces, thus avoiding unspecific tip-sample forces. Remarkably, adhesion images give only specific rupture events, becoming qualitative and quantitative molecular recognition maps obtained at reasonably fast rates, which is a great advantage compared to the force-volume modes. This procedure has been used to go further in discriminating between two similar protein molecules, avidin and streptavidin, in hybrid samples. The adhesion maps generated scanning with biotinylated probes showed features identified as avidin molecules, in the range of 40-80 pN; meanwhile, streptavidin molecules rendered 120-170 pN at the selected working conditions. The gathered results evidence that repulsive jumping force mode applying very small forces allows the identification of biomolecules through the specific rupture forces of the complexes and could serve to identify receptors on membranes or samples or be applied to design ultrasensitive detection technologies.

Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP.

Plastidic ferredoxin-NADP+ reductase (FNR) transfers two electrons from two ferredoxin or flavodoxin molecules to NADP+, generating NADPH. The forces holding the Anabaena FNR:NADP+ complex were analyzed by dynamic force spectroscopy, using WT FNR and three C-terminal Y303 variants, Y303S, Y303F, and Y303W. FNR was covalently immobilized on mica and NADP+ attached to AFM tips. Force-distance curves were collected for different loading rates and specific unbinding forces were analyzed under the Bell-Evans model to obtain the mechanostability parameters associated with the dissociation processes. The WT FNR:NADP+ complex presented a higher mechanical stability than that reported for the complexes with protein partners, corroborating the stronger affinity of FNR for NADP+. The Y303 mutation induced changes in the FNR:NADP+ interaction mechanical stability. NADP+ dissociated from WT and Y303W in a single event related to the release of the adenine moiety of the coenzyme. However, two events described the Y303S:NADP+ dissociation that was also a more durable complex due to the strong binding of the nicotinamide moiety of NADP+ to the catalytic site. Finally, Y303F shows intermediate behavior. Therefore, Y303, reported as crucial for achieving catalytically competent active site geometry, also regulates the concerted dissociation of the bipartite nucleotide moieties of the coenzyme.

Oligomerization properties of FurA from the cyanobacterium Anabaena sp. PCC 7120: direct visualization by in situ atomic force microscopy under different redox conditions.

Fur proteins are global prokaryotic transcriptional regulators. Functional studies of FurA from the cyanobacterium Anabaena sp. PCC 7120 evidenced the influence of the redox environment in the activity of the regulator and its ability to aggregate through disulphide bridges. Atomic force microscopy allows single-molecule imaging and monitorization of the status of FurA under different redox conditions mimicking a physiological environment. The estimated FurA average diameter was of 4 nm. In the absence of reducing agents, the purified FurA is mainly associated as trimers, being 40 degrees the prevalent angle alpha conformed by protein monomers. Reducing conditions induces trimer rearrangement to protein monomers and a major fraction of FurA dimers. Disruption of the dimeric assemblies and appearance of higher order aggregates, namely trimers and tetramers are induced by oxidation with diamide or hydrogen peroxide. The homogeneity of the angles exhibited by the trimeric particles, as well as the occurrence of dimers in the presence of DTT, suggests the participation of relatively specific hydrophobic interactions maintaining the dimer. Direct visualization of the regulator under liquid phase at molecular resolution unravels the importance of non-polar interactions in FurA dynamics and shows that in Anabaena disulphide bridges are not essential for the dimerization of FurA.

Atomic Force Microscopy: Single-Molecule Imaging and Force Spectroscopy in the Study of Flavoproteins Ligand Binding and Reaction Mechanisms.

Atomic force microscopy (AFM) is one of the most versatile tools currently used in nanoscience. AFM allows for performing nondestructive imaging of almost any sample in either air or liquid, regardless whether the specimen is insulating, conductive, transparent, or opaque. It also allows for measuring interaction forces between a sharp probe and a sample surface, therefore allowing to probe nanomechanical properties of the specimen by either applying a controlled force or pulling the sample. It can provide topography, mechanical, magnetic, and conductive maps for very different type of samples. Transferred to the field of biology, today, AFM is the only microscopy technique able to produce images from biomolecules to bacteria and cells with nanometric resolution in aqueous media. Here, we will focus on the biological applications of AFM to flavoproteins. Despite references in the literature are scarce in this particular field, here it is described how imaging with AFM can contribute to describe catalysis mechanisms of some flavoenzymes, how oxidation states or binding of relevant ligands influence the association state of molecules, the dynamics of functional quaternary assemblies, and even visualize structural differences of individual protein molecules. Furthermore, we will show how force spectroscopy can be used to obtain the kinetic parameters, the dissociation landscape and the mechanical forces that maintain flavoprotein complexes, including the possibility to specifically detect particular flavoproteins on a sample.