We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries.

Ammonium is one of the most important nitrogen sources for bacteria, fungi, and plants, but it is toxic to animals. The ammonium transport proteins (methylamine permeases͞ammonium transporters͞rhesus) are present in all domains of life; however, functional studies with members of this family have yielded controversial results with respect to the chemical identity (NH 4 ؉ or NH 3) of the transported species. We have solved the structure of wild-type AmtB from Escherichia coli in two crystal forms at 1.8-and 2.1-Å resolution, respectively. Substrate transport occurs through a narrow mainly hydrophobic pore located at the center of each monomer of the trimeric AmtB. At the periplasmic entry, a binding site for NH 4 ؉ is observed. Two phenylalanine side chains (F107 and F215) block access into the pore from the periplasmic side. Further into the pore, the side chains of two highly conserved histidine residues (H168 and H318) bridged by a H-bond lie adjacent, with their edges pointing into the cavity. These histidine residues may facilitate the deprotonation of an ammonium ion entering the pore. Adiabatic free energy calculations support the hypothesis that an electrostatic barrier between H168 and H318 hinders the permeation of cations but not that of the uncharged NH 3. The structural data and energetic considerations strongly indicate that the methylamine permeases͞ammonium transporters͞rhesus proteins are ammonia gas channels. Interestingly, at the cytoplasmic exit of the pore, two different conformational states are observed that might be related to the inactivation mechanism by its regulatory partner.conformational change ͉ x-ray structure

Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine.

The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A 2 to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/ acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a "sideways sliding" mechanism to explain how a conserved membrane-embedded ␣-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane.

exchangers exhibit a common Ca 2ϩ -dependent regulatory mechanism, whereby their activity requires the presence of low concentrations of Ca 2ϩ on their intracellular surface, and their activity is augmented in parallel with elevated intracellular Ca 2ϩ levels (3). This important regulatory property may permit the timely coupling of exchange function to alterations in intracellular Ca 2ϩ concentrations to meet the continuous needs for overall Ca 2ϩ balance. The general similarities of exchange function and regulatory properties within the large NCX protein family are ascribed to their conserved structural arrangements: nine predicted transmembrane (TM) segments form the ion translocation pathway and a large loop of ϳ500 amino acid residues splits TM helix-5 and -6 on the intracellular side of the molecule (4). Ca 2ϩ -dependent regulation is attributed exclusively to Ca 2ϩ interactions on the intracellular loop (5). A pair of Ca 2ϩ binding domains (CBD1 and -2), called CALX-␤ motifs, has been identified (6). Sequence analysis revealed that CBD1 has conserved Ca 2ϩ binding sites throughout the NCX family, whereas greater sequence diversity and/or Ca 2ϩ binding capabilities occurs in CBD2 (7,27). Given that CBD1 exhibits a higher Ca 2ϩ affinity than CBD2 (8), it has been suggested that CBD1 acts as the primary sensor in the pair of CBDs. Mutations of carboxylate residues at CBD1 result in a pronounced reduction of the affinity for functional Ca 2ϩ regulation (9). The Ca 2ϩ -bound structures of CBD1 of NCX1 have recently been determined by NMR, and more recently by x-ray crystallography (8,11

CaCAs are integral membrane proteins containing 10-11 predicted transmembrane helices (TMs) (1) (SI Appendix, Fig. S1). Despite the divergence in cation driving force, the superfamily is defined by the presence of two highly conserved α-repeat motifs in TMs 2-3 (α-1) and TMs 7-8 (α-2). The importance of these conserved motifs for Ca 2+ /cation exchange has been well documented in all characterized CaCA proteins (8-10), implying the conservation of the Ca 2+ /cation translocation mechanism in the CaCA superfamily. Recently, Liao et al. reported the crystal structure of the NCX_Mj protein (6). The structure shows a large cavity opening on the periplasmic surface (outward-facing conformation) with both Ca 2+ and Na + bound at the α-repeat regions, suggesting the following hypotheses: (i) a substrate alternating access mechanism in which Ca 2+ and the counter transported cation access the ion-binding sites from each side of the membrane surface; and (ii) transition between outward-facing and inward-facing conformations is required to achieve Ca 2+ /cation exchange. Although the structure of the outward-facing conformation of NCX_Mj is available, structural evidence for the conformational change essential for alternating substrate access has not yet been obtained for any CaCA member. In addition, how the CaCA superfamily proteins modify their Ca 2+ -translocation pathways to use the different cation-gradient driving forces, H + vs. Na + , remains a fundamental and interesting question.To address these important questions, here, we report the crystal structure of an inward-facing conformation of the YfkE protein. This structure, together with protein kinetic analysis, not only provides an important structural characterization of the mechanism of Ca 2+ efflux across the cell membrane but also sheds light on the different modes of energy coupling used by members of the CaCA protein superfamily. Results Ca 2+-Transport Specificity and Structural Determination of YfkE. YfkE catalyzes a H+ -coupled Ca 2+ influx in everted vesicles (SI Appendix, Fig. S2A This article is a PNAS Direct Submission.Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4KJR and 4KJS).

No abstract