An X-ray structure of the lactose permease of Escherichia coli (LacY) in an inward-facing conformation has been solved. LacY contains N-and C-terminal domains, each with six transmembrane helices, positioned pseudosymmetrically. Ligand is bound at the apex of a hydrophilic cavity in the approximate middle of the molecule. Residues involved in substrate binding and H + translocation are aligned parallel to the membrane at the same level and may be exposed to a water-filled cavity in both the inward-and outward-facing conformations, thereby allowing both sugar and H + release directly into either cavity. These structural features may explain why LacY catalyzes galactoside/H + symport in both directions utilizing the same residues. A working model for the mechanism is presented that involves alternating access of both the sugar-and H + -binding sites to either side of the membrane.

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces displayed by native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each of which is built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family display favorable behavior relative to conventional detergents, as tested on multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same global fold as the previous x-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry separated by a large interior hydrophilic cavity open only to the cytoplasmic side and containing the side chains important for sugar and H ؉ binding. To initiate transport, binding of sugar and/or an H ؉ electrochemical gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.conformation ͉ mechanism ͉ membrane protein ͉ transport ͉ x-ray structure

The bacterial melibiose permease (MelB) belongs to the glycoside-pentoside-hexuronide:cation symporter family (GPH), a part of the major facilitator superfamily (MFS). Structural information regarding GPH transporters and other Na+-coupled permeases within MFS has been lacking, although a wealth of biochemical and biophysical data are available. Here we present the 3D crystal structures of Salmonella typhimurium MelBSt in two conformations, representing an outward partially occluded and an outward inactive state of MelBSt. MelB adopts a typical MFS fold, and contains a previously unidentified cation-binding motif. Three conserved acidic residues form a pyramidal-shaped cation-binding site for Na+, Li+, or H+, which is in close proximity to the sugar-binding site. Both co-substrate-binding sites are mainly contributed by the residues from the N-terminal domain. These two structures and the functional data presented here provide mechanistic insights into Na+/melibiose symport. We also postulate a structural foundation for the conformational cycling necessary for transport catalyzed by MFS permeases in general.

Cation-coupled active transport is an essential cellular process found ubiquitously in all living organisms. Here, we present two novel ligand-free X-ray structures of the lactose permease (LacY) of Escherichia coli determined at acidic and neutral pH, and propose a model for the mechanism of coupling between lactose and H þ translocation. No sugar-binding site is observed in the absence of ligand, and deprotonation of the key residue Glu 269 is associated with ligand binding. Thus, substrate induces formation of the sugar-binding site, as well as the initial step in H þ transduction.

In eukaryotic cells, the primary cation responsible for the electrochemical gradient across membranes is Na ϩ , to which most symporters are coupled. In certain Na ϩ symporters, H ϩ and Li ϩ can substitute for Na ϩ ; examples are the human Na ϩ /nucleoside cotransporter (CNT3) (1) and the human Na ϩ /glucose cotransporter (SGLT) (2). In bacterial membranes, the electrochemical gradient is built by H ϩ , and H ϩ / solute cotransporters are most common. Bacterial melibiose permease (MelB) 2 exhibits a unique property that couples sugar transport with Na ϩ , Li ϩ , or H ϩ and selects the cation depending on the transported sugar structure (3). The multiple coupling feature of this group of transporters yields a useful tool to study the mechanism of cation/sugar symport.Bacterial MelB belongs to the glycoside/pentoside/hexuronide:cation family (4), a subgroup of the major facilitator superfamilies of membrane transport proteins, where the lactose permease (LacY) is the best characterized member (5, 6). MelB of Escherichia coli (MelB-EC) is the best characterized member among all MelB orthologues (7-16). MelB-EC catalyzes the coupled stoichiometric symport of a galactoside with a cation (Na ϩ , Li ϩ , or H ϩ ) utilizing the free energy from the downhill translocation of one cosubstrate to catalyze the translocation of the other (3,(17)(18)(19)(20), and all three cations compete for a common binding pocket (21-23).The primary sequence alignment between MelB-EC and LacY is relatively poor with ϳ37% sequence similarity and ϳ15% identity; however, membrane topology studies of MelB (24 -26) suggested a topology similar to LacY, with 12 transmembrane helices and cytoplasmically located N and C termini. A three-dimensional structure model of MelB was recently built by threading analysis (27), using a crystal structure (PDB ID 1PV6) of LacY (28,29) as the template. The model suggested a similar overall fold between these two permeases; i.e. MelB is organized in two-helix bundles connected with a central loop and separated by an internal cavity facing the cytoplasmic side. From bioinformatics data, this overall fold seems conserved among MelB orthologues (27). Moreover, this model is consistent with numerous previous biochemical/biophysical data (14, 30 -37), as well as low-resolution EM structures obtained from MelB-EC (38, 39).Diverse cation selectivity was identified in MelB orthologues (40). Although it shares high sequence identity with MelB-EC, MelB of Klebsiella pneumoniae couples melibiose transport only to H ϩ and Li

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBC's genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three m6A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring ‘the methylated-motif-site ratio’ and ‘the methylated-read ratio’, we explored the methylation status of each modified site and sequence-read to obtain the ‘precision methylome’ of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.

BackgroundTuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is one of the major causes of death in the world today. Although China has the second largest global case rate of tuberculosis, a systematic study of TB prevalence in China has not been completed. From 2006 to 2007, the base line surveillance of tuberculosis was carried out by Ministry of Health, and more than 4000 representative strains were selected from 31 provinces in China.Methodology/Principal FindingsThe aim of the present research was to survey the genotypes of representative Mycobacterium tuberculosis (M. tuberculosis) strains from China using spacer oligonucleotide typing (spoligotyping), and to analyze the relationship between genotype and drug resistance for the first time. A total of 4017 clinical isolates were collected from 2007 to 2008 throughout China. Among those M. tuberculosis isolates, 2500 (62.2%) isolates were Beijing genotypes. The percentage of Beijing genotypes in northern China was higher than in southern China (76.5% vs. 53.2%). Additionally, the frequencies of rifampin-resistant, ofloxacin-resistant and multidrug-resistant isolates were significantly higher in Beijing genotype strains than non-Beijing strains. Furthermore, a novel genotype named “China Southern genotype (CS)” was only isolated from Fujian and Guangdong provinces. Hence, it is very practical to uncover the reason for prevalence of the CS type in southern China.Conclusions/SignificanceIn conclusion, Beijing family genotypes were still the predominant genotype throughout China, which exhibited a greater correlation with rifampin-resistance, ofloxacin-resistance and MDR phenotypes than other TB spoligotypes, and some regions of China showed several unique characters in the distribution of M. tuberculosis genotypes. Our research represents an important contribution for the TB control and research community, which completes broad pictures on drug resistance levels and distribution of M. tuberculosis strain types over China.