A multipoint guidance mechanism for ß-barrel folding on the SAM complex.

Mitochondrial ß-barrel proteins are essential for the transport of metabolites, ions and proteins. The sorting and assembly machinery (SAM) mediates their folding and membrane insertion. We report the cryo-electron microscopy structure of the yeast SAM complex carrying an early eukaryotic ß-barrel folding intermediate. The lateral gate of Sam50 is wide open and pairs with the last ß-strand (ß-signal) of the substrate-the 19-ß-stranded Tom40 precursor-to form a hybrid barrel in the membrane plane. The Tom40 barrel grows and curves, guided by an extended bridge with Sam50. Tom40's first ß-segment (ß1) penetrates into the nascent barrel, interacting with its inner wall. The Tom40 amino-terminal segment then displaces ß1 to promote its pairing with Tom40's last ß-strand to complete barrel formation with the assistance of Sam37's dynamic α-protrusion. Our study thus reveals a multipoint guidance mechanism for mitochondrial ß-barrel folding.

The structure of MgtE in the absence of magnesium provides new insights into channel gating.

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Mitochondrial sorting and assembly machinery operates by ß-barrel switching.

The mitochondrial outer membrane contains so-called ß-barrel proteins, which allow communication between the cytosol and the mitochondrial interior1-3. Insertion of ß-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)4-6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8-3.2 Å. The dimeric complex contains two copies of the ß-barrel channel protein Sam50-Sam50a and Sam50b-with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the ß-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed ß-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a ß-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of ß-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for ß-barrel assembly.

Continuum modeling for neuronal lamination during cerebral morphogenesis considering cell migration and tissue growth.

For neuronal lamination during cerebral morphogenesis, later-born neurons must migrate through already-accumulated neurons. This neuronal migration is biochemically regulated by signaling molecules and mechanically affected by tissue deformation. To understand the neuronal lamination mechanisms, we constructed a continuum model of neuronal migration in a growing deformable tissue. We performed numerical analyses considering the migration promoted by signaling molecules and the tissue growth induced by neuron accumulation. The results suggest that the promoted migration and the space ensured by tissue growth are essential for neuronal lamination. The proposed model can describe the coupling of mechanical and biochemical mechanisms for neuronal lamination.

An energy landscape approach to understanding variety and robustness in tissue morphogenesis.

During morphogenesis in development, multicellular tissues deform by mechanical forces induced by spatiotemporally regulated cellular activities, such as cell proliferation and constriction. Various morphologies are formed because of various spatiotemporal combinations and sequences of multicellular activities. Despite its potential to variations, morphogenesis is a surprisingly robust process, in which qualitatively similar morphologies are reproducibly formed even under spatiotemporal fluctuation of multicellular activities. To understand these essential characteristics of tissue morphogenesis, which involves the coexistence of various morphologies and robustness of the morphogenetic process, in this study, we propose a novel approach to capture the overall view of morphogenesis from mechanical viewpoints. This approach will enable visualization of the energy landscape, which includes morphogenetic processes induced by admissible histories of cellular activities. This approach was applied to investigate the morphogenesis of a sheet-like tissue with curvature, where it deformed to a concave or convex morphology depending on the history of growth and constriction. Qualitatively different morphologies were produced by bifurcation of the valley in the energy landscape. The depth and steepness of the valley near the stable states represented the degree of robustness to fluctuations of multicellular activities. Furthermore, as a realistic example, we showed an application of this approach to luminal folding observed in the initial stage of intestinal villus formation. This approach will be helpful to understand the mechanism of how various morphologies are formed and how tissues reproducibly achieve specific morphologies.

ATP-dependent modulation of MgtE in Mg2+ homeostasis.

Magnesium is an essential ion for numerous physiological processes. MgtE is a Mg2+ selective channel involved in the maintenance of intracellular Mg2+ homeostasis, whose gating is regulated by intracellular Mg2+ levels. Here, we report that ATP binds to MgtE, regulating its Mg2+-dependent gating. Crystal structures of MgtE-ATP complex show that ATP binds to the intracellular CBS domain of MgtE. Functional studies support that ATP binding to MgtE enhances the intracellular domain affinity for Mg2+ within physiological concentrations of this divalent cation, enabling MgtE to function as an in vivo Mg2+ sensor. ATP dissociation from MgtE upregulates Mg2+ influx at both high and low intracellular Mg2+ concentrations. Using site-directed mutagenesis and structure based-electrophysiological and biochemical analyses, we identify key residues and main structural changes involved in the process. This work provides the molecular basis of ATP-dependent modulation of MgtE in Mg2+ homeostasis.MgtE is an Mg2+ transporter involved in Mg2+ homeostasis. Here, the authors report that ATP regulates the Mg+2-dependent gating of MgtE and use X-ray crystallography combined with functional studies to propose the molecular mechanisms involved in this process.

Structural basis for dynamic mechanism of nitrate/nitrite antiport by NarK.

NarK belongs to the nitrate/nitrite porter (NNP) family in the major facilitator superfamily (MFS) and plays a central role in nitrate uptake across the membrane in diverse organisms, including archaea, bacteria, fungi and plants. Although previous studies provided insight into the overall structure and the substrate recognition of NarK, its molecular mechanism, including the driving force for nitrate transport, remained elusive. Here we demonstrate that NarK is a nitrate/nitrite antiporter, using an in vitro reconstituted system. Furthermore, we present the high-resolution crystal structures of NarK from Escherichia coli in the nitrate-bound occluded, nitrate-bound inward-open and apo inward-open states. The integrated structural, functional and computational analyses reveal the nitrate/nitrite antiport mechanism of NarK, in which substrate recognition is coupled to the transport cycle by the concomitant movement of the transmembrane helices and the key tyrosine and arginine residues in the substrate-binding site.

Structural basis for ion selectivity revealed by high-resolution crystal structure of Mg2+ channel MgtE.

Magnesium is the most abundant divalent cation in living cells and is crucial to several biological processes. MgtE is a Mg(2+) channel distributed in all domains of life that contributes to the maintenance of cellular Mg(2+) homeostasis. Here we report the high-resolution crystal structures of the transmembrane domain of MgtE, bound to Mg(2+), Mn(2+) and Ca(2+). The high-resolution Mg(2+)-bound crystal structure clearly visualized the hydrated Mg(2+) ion within its selectivity filter. Based on those structures and biochemical analyses, we propose a cation selectivity mechanism for MgtE in which the geometry of the hydration shell of the fully hydrated Mg(2+) ion is recognized by the side-chain carboxylate groups in the selectivity filter. This is in contrast to the K(+)-selective filter of KcsA, which recognizes a dehydrated K(+) ion. Our results further revealed a cation-binding site on the periplasmic side, which regulate channel opening and prevents conduction of near-cognate cations.

Phenol-oxidizing enzyme expression in Lentinula edodes by the addition of sawdust extract, aromatic compounds, or copper in liquid culture media.

This study examined how the addition of a sawdust extract from Castanopsis cuspidata, several aromatic compounds, and copper affected the expression of a phenol-oxidizing enzyme in the white-rot basidiomycete, Lentinula edodes. Compared to liquid media that had not been supplemented with sawdust extract (MYPG), MYPG containing low (MYPG-S100) or high (MYPG-S500) concentrations of sawdust extract had a marked effect on the promotion of mycelial growth. No manganese peroxidase (MnP) production was observed in either MYPG or MYPG-S100 media until 35 days after inoculation. However, MnP production was enhanced by culture in MYPG-S500, with a marked increase observed suddenly at 14 days after inoculation. Northern blot analysis revealed that the transcription of the lemnp2 gene coding extracellular MnP was initially observed at detectable levels at day 10 after the initial inoculation of MYPG-S500, increasing gradually thereafter until days 22-25. However, laccase (Lcc) production was not observed in any of the media until 35 days after inoculation. Addition of 10 mM aromatic compounds - 1,2-benzenediol, 2-methoxyphenol, hydroquinone, and 4-anisidine--into the MYPG-S500 medium completely inhibited MnP production and did not enhance any Lcc production. While the addition of 1 or 2 mM Cu2+ (CuSO4 x 5H2O) to MYPG-S500 medium completely inhibited MnP production, this Cu2+ addition caused a marked increase in Lcc production at 17 and 6 days after the addition, respectively.

Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins.

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, the 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (Phos-tag), which is linked to a highly cross-linked 4% (w/v) agarose. The affinity gel (Phos-tag agarose) was prepared by the quantitative reaction of N-hydroxysuccinimide-activated Sepharose and a Phos-tag derivative having a 2-aminoethylcarbamoyl group in dry CH3CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos-tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, alpha(s1)-casein, and beta-casein at physiological pH.

Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex.

Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex (Zn2L3+) has been studied (L = alkoxide species of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol). Potentiometric pH titration study disclosed a 1 : 1 phenyl phosphate complexation with Zn2L3+ in aqueous solution. The dissociation constant (= [Zn2L3+][PhOPO3(2-)]/[Zn2L3+-PhOPO3(2-)]) is an extremely small value of 2.5 x 10(-8) mol dm(-3) at 25 degrees C with I = 0.10 (NaNO3). The X-ray crystal analysis of the dizinc(II) complex with p-nitrophenyl phosphate showed that the phosphate dianion binds as a bridging ligand to the two zinc(II) ions.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule.

This paper introduces a simple, rapid, and sensitive procedure for the analysis of phosphorylated compounds (ROPO(3) (2-)) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The method is based on a characteristic mass shift and a total-charge change (from -2 to +1) of the phosphate residue due to complexation of ROPO(3) (2-) with a dinuclear zinc(II) complex (1,3-bis[bis(2-pyridinylmethyl)amino]-2-propanolato dizinc(II) complex, Zn(2)L(3+)) in aqueous solution at physiological pH. Furthermore, the use of single zinc-isotope derivatives ((64)Zn(2)L(3+) and (68)Zn(2)L(3+)) enables improvement of the sensitivity and accuracy of the analysis.