幽门螺杆菌Lpp20蛋白的生物信息学分析

目的：分析幽门螺杆菌Lpp20蛋白的主要化学和免疫学分子特征,为基因工程疫苗和诊断抗原的研究奠定基础。方法：根据Lpp20蛋白的氨基酸序列,应用生物信息学工具分析其蛋白序列,预测其信号肽、跨膜区、疏水性、二级结构、三级结构等性质。结果：Lpp20蛋白具有一段信号肽、脂蛋白信号肽酶切位点及脂盒模体,没有跨膜区,可能是一个外周膜蛋白;Lpp20蛋白的二级结构以α螺旋为主,其三级结构为一个致密的球状。结论：为基于幽门螺杆菌Lpp20蛋白的疫苗开发打下了基础。     

超高压处理对副溶血性弧菌的影响研究

摘要：【目的】探讨超高压致死微生物的机理。【方法】本文以副溶血性弧菌为对象,研究了超高压处理对副溶血性弧菌的灭菌效果、对副溶血性弧菌细胞超微结构、细胞无机盐离子含量以及细胞膜蛋白的影响。【结果】结果表明,在20℃下分别经100、200 MPa高压处理10min后,副溶血性弧菌致死率为40%、84.7%,经300 MPa及以上的压力处理,副溶血性弧菌的致死率为100%。超高压处理对细菌细胞形态结构造成明显的损伤：局部细胞壁遭到破坏,出现缺口;胞质内含物结构紊乱,出现泄漏,细胞中部出现透电子区;细胞结构不完整     

八氢番茄红素合成酶(PSY)的生物信息学分析

目的:通过生物信息学方法对八氢番茄红素合成酶基因(PSY)及氨基酸序列分析,并构建三维结构。方法:运用生物信息学方法对八氢番茄红素合成酶基因及其蛋白质序列的理化性质、亲/疏水性、信号肽、跨膜结构域、糖基化位点,磷酸化位点,二级结构,功能结构域和三级结构进行预测分析。结果:PSY基因含1239bp的开放阅读框,编码氨基酸数为412,为碱性不稳定蛋白;八氢番茄红素合成酶富含Arg、Leu、Ala、Ser、Val等氨基酸,为亲水性蛋白质;PSY为非跨膜蛋白,不含信号肽,具有多个磷酸化位点,α螺旋和无规卷曲是其主要结构元件。结论:用同源建模的方法构建其三维结构,得到合理模型,为采用生物工程提高番茄红素产量提供理论依据。     

副溶血弧菌SH112株OmpA蛋白的高效表达及免疫学特性

【目的】我们前期研究表明副溶血弧菌SH112株的OmpA蛋白在该菌的致病过程中发挥重要作用,是亚单位疫苗研制的潜在靶标抗原。本研究进一步对ompA(VPA1186)基因进行克隆表达,并研究其免疫学特性。【方法】扩增去除信号肽序列的成熟外膜蛋白OmpA的基因片段,定向克隆至表达载体,基因测序后对其编码蛋白质进行生物信息学分析。重组蛋白His-OmpA经纯化后,免疫ICR小鼠制备鼠多抗血清。Western blotting检测该蛋白的免疫原性及鼠多抗血清的特异性。动物实验验证其免疫保护率。【结果】成功表达分子量约为40.0 kDa的重组蛋白His-OmpA。制备的鼠多抗血清ELISA效价可达1∶50000以上。Westernblotting检测结果显示,该血清可与His-OmpA蛋白、总外膜蛋白和全菌蛋白发生特异性反应,说明所表达的目的蛋白保持原蛋白的免疫原性。此外,该高免血清可与其他主要血清型的副溶血弧菌发生特异性交叉反应,而与其他非副溶血弧菌菌株无交叉反应,表明该血清特异性较高,且提示OmpA蛋白可能是副溶血弧菌属的共同保护性抗原。小鼠免疫保护实验结果表明,该蛋白可提供约35%的免疫保护率。【结论】OmpA蛋白可作为诊断副溶血弧菌感染和亚单位疫苗研制的靶蛋白,为进一步开展该蛋白的功能研究提供了参考。     

副溶血性弧菌全基因组DNA芯片的研制和质量评价

【目的】研制副溶血性弧菌全基因组芯片,建立芯片杂交方法,并对芯片质量进行评价。【方法】利用副溶血性弧菌全基因组序列,挑选出4770条基因,PCR扩增各基因并将PCR产物纯化,点样制备芯片;设计了两个质控杂交组合,采用双色荧光杂交策略,对芯片质量进行评价;PCR方法验证部分芯片结果。【结果】芯片杂交与理论预期结果以及PCR验证结果完全一致。【结论】成功的研制了一批质量良好的副溶血性弧菌全基因组DNA芯片,并建立了基于DNA芯片的副溶血性弧菌比较基因组学技术平台,建立了一套系统的芯片数据分析的标准方法。     

不同温度条件下副溶血性弧菌tdh基因表达及其代谢组响应

【目的】采用RT-PCR的方法分析致病性副溶血性弧菌毒力基因表达,并应用代谢组学的方法研究毒力基因不同表达水平下致病性副溶血性弧菌代谢组的响应。【方法】本文以致病性副溶血性弧菌(Vibrio parahaemolyticus)ATCC33846为材料,分别提取不同温度(4、25和37℃)下菌体总RNA和代谢组。采用相对定量的方法检测副溶血性弧菌tdh基因在不同温度条件下的表达差异,同时应用超高压液相色谱-四级杆飞行时间质谱联用仪(UPLC/Q-TOF-MS)系统为工作平台检测其代谢组。采用主成分分析法(principal component analysis,PCA)比较副溶血性弧菌代谢组轮廓差异,并通过皮尔森和斯皮尔曼相关性分析法分析代谢组与tdh基因表达之间相关性。【结果】结果表明,不同温度条件下tdh基因表达强弱的排列顺序25℃4℃37℃;在tdh基因不同表达水平下发生显著性(P0.05)变化的主要代谢物是有机酸、氨基酸、醇、酮、酯;共得到11种代谢物与tdh基因表达高度相关(相关性系数︱r︱=1,P0.05),其中3种为负相关,8种为正相关,且醇类代谢物与tdh基因表达的正相关性最显著。【结论】本研究发现副溶血性弧菌代谢组与毒力基因表达存在一定的相关性,有望为副溶血性弧菌致病机理的深入探究提供一定的理论支持。     

信号分子AI-2的体外合成及其对副溶血性弧菌四环素耐药性的调控作用

【背景】抗菌药的过度使用引起细菌耐药性日益严重,作为重要的食源性致病菌,副溶血性弧菌也表现出一定程度的耐药性。群体感应系统可以调控细菌的耐药性,为研究副溶血性弧菌的耐药机制和控制技术提供新的途径。【目的】探讨群体感应信号分子AI-2 (autoinducer-2)对海产品中分离的副溶血性弧菌四环素耐药性的调控作用。【方法】通过原核表达制备AI-2合成关键酶——S-核糖同型半胱氨酸酶(S-ribosylhomocysteinase, LuxS)和S-腺苷同型半胱氨酸核苷酶(S-adenosylhomocysteinenucleosidase,Pfs),体外合成AI-2,通过菌落计数法分析AI-2对副溶血性弧菌在四环素亚抑菌浓度下耐受性的影响,采用逆转录实时荧光定量PCR法测定不同浓度AI-2对副溶血性弧菌四环素耐药基因转录水平的影响。【结果】通过原核表达获得LuxS和Pfs,作用于底物S-腺苷同型半胱氨酸能合成具有生物活性的AI-2,其荧光强度约为阳性对照的6倍。在四环素亚抑菌浓度下,AI-2能显著促进副溶血性弧菌的生长,6、15、30μmol/L浓度AI-2能不同程度地提高副溶血性弧菌四环素耐药基因的转录水平。【结论】AI-2能增强副溶血性弧菌对四环素的耐受作用,为解析副溶血性弧菌的耐药机制、研制以AI-2为靶点的副溶血性弧菌耐药性控制技术提供基础。     

高粱SUT基因家族的生物信息学分析

蔗糖转运蛋白(sucrose transporters,SUTs)属于跨膜转运蛋白,大多数参与蔗糖的吸收和转运。迄今为止,对高粱蔗糖转运蛋白知之甚少,为进一步研究高粱蔗糖转运蛋白家族(SbSUTs),本研究利用生物信息学方法对SbSUTs的6个成员(编号SbSUT1~SbSUT6)进行蛋白理化性质、基因结构、蛋白结构、同源性及系统进化树构建等分析。结果表明:SbSUTs是一种无信号肽、定位于质膜和叶绿体类囊膜上的疏水性膜蛋白;SbSUTs均具有GPH结构功能域,是高度保守的蛋白;α-螺旋和无规卷曲是主要的二级结构元件,其三级结构较为相似。本研究为探究SbSUTs蛋白家族在高粱的蔗糖吸收及转运中的功能提供理论依据。     

副溶血性弧菌基因敲除方法的建立及应用

目的摸索出一套副溶血性弧菌基因敲除的可靠方案,副溶血性弧菌致病相关基因的敲除对深入研究其致病机制有重要意义。方法通过融合PCR技术将目的基因上下游同源臂融合并克隆到自杀载体pDS132上,将重组质粒转化大肠杆菌S17λpir中,再接合转移到副溶血性弧菌菌株内,经pDS132质粒上sacB基因的反向筛选得到突变株。结果成功构建了副溶血性弧菌RIMD2210633菌株ΔopaR,ΔtoxR和ΔaphA三个基因突变株。结论通过自杀载体同源重组成功获得精确敲除的无痕突变株更有利于基因功能的研究,使后续副溶血性弧菌突变株与野生株的对比研究成为可能。     

基于生物信息学方法预测人线粒体转录终止因子3蛋白的结构与功能

目的:基于生物信息学预测人线粒体转录终止因子3(hMTERF3)蛋白的结构与功能。方法:利用GenBank、Uniprot、ExPASy、SWISS-PROT数据库资源和不同的生物信息学软件对hMTERF3蛋白进行系统研究,包括hMTERF3的理化性质、跨膜区和信号肽、二级结构功能域、亚细胞定位、蛋白质的功能分类预测、同源蛋白质多重序列比对、系统发育树构建、三级结构同源建模。结果:软件预测hMTERF3蛋白的相对分子质量为47.97×103,等电点为8.60,不具信号肽和跨膜区;二级结构分析显示主要为螺旋和无规则卷曲,包含6个MTERF基序,三级结构预测结果与二级结构预测结果相符;亚细胞定位分析结果显示该蛋白定位于人线粒体;功能分类预测其为转运和结合蛋白,参与基因转录调控;同源蛋白质多重序列比对和进化分析显示,hMTERF3蛋白与大鼠、小鼠等哺乳动物的MTERF3蛋白具有高度同源性,在系统发育树上聚为一类。结论:hMTERF3蛋白的生物信息学分析为进一步开展对该蛋白的结构和功能的实验研究提供了理论依据。     

Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.     

Roles of the Protruding Loop of Factor B Essential for the Localization of Lipoproteins (LolB) in the Anchoring of Bacterial Triacylated Proteins to the Outer Membrane

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.     

Structural Investigation of the Interaction between LolA and LolB Using NMR

Lipoproteins that play critical roles in various cellular functions of Gram-negative bacteria are localized in the cells inner and outer membranes. Lol proteins (LolA, LolB, LolC, LolD, and LolE) are involved in the transportation of outer membrane-directed lipoproteins from the inner to the outer membrane. LolA is a periplasmic chaperone that transports lipoproteins, and LolB is an outer membrane receptor that accepts lipoproteins. To clarify the structural basis for the lipoprotein transfer from LolA to LolB, we examined the interaction between LolA and mLolB, a soluble mutant of LolB, using solution NMR spectroscopy. We determined the interaction mode between LolA and mLolB with conformational changes of LolA. Based upon the observations, we propose that the LolA·LolB complex forms a tunnel-like structure, where the hydrophobic insides of LolA and LolB are connected, which enables lipoproteins to transfer from LolA to LolB.Gram-negative bacteria express lipid-modified proteins, lipoproteins, which are anchored to the cellular membrane via acyl chains attached to N-terminal cysteine residues of the lipoproteins. Putative lipoproteins have been found in various bacteria. For example, Escherichia coli has at least 90 types of lipoproteins (1), and the Lyme disease spirochete Borrelia burgdorferi has 105 putative lipoproteins (2). Although little is known about the functions of the majority of lipoproteins, some of the lipoproteins play essential roles in various cellular functions of Gram-negative bacteria, such as cell surface structure stabilization, cell shape maintenance, substrate transport, cell growth, and cell signaling (3).Lipoproteins are located at three cellular membrane sites; they are the periplasmic side of the inner membrane, the periplasmic side of the outer membrane, and the outside of the outer membrane (4). In E. coli most of the lipoproteins are anchored to the periplasmic side of the outer membrane, whereas others are anchored to that of the inner membrane (1). Therefore, the transportation of the lipoproteins to the outer membrane is essential for E. coli.Five Lol proteins, LolA, LolB, LolC, LolD, and LolE, play central roles in the outer membrane-directed lipoprotein localization. The Lol·CDE complex, which is anchored to the inner membrane, transfers the lipoproteins from the membrane to a soluble monomer periplasmic protein, LolA (182 amino acids) in an ATP-dependent manner (5–7). LolA transports the lipoproteins from the inner membrane through the periplasmic space to the outer membrane and transfers them to an outer membrane lipoprotein, LolB (186 amino acids). LolB is anchored to the membrane by acyl chains attached to its N-terminal cysteine, and it finally inserts the lipoproteins into the outer membrane (8–10).Among the Lol proteins the crystal structures of LolA and LolB have been solved. As for LolB, the soluble mutant of LolB, mLolB, in which the N-terminal cysteine residue was replaced with an alanine residue, was used for the structural analysis. Although LolA and mLolB share only 8% primary sequence identity, their tertiary structures are similar to each other (11). The structures of both LolA and mLolB resemble an open β-barrel with a lid. The convex side of the β-barrel is fully solvent-exposed, whereas the concave side is partly exposed (supplemental Fig. S1).The open β-barrels of LolA and LolB comprise 11 antiparallel β-strands (β1–β11) and an extra β-strand, β12 for LolA and β11′ for LolB. The lid is composed of three α-helices (α1–α3) and is embedded in the concave side of the β-barrel. The concave sides of LolA and LolB contain many hydrophobic residues. Therefore, this concave side of the proteins is speculated to be the binding site for the hydrophobic acyl chains of lipoproteins. Interestingly, one of the crystal structures of LolB accommodated a molecule of polyethylene glycol 2000 monomethyl ether, PEGMME2000, on the hydrophobic surface of the concave side (supplemental Fig. S1).The specific interaction between LolA and LolB is a decisive step in correctly sorting lipoproteins from LolA via LolB to the outer membrane. However, the structural aspects of the interaction, which would clarify how LolA transfers lipoproteins to LolB, remain unknown. To address this issue, we focused on the interaction between LolA and LolB.Here we investigated the interaction of LolA with LolB by NMR spectroscopy. We used LolA with a His₆ tag and mLolB, which retain the biological activities similar to those of the wild type protein (8, 12). By exploiting the cross-saturation and paramagnetic relaxation enhancement (PRE)² techniques, we successfully determined the interfacial residues of LolA and mLolB and the relative orientation of the two molecules in the complex. In addition, we identified the binding sites of an acyl chain analogue, decanoate, on LolA and mLolB. The results obtained from the present study not only explain how LolA might achieve lipoprotein transfer to LolB but also may provide new insights into the structural and functional aspects of other fatty acid-binding proteins.     

Real time analysis of lipoprotein transfer from LolA to LolB by means of surface plasmon resonance

Lipoproteins of Escherichia coli are sorted to the outer membrane through a pathway composed of five Lol proteins. LolA transports lipoproteins released from the inner membrane by LolCDE to LolB on the outer membrane via the periplasm. Interaction between LolA and LolB was speculated to be strong when LolA binds lipoprotein. However, due to a lack of a sensitive method, the kinetics of this reaction have not been examined in detail. We report here the detection of lipoprotein transfer in real time by means of surface plasmon resonance. The kinetic parameters of lipoprotein transfer were determined with wild-type LolA and a mutant defective in it.

Structured summary

MINT-7259948: mlolB (uniprotkb:P61320) binds (MI:0407) to pal (uniprotkb:P0A912) by surface plasmon resonance (MI:0107)     

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 beta barrel and an alpha-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.     

A mutation in the membrane subunit of an ABC transporter LolCDE complex causing outer membrane localization of lipoproteins against their inner membrane-specific signals

Lipoproteins in Gram-negative bacteria are anchored to the inner or outer membrane via fatty acids attached to the N-terminal cysteine. The residue at position 2 determines the membrane specificity. An ATP binding cassette transporter LolCDE complex releases lipoproteins with residues other than aspartate at position 2 from the inner membrane, whereas those with aspartate at position 2 are rejected by LolCDE and therefore remain in the inner membrane. For further understanding of this rejection mechanism, a novel strategy was developed to select mutants in which lipoproteins with aspartate at position 2 are released. The isolated mutants carried an alanine to proline mutation at position 40 of LolC, a membrane subunit of the LolCDE complex. A significant portion of an inner membrane lipoprotein, L10P(DQ), was localized to the outer membrane when the LolC mutant was expressed. Periplasmic chaperone LolA formed a complex with the released L10P(DQ), which was subsequently incorporated into the outer membrane in a LolB-dependent manner, indicating that neither LolA nor LolB rejects lipoproteins with aspartate at position 2. The amount of the LolC mutant co-purified with LolD and LolE after membrane solubilization was reduced significantly. Taken together, these results indicate that the mutation causes destabilization of the LolCDE complex and concomitantly prevents the accurate recognition of lipoprotein-sorting signals.     

Characterization of the Pseudomonas aeruginosa Lol system as a lipoprotein sorting mechanism

Escherichia coli lipoproteins are localized to either the inner or the outer membrane depending on the residue that is present next to the N-terminal acylated Cys. Asp at position 2 causes the retention of lipoproteins in the inner membrane. In contrast, the accompanying study (9) revealed that the residues at positions 3 and 4 determine the membrane specificity of lipoproteins in Pseudomonas aeruginosa. Since the five Lol proteins involved in the sorting of E. coli lipoproteins are conserved in P. aeruginosa, we examined whether or not the Lol proteins of P. aeruginosa are also involved in lipoprotein sorting but utilize different signals. The genes encoding LolCDE, LolA, and LolB homologues were cloned and expressed. The LolCDE homologue thus purified was reconstituted into proteoliposomes with lipoproteins. When incubated in the presence of ATP and a LolA homologue, the reconstituted LolCDE homologue released lipoproteins, leading to the formation of a LolA-lipoprotein complex. Lipoproteins were then incorporated into the outer membrane depending on a LolB homologue. As revealed in vivo, lipoproteins with Lys and Ser at positions 3 and 4, respectively, remained in proteoliposomes. On the other hand, E. coli LolCDE released lipoproteins with this signal and transferred them to LolA of not only E. coli but also P. aeruginosa. These results indicate that Lol proteins are responsible for the sorting of lipoproteins to the outer membrane of P. aeruginosa, as in the case of E. coli, but respond differently to inner membrane retention signals.     

Mechanisms underlying energy-independent transfer of lipoproteins from LolA to LolB, which have similar unclosed {beta}-barrel structures

The Lol system, comprising five Lol proteins, transfers lipoproteins from the inner to the outer membrane of Escherichia coli. Periplasmic LolA accepts lipoproteins from LolCDE in the inner membrane and immediately transfers them to LolB, a receptor anchored to the outer membrane. The unclosed beta-barrel structures of LolA and LolB are very similar to each other and form hydrophobic cavities for lipoproteins. The lipoprotein transfer between these similar structures is unidirectional and very efficient, but requires no energy input. To reveal the mechanisms underlying this lipoprotein transfer, Arg and Phe at positions 43 and 47, respectively, of LolA were systematically mutagenized. The two residues were previously found to affect abilities to accept and transfer lipoproteins. Substitution of Phe-47 with polar residues inhibited the ability to accept lipoproteins from the inner membrane. No derivatives caused periplasmic accumulation of lipoproteins. In contrast, many Arg-43 derivatives caused unusual periplasmic accumulation of lipoproteins to various extents. However, all derivatives, except one having Leu instead of Arg, supported the growth of cells. All Arg-43 derivatives retained the ability to accept lipoproteins from the inner membrane, whereas their abilities to transfer associated lipoproteins to LolB were variously reduced. Assessment of the intensity of the hydrophobic interaction between lipoproteins and Arg-43 derivatives revealed that the LolA-lipoprotein interaction should be weak, otherwise lipoprotein transfer to LolB is inhibited, causing accumulation of lipoproteins in the periplasm.     

Sorting of lipoproteins to the outer membrane in E. coli

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane. The structures of LolA and LolB are remarkably similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta-barrel and an alpha-helical lid. Structural differences between the two proteins reveal the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB. Strong inner membrane retention of lipoproteins occurs with Asp at position 2 and a few limited residues at position 3. The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing their retention in the inner membrane. The positive charge of phosphatidylethanolamine and the negative charge of Asp at position 2 are essential for Lol avoidance. The Lol avoidance signal is speculated to cause the formation of a tight lipoprotein-phosphatidylethanolamine complex that has five acyl chains and therefore cannot be recognized by LolCDE.