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.

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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.

Magnesium is the most abundant divalent cation in living cells and is crucial to several biological processes. MgtE is a Mg2+ channel distributed in all domains of life that contributes to the maintenance of cellular Mg2+ homeostasis. Here we report the high-resolution crystal structures of the transmembrane domain of MgtE, bound to Mg2+, Mn2+ and Ca2+. The high-resolution Mg2+-bound crystal structure clearly visualized the hydrated Mg2+ 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 Mg2+ 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.

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.

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.

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.

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