Protein O-Fucosyltransferase 1 Undergoes Interdomain Flexibility in Solution

Lira‐Navarrete, Erandi; Pallarés, María Carmen; Castello, Fabio; Ruedas-Rama, María J.; Orte, Ángel; Lostao, Anabel; Hurtado‐Guerrero, R.

doi:10.3390/molecules26082105

Cited by 5 publications

(5 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Despite the great power of enzymatic processes, their potential has not been fully exploited, mainly due to the limited understanding of the mechanisms regarding the assembly and dissociation between reacting molecules. Recent advances in imaging methods have demonstrated that it is possible to make direct observations of the dynamic behavior of single molecules [3,4] and to determine the mechanisms of action at the single-molecule level [5][6][7]. Among the available singlemolecule methods, those designed to measure forces between molecules or within an individual molecule stand out [8].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…GTs have been well-characterized via both molecular dynamics simulations and static crystal structures. ,,– However, experiments designed to report on in-solution dynamics, such as 19 F NMR spectroscopy for PimA, single-molecule Förster resonance energy transfer for Fut1, and intrinsic tryptophan fluorescence for HepI, , are less common. This gap in characterization may explain the difficulties faced in identifying potent inhibitors for this class of enzymes.…”

mentioning

confidence: 99%

HDX-MS Reveals Substrate-Dependent, Localized EX1 Conformational Dynamics in the Retaining GT-B Glycosyltransferase, MshA

Karki,

Hennek,

Chen

et al. 2023

Biochemistry

View full text Add to dashboard Cite

Glycosyltransferases (GTs) are well-characterized with respect to static 3D structures and molecular dynamics simulations, but there is a lack of reports on in-solution dynamics on time scales relevant to turnover. Here, backbone amide hydrogen/deuterium exchange followed by mass spectrometry (HDX-MS) was used to investigate the in-solution dynamics of the model retaining GT MshA from Corynebacterium glutamicum (CgMshA). CgMshA has a GT-B fold and catalyzes the transfer of N-acetyl-glucosamine (GlcNAc) from UDP−GlcNAc to L-myoinositol-1-phosphate in the first step in mycothiol biosynthesis. HDX-MS results identify several key regions of conformational changes in response to UDP−GlcNAc binding, including residues 159−198 in the N-terminal domain and residues 323−354 in the C-terminal domain. These regions also exhibited substratedependent EX1 exchange kinetics consistent with conformational tension on the milliseconds to seconds time scale. A potential source of this conformational change is the flexible β4/α5 loop in the C-terminal domain, which sits at the interface of the two domains and likely interacts with the GlcNAc ring of UDP−GlcNAc. In contrast to UDP−GlcNAc, the UDP−CgMshA product complex exhibited severe decreases in deuterium incorporation, suggesting a less dynamic conformation. The HDX-MS results are complemented by solvent viscosity effects of 1.8−2.3 on the CgMshA k cat value, which are consistent with product release as a ratedetermining step and possibly a direct role for protein dynamics in catalysis. The identification of in-solution dynamics that are sensitive to substrate binding allows for the proposal of a more detailed mechanism in CgMshA including conformation tension between the donor sugar and the flexible C-terminal domain β4/α5 loop providing sufficient conformational sampling for substrateassisted catalysis to occur.

show abstract

“…On the contrary, 2D images for GvFFTR and CaFFTR2 homodimers, despite displaying some particularly preferred topologies, exhibit a range of them, with some features appearing more elongated and others slightly more compact ( Figure 2 a,b; see 2D images for homodimers). Considering that (i) the protein-mica interactions are weaker than covalent or other protein:ligand interactions, (ii) AFM imaging enables transient dynamical conformations to occur without losing protein functionality, structure and quaternary conformations [ 21 , 25 ], and (iii) conformations visualized agree with other parallel single molecule studies as small angle X-ray scattering (SAXS) experiments [ 26 ] and single-molecule fluorescence resonance energy transfer (SMF-FRET) measurements [ 27 ], the AFM images here presented must reflect homodimers with a slightly different relative disposition of their disulfide domains relative to the main protein core ( Figure 5 ). Nonetheless, all GvFFTR and GaFFTR2 images clearly reflect FR conformations, in agreement with the evaluated oxidized states.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, mica surface chemistry hardly alters the morphology, conformation, or association of the molecules, and in general, only causes the molecules, or their complexes, to be weakly immobilized by some area of their surface, preferably mainly by electrostatic adsorption through their exposed hydroxyl groups [ 28 , 29 ]. Therefore, imaging of enzymes by AFM at nearly physiologically relevant conditions, in the presence of reactants, products, or partners, is nowadays even used to understand catalytic pathways that occur through the formation of specific transient quaternary organizations [ 21 , 30 ], or even through large conformational changes upon ligand binding [ 26 , 27 ]. Nonetheless, there have been few studies evaluating protein morphology changes in detail [ 20 , 31 , 32 , 33 , 34 , 35 , 36 ].…”

Section: Discussionmentioning

confidence: 99%

Atomic Force Microscopy to Elicit Conformational Transitions of Ferredoxin-Dependent Flavin Thioredoxin Reductases

et al. 2021

Self Cite

View full text Add to dashboard Cite

Flavin and redox-active disulfide domains of ferredoxin-dependent flavin thioredoxin reductase (FFTR) homodimers should pivot between flavin-oxidizing (FO) and flavin-reducing (FR) conformations during catalysis, but only FR conformations have been detected by X-ray diffraction and scattering techniques. Atomic force microscopy (AFM) is a single-molecule technique that allows the observation of individual biomolecules with sub-nm resolution in near-native conditions in real-time, providing sampling of molecular properties distributions and identification of existing subpopulations. Here, we show that AFM is suitable to evaluate FR and FO conformations. In agreement with imaging under oxidizing condition, only FR conformations are observed for Gloeobacter violaceus FFTR (GvFFTR) and isoform 2 of Clostridium acetobutylicum FFTR (CaFFTR2). Nonetheless, different relative dispositions of the redox-active disulfide and FAD-binding domains are detected for FR homodimers, indicating a dynamic disposition of disulfide domains regarding the central protein core in solution. This study also shows that AFM can detect morphological changes upon the interaction of FFTRs with their protein partners. In conclusion, this study paves way for using AFM to provide complementary insight into the FFTR catalytic cycle at pseudo-physiological conditions. However, future approaches for imaging of FO conformations will require technical developments with the capability of maintaining the FAD-reduced state within the protein during AFM scanning.

show abstract

Protein O-Fucosyltransferase 1 Undergoes Interdomain Flexibility in Solution

Cited by 5 publications

References 52 publications

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

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

HDX-MS Reveals Substrate-Dependent, Localized EX1 Conformational Dynamics in the Retaining GT-B Glycosyltransferase, MshA

Atomic Force Microscopy to Elicit Conformational Transitions of Ferredoxin-Dependent Flavin Thioredoxin Reductases

Contact Info

Product

Resources

About