Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

Electron-bifurcation is a fundamental energy conservation mechanism in nature. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic primarily due to the lack of structural information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure is a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC heterotrimers electrically connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and proton reduction. Symmetry expansion identified two conformations of a flexible iron-sulfur cluster domain: a “closed bridge” and an “open bridge” conformation, where a Zn2+ site may act as a “hinge” allowing domain movement. Based on these structural revelations, we propose two new mechanisms of electron-bifurcation in HydABC.

Cryo-EM structures reveal the electromotility mechanism of prestin, the cochlear amplifier

Outer hair cell electromotility, driven by prestin, is essential for mammalian cochlear amplification. Here, we report the cryo-EM structures of thermostabilized human prestin (hPresTS), complexed with chloride, sulfate, or salicylate at 3.52–3.61 Å resolutions, revealing a crossed dimeric arrangement. The central positively-charged cavity allows flexible binding of various anion species, resulting in distinct modulations of nonlinear capacitance (NLC), playing an important role in electromotility. Comparisons of these hPresTS structures suggest rigid-body movement between the core and gate domains, and provide mechanistic insight into prestin inhibition by salicylate. Mutations at the dimeric interface severely diminished NLC, suggesting that stabilization of the gate domain facilitates core domain movement, thereby contributing to the expression of NLC. These findings advance our understanding of the molecular mechanism underlying mammalian cochlear amplification.

Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

Electron-bifurcation is a fundamental energy conservation mechanism in nature. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic primarily due to the lack of structural information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure is a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC trimers electrically connected via a [4Fe-4S] cluster, forming a bus-bar system. Symmetry expansion identified two conformations: a “closed bridge” and an “open bridge” conformation, where a Zn2+ site may act as a “hinge” allowing domain movement. Based on these structural revelations, we propose two new mechanisms of electron-bifurcation in HydABC.

Mn ions' site and valence in PbTiO3 based on the native vacancy defects

Mn ions' doping site and valence were studied in PbTiO3 (PT) with the native vacancy defects by the first-principles calculations. Firstly, the native vacancy defects of Pb, O and Ti in PT were investigated and it was found that Pb vacancy is preferred to others. And then the growth of Mn doped PT should be preferred to Mn ion substituting for an A-site Pb ion with +3 valence when Pb is deficient under equilibrium conditions driven solely by minimization of the formation energy, and this could result in a larger lattice distortion of PT. In addition, when Mn enters the Pb site, the electronegativity of O becomes weaker which makes the domain movement easier in PT to improve the performance of PT, while Mn ion substitution for a B-site Ti ion is the opposite.

Conformational flexibility within the small domain of human serine racemase

Serine racemase (SR) is a pyridoxal 5′-phosphate (PLP)-containing enzyme that converts L-serine to D-serine, an endogenous co-agonist for the N-methyl-D-aspartate receptor (NMDAR) subtype of glutamate ion channels. SR regulates D-serine levels by the reversible racemization of L-serine to D-serine, as well as the catabolism of serine by α,β-elimination to produce pyruvate. The modulation of SR activity is therefore an attractive therapeutic approach to disorders associated with abnormal glutamatergic signalling since it allows an indirect modulation of NMDAR function. In the present study, a 1.89 Å resolution crystal structure of the human SR holoenzyme (including the PLP cofactor) with four subunits in the asymmetric unit is described. Comparison of this new structure with the crystal structure of human SR with malonate (PDB entry 3l6b) shows an interdomain cleft that is open in the holo structure but which disappears when the inhibitor malonate binds and is enclosed. This is owing to a shift of the small domain (residues 78–155) in human SR similar to that previously described for the rat enzyme. This domain movement is accompanied by changes within the twist of the central four-stranded β-sheet of the small domain, including changes in the φ–ψ angles of all three residues in the C-terminal β-strand (residues 149–151). In the malonate-bound structure, Ser84 (a catalytic residue) points its side chain at the malonate and is preceded by a six-residue β-strand (residues 78–83), but in the holoenzyme the β-strand is only four residues (78–81) and His82 has φ–ψ values in the α-helical region of the Ramachandran plot. These data therefore represent a crystallographic platform that enables the structure-guided design of small-molecule modulators for this important but to date undrugged target.

Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homologue with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified two outward- and one inward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by Cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.