A Structural Model for the Osmosensor, Transporter, and Osmoregulator ProP of<i>Escherichia coli</i>

Wood, Janet M.; Culham, Doreen E.; Hillar, Alexander; Vernikovska, Yaroslava I.; Liu, Feng; Boggs, Joan M.; Keates, Robert A. B.

doi:10.1021/bi047383o

Cited by 43 publications

(103 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the future, this issue will be examined quantitatively, using new tools for substrate binding experiments that could be useful in a wide range of pH conditions. In summary, the results of the kPROT analysis and the successful construction of a 3D model for MdfA strongly support the prediction that MFS transporters generally have a similar fold in the membrane (21)(22)(23)(24)(25). The 3D model of MdfA, as presented here, further substantiates important previous predictions regarding the mechanism underlying Mdr transport.…”

Section: Discussionsupporting

confidence: 85%

3D Model of the Escherichia coli Multidrug Transporter MdfA Reveals an Essential Membrane-Embedded Positive Charge

et al. 2005

View full text Add to dashboard Cite

MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and GlpT. However, because of poor sequence homology, between LacY, GlpT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of GlpT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and GlpT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.

show abstract

Section: Discussionsupporting

confidence: 85%

3D Model of the Escherichia coli Multidrug Transporter MdfA Reveals an Essential Membrane-Embedded Positive Charge

et al. 2005

View full text Add to dashboard Cite

show abstract

“…The osmolality at which ProP activity is half-maximal (⌸ 1/2 /RT) depends on the structure of the transporter (21,27,28,34,48) and the solvent to which it is exposed (42). The positive correlations among growth medium osmolality, the CL content of E. coli, and ⌸ 1/2 /RT for ProP suggested that ⌸ 1/2 /RT is also elevated by a CL-rich membrane environment (7).…”

Section: Subcellular Locations Of CL Lacymentioning

confidence: 99%

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

Romantsov

Stalker

Culham

et al. 2008

Journal of Biological Chemistry

Self Cite

125

View full text Add to dashboard Cite

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiledcoil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.Osmotic pressure changes elicit transmembrane water fluxes that concentrate or dilute the cytoplasm of living cells, disrupting their structure and function. Cells respond by actively adjusting the distributions of selected solutes across the cytoplasmic membrane and water follows, restoring cellular hydration and volume (1). Bacteria can use K ϩ as an osmoregulatory solute but they prefer protein-stabilizing organic osmolytes like proline, glycine, glycine betaine, and ectoine (1, 2). These compounds are also called osmoprotectants because, when provided exogenously, they stimulate bacterial growth in high (but not low) osmotic pressure media. Well characterized, functionally redundant transporters, enzymes, and channels modulate the osmolyte composition of Gram-negative bacterium Escherichia coli (3-5). We are exploiting that system to learn how osmotic pressure is sensed, how resulting signals are transduced, and how cells respond by modulating their own structure, growth, and division.The mole fractions of the major phospholipids in the cytoplasmic membrane of E. coli are usually cited as 0.75 for phosphatidylethanolamine (PE), 3 0.20 for phosphatidylglycerol (PG), and 0.05 for ...

show abstract

“…[11][12][13][14][15]54,55 Nevertheless, significant sequence homology with LacY has been found with only a few bacterial transporters grouped in the Oligosaccharide/H + Symport subfamily. 3 A BLAST search was performed using the translated genomic databases starting from the amino acid sequence of the maltose permease (MaltP) homolog from the deep sea alkalophile B. halodurans ,56 a homolog of LacY with conservation of the most essential amino acid residues (17% identity, see Table 1).…”

Section: Eukaryotic Homologsmentioning

confidence: 99%

“…8,9 As summarized in a recent review, 10 a large amount of biochemical and biophysical data have been obtained for LacY over the last 20 years, much of which is confirmed by the crystal structure. 5 LacY has been used to model tertiary structures of different MFS transporters, [11][12][13][14][15][16] but significant homology of amino acid residues involved in substrate binding and H + translocation is apparent for only a few prokaryotic transporters, [17][18][19][20][21] The availability of a LacY X-ray structure allows homology threading of MFS sugar transporters that have amino acid sequences similar to LacY. The process yields a predicted low resolution structure 22 in which accuracy depends upon the level of homology between proteins, and together with sequence alignment, may provide a powerful tool for predicting amino acid residues that may be mechanistically important.…”

Section: Introductionmentioning

confidence: 99%

Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease

Kasho

Смирнова

Kaback

2006

Journal of Molecular Biology

View full text Add to dashboard Cite

Certain prokaryotic transport proteins similar to the lactose permease of Escherichia coli (LacY) have been identified by BLAST searches from available genomic databanks. These proteins exhibit conservation of amino acid residues that participate in sugar binding and H + translocation in LacY. Homology threading of prokaryotic transporters based on the X-ray structure of LacY (PDB ID: 1PV7) and sequence similarities reveals a common overall fold for sugar transporters belonging to the Major Facilitator Superfamily (MFS) and suggest new targets for study. Evolution-based searches for sequence similarities also identify eukaryotic proteins bearing striking resemblance to MFS sugar transporters. Like LacY, the eukaryotic proteins are predicted to have 12 transmembrane domains (TMDs), and many of the irreplaceable residues for sugar binding and H + translocation in LacY appear to be largely conserved. The overall size of the eukaryotic homologs is about twice that of prokaryotic permeases with longer N and C termini and loops between TMDs III-IV and VI-VII. The human gene encoding protein FLJ20160 (NM_017694) consists of six exons located on more than 60,000 bp of DNA sequences and requires splicing to produce mature mRNA. Cellular localization predictions suggest membrane insertion with possible proteolysis at the N terminus, and expression studies with the human protein FJL20160 demonstrate membrane insertion in both E. coli and Pichia pastoris. Widespread expression of the eukaryotic sugar transport candidates suggests an important role in cellular metabolism, particularly in brain and tumors. Homology is observed in the TMDs of both the eukaryotic and prokaryotic proteins that contain residues involved in sugar binding and H + translocation in LacY.

show abstract

A Structural Model for the Osmosensor, Transporter, and Osmoregulator ProP ofEscherichia coli

Cited by 43 publications

References 68 publications

3D Model of the Escherichia coli Multidrug Transporter MdfA Reveals an Essential Membrane-Embedded Positive Charge

3D Model of the Escherichia coli Multidrug Transporter MdfA Reveals an Essential Membrane-Embedded Positive Charge

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease

Contact Info

Product

Resources

About