Naoko Yokota

Crystal structures of bacterial lipoprotein localization factors, LolA and LolB

Lipoproteins having a lipid‐modified cysteine at the N‐terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram‐negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed β barrel and an α‐helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy‐independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.

Characterization of the LolA-LolB System as the General Lipoprotein Localization Mechanism of Escherichia coli

The major outer membrane lipoprotein (Lpp) ofEscherichia coli requires LolA for its release from the cytoplasmic membrane, and LolB for its localization to the outer membrane. We examined the significance of the LolA-LolB system as to the outer membrane localization of other lipoproteins. All lipoproteins possessing an outer membrane-directed signal at the N-terminal second position were efficiently released from the inner membrane in the presence of LolA. Some lipoproteins were released in the absence of externally added LolA, albeit at a slower rate and to a lesser extent. This LolA-independent release was also strictly dependent on the outer membrane sorting signal. A lipoprotein-LolA complex was formed when the release took place in the presence of LolA, whereas lipoproteins released in the absence of LolA existed as heterogeneous complexes, suggesting that the release and the formation of a complex with LolA are distinct events. The release of LolB, an outer membrane lipoprotein functioning as the receptor for a lipoprotein-LolA complex, occurred with a trace amount of LolA, and therefore was extremely efficient. The LolA-dependent release of lipoproteins was found to be crucial for the specific incorporation of lipoproteins into the outer membrane, whereas lipoproteins released in the absence of LolA were nonspecifically and inefficiently incorporated into the membrane. The outer membrane incorporation of lipoproteins including LolB per se was dependent on LolB in the outer membrane. From these results, we conclude that lipoproteins in E. coli generally utilize the LolA-LolB system for efficient release from the inner membrane and specific localization to the outer membrane.

Genetic analyses of the in vivo function of LolA, a periplasmic chaperone involved in the outer membrane localization of Escherichia coli lipoproteins

The major outer membrane lipoprotein (Lpp) of Escherichia coli is released from the cytoplasmic membrane into the periplasm as a complex with LolA, a periplasmic chaperone, prior to the localization in the outer membrane. To determine whether or not LolA is generally involved in the outer membrane localization of lipoproteins in vivo, the chromosomal lolA gene was manipulated so as to be controlled by the lac promoter‐operator. Depletion of LolA caused a severe growth defect, and impaired the outer membrane localization of Lpp and Pal, another outer membrane lipoprotein. Although LolA depletion did not immediately arrest the growth of cells lacking Lpp, disruption of the chromosomal lolA gene was lethal to the lpp − strain, indicating that LolA is generally required for the outer membrane localization of lipoproteins, and therefore essential irrespective of the presence or absence of Lpp.

Opening and closing of the hydrophobic cavity of LolA coupled to lipoprotein binding and release.

Outer membrane-specific lipoproteins of Escherichia coli are released from the inner membrane through the action of Lol-CDE, which leads to the formation of a complex between the lipoprotein and LolA, a periplasmic chaperone. LolA then transfers lipoproteins to LolB, a receptor in the outer membrane. The structures of LolA and LolB are very similar, having an incomplete β-barrel covered with an α-helical lid forming a hydrophobic cavity inside. The cavity of LolA, but not that of LolB, is closed and thus inaccessible to the bulk solvent. Previous studies suggested that Arg at position 43 of LolA is critical for maintaining this closed structure. We show here, through a crystallographic study, that the cavity of the LolA(R43L) mutant, in which Leu replaces Arg-43, is indeed open to the external milieu. We then found that the binding of a fluorescence probe distinguishes the open/close state of the cavity. Furthermore, it was revealed that the hydrophobic cavity of LolA opens upon the binding of lipoproteins. Such a liganded LolA was found to be inactive in the release of lipoproteins from the inner membrane. On the other hand, the liganded LolA became fully functional when lipoproteins were removed from LolA by detergent treatment or transferred to LolB. Free LolA thus formed was inaccessible to a fluorescence probe. These results, taken together, reveal the LolA cycle, in which the hydrophobic cavity undergoes opening and closing upon the binding and release of lipoproteins, respectively.

MYU, a Target lncRNA for Wnt/c-Myc Signaling, Mediates Induction of CDK6 to Promote Cell Cycle Progression.

Aberrant activation of Wnt/β-catenin signaling is a major driving force in colon cancer. Wnt/β-catenin signaling induces the expression of the transcription factor c-Myc, leading to cell proliferation and tumorigenesis. c-Myc regulates multiple biological processes through its ability to directly modulate gene expression. Here, we identify a direct target of c-Myc, termed MYU, and show that MYU is upregulated in most colon cancers and required for the tumorigenicity of colon cancer cells. Furthermore, we demonstrate that MYU associates with the RNA binding protein hnRNP-K to stabilize CDK6 expression and thereby promotes the G1-S transition of the cell cycle. These results suggest that the MYU/hnRNP-K/CDK6 pathway functions downstream of Wnt/c-Myc signaling and plays a critical role in the proliferation and tumorigenicity of colon cancer cells.

Highly enantioselective reduction of symmetrical diacetylaromatics with baker's yeast

Abstract Asymmetric reduction of symmetrical diacetylaromatics ( 1a , 1b , and 1d-g ) with bakers yeast ( Saccharomyces cerevisiae ) provided the corresponding alcohols of high enantiomeric purity. By choosing appropriate reaction conditions, the products were preferentially the monoalcohols over the diols.

Large‐scale preparation of the homogeneous LolA–lipoprotein complex and efficient in vitro transfer of lipoproteins to the outer membrane in a LolB‐dependent manner

An ATP‐binding cassette transporter LolCDE complex of Escherichia coli releases lipoproteins destined to the outer membrane from the inner membrane as a complex with a periplasmic chaperone, LolA. Interaction of the LolA–lipoprotein complex with an outer membrane receptor, LolB, then causes localization of lipoproteins to the outer membrane. As far as examined, formation of the LolA–lipoprotein complex strictly depends on ATP hydrolysis by the LolCDE complex in the presence of LolA. It has been speculated, based on crystallographic and biochemical observations, that LolA undergoes an ATP‐dependent conformational change upon lipoprotein binding. Thus, preparation of a large amount of the LolA–lipoprotein complex is difficult. Moreover, lipoproteins bound to LolA are heterogeneous. We report here that the coexpression of LolA and outer membrane‐specific lipoprotein Pal from a very efficient plasmid causes the unusual accumulation of the LolA–Pal complex in the periplasm. The complex was purified to homogeneity and shown to be a functional intermediate of the lipoprotein localization pathway. In vitro incorporation of Pal into outer membranes revealed that a single molecule of LolB catalyzes the incorporation of more than 100 molecules of Pal into outer membranes. Moreover, the LolB‐dependent incorporation of Pal was not affected by excess‐free LolA, indicating that LolB specifically interacts with liganded LolA. Finally, the LolB depletion caused the accumulation of a significant amount of Pal in the periplasm, thereby establishing the conditions for preparation of the homogeneous LolA–lipoprotein complex.

Crystallization and preliminary crystallographic study of the outer-membrane lipoprotein receptor LolB, a member of the lipoprotein localization factors

The Lol system mediates the translocation of the water-insoluble outer-membrane lipoprotein across the periplasm of Gram-negative bacteria depending on the sorting signal. The outer-membrane lipoprotein receptor LolB (21.2 kDa) is a member of the Lol system. A soluble mutant of LolB (mLolB) from Escherichia coli was crystallized in two forms. Monoclinic crystals diffract X-rays to 1.9 A resolution and belong to space group P2(1), with unit-cell parameters a = 37.2, b = 112.4, c = 47.8 A, beta = 111.4 degrees. The V(M) value is most likely to be 2.2 A(3) Da(-1), assuming the presence of two molecules in the asymmetric unit. Hexagonal crystals diffract X-rays to 2.2 A resolution and belong to space group P6(3)22, with unit-cell parameters a = b = 71.4, c = 133.9 A. The V(M) value is determined as 2.3 A(3) Da(-1), assuming a single molecule in the asymmetric unit. A four-wavelength data set was collected from a monoclinic crystal of selenomethionylated mLolB in order to perform MAD phasing. The quality of the initial electron-density map was sufficient to build a molecular model.