The bacterial lipocalins were discovered in 1995 and first reviewed in the year 2000. In the subsequent 5 years, two important developments have been made. First, an explosion of molecular sequence information from microbial genome projects has uncovered more than 90 bacterial lipocalin sequences. The phylogenetic distribution indicates that lipocalins are absent from the archaebacteria and nonmycolic acid-producing Gram-positive bacteria, which lack a permeability barrier located extrinsic to the inner (cytoplasmic) membrane, a point of contrast with the lipocalin-encoding Gram-negative bacteria and mycolic acid-producing actinobacteria. This observation strongly supports the conclusion that bacterial lipocalins originated in association with the development of the bacterial outer membrane structure. Combined with the recent finding that the integral outer membrane enzyme PagP displays a compact lipocalin-like 8-stranded anti-parallel β-barrel fold, complete with an internal lipid-binding pocket, it becomes difficult to ignore the possibility that lipocalins might share a common origin with bacterial β-barrel membrane proteins. Second, the high resolution crystal structure of the prototypical bacterial lipocalin Blc, an outer membrane lipoprotein from E. coli, has now been solved. A recent reappraisal of the crystal packing contacts in this structure have led to the conclusion that Blc is a functional dimer with a lipid acyl-chain binding-site buried at the subunit interface. Tryptophan fluorescence-quenching experiments indicate micromolar affinities for various fatty acids and phospholipids, but nanomolar affinities for lysophospholipids. Outer membrane enzymes like PagP generate lysophospholipids as enzymatic products, suggesting that Blc might fulfil a role in outer membrane biogenesis or repair related to lysophospholipid metabolism.

Introduction

The lipocalin protein family consists mainly of small extracellular proteins that bind hydrophobic ligands and fulfill numerous biological functions including ligand transport, cryptic coloration, sensory transduction, the biosynthesis of prostaglandins, and the regulation of cellular homeostasis and immunity. The lipocalins can be structurally characterized as an 8-stranded antiparallel β-barrel followed by a C-terminal α-helix, as exemplified by serum retinol binding protein.¹ The lipocalin fold is widely distributed among vertebrates and a few have been isolated from invertebrates and plants. Until 1995, when the first bacterial lipocalins were identified,² the family was thought to be restricted to eukaryotes. The recent crystal structure of a bacterial lipocalin suggests that phospholipid derivatives are likely ligands.³ The existence of bacterial lipocalins provides insight into the origins of the lipocalin family and, combined with the powerful tools of bacterial genetics, provides fertile ground for the investigation of lipocalin structure and function.

The bacterial lipocalin Blc was first identified as an outer membrane lipoprotein of E. coli in 1995.² Approximately 100 lipoproteins with varied structures and functions are encoded within the E. coli genome.⁴ Export of bacterial lipoproteins across the inner membrane is directed by a sec-dependent type-II signal peptide, which converts the amino terminal cysteine into N-acyl-S-sn-1,2-diacylglycerylcysteine. The lipidated protein remains anchored to the periplasmic surface of the inner membrane until it encounters the LolCDE ATP-binding cassette transport system, which can actively disengage the lipoprotein from the membrane and complex it with the periplasmic chaperone LolA. The LolA/lipoprotein complex moves across the periplasmic space and docks in the outer membrane with LolB, which is also a lipoprotein, to allow delivery of the lipoprotein from LolA into the periplasmic leaflet of the outer membrane.⁵ The outer membrane β-barrel proteins are similarly exported across the inner membrane in a process that subsequently employs dedicated chaperones to move them through the periplasmic space before folding into the outer membrane.⁶ Lipoproteins and integral membrane β-barrel proteins are the two major classes of proteins present in the bacterial outer membrane. Additionally, the outer membranes of Gram-negative bacteria are distinguished by an asymmetric lipid organization, with the inner and outer leaflets composed of phospholipids and lipopolysaccharides (LPS), respectively.

β-barrel membrane proteins are not normally found in the inner membrane, although there is no physical reason why they could not assemble in that location. The β-barrel structure is very effective at creating pores that would dissipate the proton motive force, suggesting that natural selection acts against their inner membrane localization. Indeed, several protein toxins act by forming β-barrel structures within energy-transducing membrane systems.⁷ The β-barrel structure satisfies the requirement of providing continuous hydrogen bonding between the amide nitrogen proton and carbonyl oxygen of the peptide bonds, which are otherwise too polar to interact stably with the hydrocarbon chains in a lipid bilayer membrane. Roughly 8 amino acids are sufficient to span the membrane in the extended β-conformation, and half of these residues project into the mostly polar β-barrel interior region.⁸ Only those residues whose registration projects them into the lipid milieu of the membrane need to possess hydrophobic side-chains. Consequently, β-barrel membrane proteins are only moderately hydrophobic, a fact that facilitates their coursing through the inner membrane and periplasmic space.⁹ Nature's other solution to provide continuous peptide hydrogen bonding is the transmembrane α-helix, which demands roughly 20 hydrophobic amino acid side chains in order to span the membrane.¹⁰ The greater hydrophobicity of transmembrane α-helical proteins dictates that their targeting to the inner membrane is essentially irreversible, a fact that explains why no transmembrane α-helical proteins are found in the outer membrane. The peptidoglycan exoskeleton that is sandwiched between the inner and outer membranes is believed to effectively block wholesale membrane exchange by vesicular trafficking. Consequently, outer membrane biogenesis likely depends on soluble periplasmic lipid-transfer proteins and/or localized contact bridges between inner and outer membranes.

The lipocalins represent a family of β-barrel proteins that are adapted to bind hydrophobic ligands, essentially chaperoning them through the aqueous compartments of the cell.¹¹ Accumulating evidence supports the hypothesis that lipocalins originated in association with the development of bacterial outer membrane systems, and that bacterial lipocalins exert a function in the maintenance of the outer membrane structure. Here we update the phylogenetic distribution of bacterial lipocalins and describe some interesting new relationships between their structure and function in the outer membrane. Some aspects of bacterial lipocalin structure-function relationships that have been reviewed previously are not addressed again here.¹² We suggest that interested readers consult this earlier article to gain a fuller picture of the bacterial lipocalin story.

Phylogenetic Distribution of Bacterial Lipocalins

Not All Bacterial Lipocalins Are Bacterial

BLAST searches using E. coli Blc as a probe can usually discern between bacterial and eukaryotic lipocalins, because the latter sequences display a number of obvious insertions/ deletions. For example, apolipoprotein D (ApoD) and lazarillo, members of a group of eukaryotic lipocalins closely related to Blc, possess an insertion between β-strands G and H that is absent from Blc.¹² However, several eukaryotic lipocalins cannot easily be distinguished from the bacterial homologues (Table 1). Most striking among these are a group of six bacterial-like lipocalins from plants. Chromosome 5 of Arabidopsis thaliana encodes a lipocalin that appears to contain a single 84 nucleotide intron with the typical 5'-donor and 3'-acceptor splice sites. Removal of the intron yields a 186 amino acid polypeptide, which exhibits greatest similarity with the bacterial lipocalins. Plant genomes are unique among eukaryotes in that some encode most of the enzymes needed to make the hydrophobic anchor of Gram-negative LPS known as lipid A or endotoxin.¹⁵ Green plants are thought to have acquired plastids by a recent endosymbiosis with Gram-negative cyanobacteria, and might consequently have retained some outer membrane components.¹⁶ These same components were apparently lost from the mitochondrion after it was derived by an earlier endosymbiosis with a Gram-negative α-proteobacterium.¹⁷ Indeed, at least one lipocalin is encoded in the genome of the cyanobacterium G. violaceus. The six bacterial-like lipocalins of plants lack any obvious signal sequence, suggesting they are soluble cytosolic proteins. The eukaryotic acquisition of bacterial genes that code for cytosolic functions is now thought to be an inevitable consequence of endosymbiosis.¹⁸ Another bacterial-like lipocalin is derived from the marine yeast D. hansenii, and this protein exhibits a typical eukaryotic signal peptide that likely targets it to the endoplasmic reticulum.

Table 1

Listing of all Clade I lipocalin sequences (2005)^a.

Two Sub-Clades of Bacterial Lipocalins

The bacterial lipocalins are defined as belonging to Clade I,¹⁹ but we can divide them further into two groups distinguished by the absence (Clade IA) or presence (Clade IB) of a lipocalin subgroup that displays a pair of cysteine residues flanking β-strands B and C in an apparent disulfide-bonding configuration (Fig. 1). The bacterial-like lipocalins from plants belong to a subgroup within Clade IB that appears to have lost this cysteine pair, whereas the D. hansenii lipocalin displays the apparent disulfide. These observations can be rationalized with the reducing cytosolic and oxidizing extracellular environments to which these lipocalins are targeted. Inexplicably, some cytosolic lipocalins within Clade IB still retain this pair of cysteine residues. The two nonconsecutive disulfides found in the closely related group of eukaryotic lipocalins that include ApoD and lazarillo each appear to have their own antecedents within the B. fungorum2 and G. violaceus lipocalins from Clade IB (Fig. 2). Thus, it appears that Clade IB lipocalins are more closely related to the eukaryotic lipocalins than are those from Clade IA. An interesting feature within Clade IA is the N-terminal fusion with a short-chain dehydrogenase/reductase domain in two Bacteroides lipocalins. Similar domains are employed for carbohydrate epimerization during the synthesis of sugars needed for LPS biosynthesis.²⁰ Intriguingly, the Chlorobium genome reveals examples of both Clade IA and Clade IB bacterial lipocalins, suggesting that an early gene duplication event produced these two extant groups.

Figure 1

Evolutionary relationships between bacterial lipocalins and their closest eukaryotic descendants. Signal peptides were removed from the lipocalin sequences listed in (Table 1). Partial sequences were excluded from the multiple sequence alignment, which (more...)

Figure 2

Relationships between apparent disulfide bonding patterns in lazarillo, ApoD, B. fungorum2, and G. violaceus lipocalins. Amino acid residues in the surface loop between β-strands G and H are shown for lazarillo, and as a consensus sequence for (more...)

Bacterial Lipocalin Structure and Function

Structural Similarities between PagP and Retinol Binding Protein

A striking similarity is seen between the structures of the β-barrel outer membrane protein PagP and the prototypic lipocalin serum retinol-binding protein (RBP).^25,26 Both PagP and RBP are 8-stranded antiparallel β-barrels with a deep lipid-binding pocket located inside one end of the barrel and a flanking α-helix at the other end (Fig. 3A and B). The structures superpose with the proper alignment of the β-strands (i.e., A-H of PagP superposes with A-H of RBP) with an r.m.s.d. of 2.9 Å over 93 Cα atoms. A structural similarity between the outer membrane protein OmpA and the lipocalins has also been noted, and several of the key differences between these proteins was pointed out, including the length of the barrels and the distinctly different protein interiors.²⁷ However, our case for similarity is greater because the PagP and RBP barrels have a higher percentage of alignable positions, and have similar inner lipid-binding pockets. Furthermore, in the lipocalins, a long loop spans strands A and B and typically forms a lid that partially closes the ligand-binding site.¹¹ It is intriguing that the long L1 loop from PagP is at an equivalent position and is strongly implicated in the function of this enzyme,^28,29 however, additional structural data will be required to test whether this loop interacts directly with ligand and/or the upper region of the barrel.

Figure 3

Comparison of PagP and serum retinol-binding protein. PagP (A) is shown in green (Protein Data Bank code 1THQ), and RBP (B) is shown in red (Protein Data Bank code 1AQB). The lauroyldimethylamine-N-oxide and retinol ligands are represented as spheres (more...)

We cannot rule out the possibility that this structural similarity represents a case of convergent evolution. However, based on the absence of lipocalins from those bacteria that also lack β-barrel membrane proteins, we cannot currently exclude the hypothesis that PagP and lipocalins share a common ancestor. This hypothesis is supported by the observation that many bacterial lipocalins are anchored in the outer membrane as lipoproteins.² The lipocalin-lipoprotein might represent an intermediate state in the adaptation between membrane-bound and soluble globular domains. Outer membrane β-barrel domains should be suitably adapted to occupy both soluble and membrane-bound conformations because both environments are encountered during the outer membrane assembly process.⁶

Structure and Function of the Bacterial Lipocalin Blc

E. coli Blc was recently cocrystallized in the presence of the 18-carbon unsaturated fatty acid known as vaccenic acid, and data collected at 1.8 Å resolution (V. Campanacci, R.E. Bishop, L. Reese, S. Blangy, M. Tegoni, and C. Cambillau; submitted). The complex is isostructural with that of native Blc and two monomers are contained in the asymmetric unit. The Blc monomer has a typical lipocalin fold consisting of a β-barrel with eight anti-parallel strands and an a-helix at the C-terminus. Inside the cavity of monomer B, a well defined elongated electron density could easily accommodate a vaccenic acid molecule (Fig. 4A). The molecule is bent at the position of the cis-double bond (Z11-12). Careful examination of the relationship between monomers A and B revealed that they interact tightly together: the interaction covers 786 Ų and 825 Ų of water accessible surface (WAS) area, on monomer A and B, respectively. Given that the total WAS area of each monomer is 7800 Ų, the buried WAS area represents ˜10% of the total, a value indicating that dimerization is not a crystallization artefact.³⁰ For comparison, these values of interacting surface area are comparable to those observed in immunoglobulin fragments/protein complexes.³¹ The interaction between the two monomers was present and essentially identical in the original native structure, but it escaped earlier observation and was not described.³

Figure 4

X-ray structure of Blc in complex with vaccenic acid. A) Ribbon view of Blc with vaccenic acid inside (spheres). Monomer B (left) is pink; monomer A (right) is rainbow colored from N- (blue) to C-terminus (red). B) Compact view of the Blc dimer attached (more...)

The comparison of subunits A and B indicate significant conformational differences that might be necessary for dimerization. In order to fit within the other monomer, strands F and G, and particularly the loops between them, move up to 5 Šin one monomer relative to the other. The dimer interface involves in large part these loops, and other residues among which the aromatics Tyr113, Tyr137, and several surface-exposed phenylalanines including Phe53 A & B, Phe108 A & B, Phe109 A & B and Phe112 A & B together form an inter-dimer hydrophobic core. The presence of many exposed phenylalanines is an uncommon feature at the surface of a monomeric globular protein, and has to be regarded as a hallmark of protein-protein or protein-lipid interactions. Both subunits are involved in the binding site of Blc, accounting for the stoichiometry of one vaccenic acid molecule per dimer. The vaccenic acid interacts with both subunits and covers 89 Ų and 171 Ų of the WAS area of monomers A and B, respectively.

It should be stressed that Blc is anchored to membranes by a covalently attached lipid modification at the amino terminus of the protein. The manner in which the lipid-anchor of each monomer is arranged relative to the other is a critical issue. The Blc construct employed has the signal peptide and the first four residues (CSSP) removed and replaced by the Gateway ATTB1 sequence.³ We have modelled into the dimeric structure the four original residues of Blc, as well as the major part of the anchoring lipid, S-sn-1,2-diacylglycerylcysteine, that we included at each N-terminal cysteine. This model reveals that the lipid-anchors of each of the 2 monomers are close to each other in the dimer, and are located on the same face in a geometry compatible with the insertion and stabilization of the dimer into the membrane (Fig. 4B). The binding site for vaccenic acid is located opposite to the membrane insertion site where it is expected to face the periplasmic space.

Tryptophan fluorescence quenching studies indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar Kd range. An exposed and unfilled pocket seemingly suited to bind a polar group extending from the fatty acid prompted investigation of lysophospholipids (LPLs), which were found to bind in a nanomolar Kd range. Favorable steric and electrostatic interactions are observed when a glycerophosphoethanolamine moiety is modeled into this region of the structure. Given the high affinity of Blc for LPLs, it seems likely that Blc might fulfil a role in cell envelope LPL transport. Although LPLs are key inner membrane intermediates of phospholipid metabolism, we do not know of any evidence to indicate that they are exported to the outer membrane. However, exogenously supplied LPLs can be taken up by deep-rough LPS mutants and converted by reacylation into glycerophospholipids using inner membrane-associated enzymes.^32-34 To date, no accessory factors needed for LPL uptake have been identified. In wild-type cells, at least two enzymes can generate LPLs in the outer membrane, namely, the phospholipase OMPLA³⁵ and the lipid A palmitoyltransferase PagP.³⁶ Both enzymes preferentially generate the sn-1 LPL regioisomers, but these are known to spontaneously rearrange into the more stable sn-2 LPLs.³⁷ Only sn-2 LPLs were tested for binding to Blc.

The LPL products sn-2-lysophosphatidic acid and sphingosine-1-phosphate, together with the structurally related platelet activating factors, have been shown to function as potent biological mediators.^38,39 In eukaryotic cells, sn-2 LPLs can be generated by phospholipases that mobilize arachidonic acid for eicosanoid-mediated signal transduction. Additionally, sn-2 LPLs are enzymatic products of the lecithin:cholesterol acyltransferase LCAT, which is, together with ApoD, a key component of the plasma high density lipoprotein particle. Mammalian ApoD and Drosophila lazarillo are the closest eukaryotic homologues of Blc and the only eukaryotic lipocalins that, like Blc, are anchored to lipid membranes. The ligand for lazarillo is unknown, but ApoD binds a variety of ligands including arachidonic acid.⁴⁰ Perhaps the high affinity of Blc for LPLs might help shed some light on the enigmatic functions of lazarillo and ApoD.

Conclusions

The bacterial lipocalin Blc is distinguished from eukaryotic lipocalins by its dimeric organization with a lipid-binding pocket at the subunit interface and by its high affinity for LPLs. We have indicated that the structural and functional organization of Blc shares some common features with β-barrel membrane proteins. The structural similarity between PagP and lipocalins is illustrative of this point, but, in the absence of measurable amino acid sequence identity, structural similarity does not by any means indicate common ancestry. In fact, we would normally regard the similarity between PagP and lipocalins as an example of convergent evolution, and other similarities between lipocalins and a polyisoprenoid-binding protein, for example,⁴¹ indicate that this structural organization has probably arisen independently on more than one occasion in the past; even the lipoprotein chaperones LolA and LolB are lipid-interactive β-barrel proteins.⁵ However, the tight association of bacterial lipocalins with those organisms that encode outer membrane β-barrel proteins like PagP makes it difficult to pronounce a verdict of convergent evolution. Combined with the functional association of bacterial lipocalins with outer membranes, we cannot ignore the possibility that lipocalins could have either engendered β-barrel membrane proteins, or vice versa. Whatever the case may be, bacterial lipocalins promise to reveal molecular details of lipid membrane biogenesis relevant to both bacteria and higher organisms, and we hope our present insight into their plausible origins will serve to guide experiments aimed at addressing this subject.

Acknowledgements

Work in the laboratories of REB, GGP, and ERMT was supported by operating grants from the Canadian Institutes of Health Research. Work in the laboratory of CC was supported by the French Ministry of Industry (grant ASG) and the Marseille-Nice Genopole.

References

1.

Newcomer ME, Jones TA, Aqvist J. et al. The three-dimensional structure of retinol-binding protein. Embo J. 1984;3:1451–1454. [PMC free article: PMC557543] [PubMed: 6540172]

2.

Bishop RE, Penfold SS, Frost LS. et al. Stationary phase expression of a novel Escherichia coli outer membrane lipoprotein and its relationship with mammalian apolipoprotein D. Implications for the origin of lipocalins. J Biol Chem. 1995;270:23097–23103. [PubMed: 7559452]

3.

Campanacci V, Nurizzo D, Spinelli S. et al. The crystal structure of the Escherichia coli lipocalin Blc suggests a possible role in phospholipid binding. FEBS Lett. 2004;562:183–188. [PubMed: 15044022]

4.

Gonnet P, Rudd KE, Lisacek F. Fine-tuning the prediction of sequences cleaved by signal peptidase II: A curated set of proven and predicted lipoproteins of Escherichia coli K-12. Proteomics. 2004;4:1597–1613. [PubMed: 15174130]

5.

Tokuda H, Matsuyama S. Sorting of lipoproteins to the outer membrane in E. coli. Biochim Biophys Acta. 2004;1693:5–13. [PubMed: 15276320]

6.

Mogensen JE, Otzen DE. Interactions between folding factors and bacterial outer membrane proteins. Mol Microbiol. 2005;57:326–346. [PubMed: 15978068]

7.

Montoya M, Gouaux E. Beta-barrel membrane protein folding and structure viewed through the lens of alpha-hemolysin. Biochim Biophys Acta. 2003;1609:19–27. [PubMed: 12507754]

8.

Schulz GE. The structure of bacterial outer membrane proteins. Biochim Biophys Acta. 2002;1565:308–317. [PubMed: 12409203]

9.

Tamm LK, Hong H, Liang B. Folding and assembly of beta-barrel membrane proteins. Biochim Biophys Acta. 2004;1666:250–263. [PubMed: 15519319]

10.

White SH, Ladokhin AS, Jayasinghe S. et al. How membranes shape protein structure. J Biol Chem. 2001;276:32395–32398. [PubMed: 11432876]

11.

Flower DR, North AC, Sansom CE. The lipocalin protein family: Structural and sequence overview. Biochim Biophys Acta. 2000;1482:9–24. [PubMed: 11058743]

12.

Bishop RE. The bacterial lipocalins. Biochim Biophys Acta. 2000;1482:73–83. [PubMed: 11058749]

13.

Faller M, Niederweis M, Schulz GE. The structure of a mycobacterial outer-membrane channel. Science. 2004;303:1189–1192. [PubMed: 14976314]

14.

Nikaido H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev. 2003;67:593–656. [PMC free article: PMC309051] [PubMed: 14665678]

15.

Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635–700. [PMC free article: PMC2569852] [PubMed: 12045108]

16.

Delwiche CF, Palmer JD. The origin of plastids and their spread via secondary symbiosis. Pl Syst Evol. 1997;11(Suppl):53–86.

17.

Gray MW, Burger G, Lang BF. Mitochondrial evolution. Science. 1999;283:1476–1481. [PubMed: 10066161]

18.

Doolittle WF. You are what you eat: A gene transfer ratchet could account for bacterial genes in eukaryotic nuclear genomes. Trends Genet. 1998;14:307–311. [PubMed: 9724962]

19.

Gutierrez G, Ganfornina MD, Sanchez D. Evolution of the lipocalin family as inferred from a protein sequence phylogeny. Biochim Biophys Acta. 2000;1482:35–45. [PubMed: 11058745]

20.

Breazeale SD, Ribeiro AA, Raetz CR. Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli. Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose. J Biol Chem. 2002;277:2886–2896. [PubMed: 11706007]

21.

Rivera MC, Lake JA. The ring of life provides evidence for a genome fusion origin of eukaryotes. Nature. 2004;431:152–155. [PubMed: 15356622]

22.

Golding GB, Gupta RS. Protein-based phylogenies support a chimeric origin for the eukaryotic genome. Mol Biol Evol. 1995;12:1–6. [PubMed: 7877484]

23.

Gupta RS. Protein phylogenies and signature sequences: A reappraisal of evolutionary relationships among archaebacteria, eubacteria, and eukaryotes. Microbiol Mol Biol Rev. 1998;62:1435–1491. [PMC free article: PMC98952] [PubMed: 9841678]

24.

Woese CR. Bacterial evolution. Microbiol Rev. 1987;51:221–271. [PMC free article: PMC373105] [PubMed: 2439888]

25.

Ahn VE, Lo EI, Engel CK. et al. A hydrocarbon ruler measures palmitate in the enzymatic acylation of endotoxin. Embo J. 2004;23:2931–2941. [PMC free article: PMC514935] [PubMed: 15272304]

26.

Bishop RE. The lipid A palmitoyltransferase PagP: Molecular mechanisms and role in bacterial pathogenesis. Mol Microbiol. 2005;57:900–912. [PubMed: 16091033]

27.

Pautsch A, Schulz GE. High-resolution structure of the OmpA membrane domain. J Mol Biol. 2000;298:273–282. [PubMed: 10764596]

28.

Hwang PM, Choy WY, Lo EI. et al. Solution structure and dynamics of the outer membrane enzyme PagP by NMR. Proc Natl Acad Sci USA. 2002;99:13560–13565. [PMC free article: PMC129713] [PubMed: 12357033]

29.

Hwang PM, Bishop RE, Kay LE. The integral membrane enzyme PagP alternates between two dynamically distinct states. Proc Natl Acad Sci USA. 2004;101:9618–9623. [PMC free article: PMC470724] [PubMed: 15210985]

30.

Miller S, Lesk AM, Janin J. et al. The accessible surface area and stability of oligomeric proteins. Nature. 1987;328:834–836. [PubMed: 3627230]

31.

Desmyter A, Spinelli S, Payan F. et al. Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology. J Biol Chem. 2002;277:23645–23650. [PubMed: 11960990]

32.

McIntyre TM, Bell RM. Escherichia coli mutants defective in membrane phospholipid synthesis: Binding and metabolism of 1-oleoylglycerol 3-phosphate by a plsB deep rough mutant. J Bacteriol. 1978;135:215–226. [PMC free article: PMC224810] [PubMed: 353031]

33.

Hsu L, Jackowski S, Rock CO. Uptake and acylation of 2-acyl-lysophospholipids by Escherichia coli. J Bacteriol. 1989;171:1203–1205. [PMC free article: PMC209723] [PubMed: 2644228]

34.

Hsu L, Jackowski S, Rock CO. Isolation and characterization of Escherichia coli K-12 mutants lacking both 2-acyl-glycerophosphoethanolamine acyltransferase and acyl-acyl carrier protein synthetase activity. J Biol Chem. 1991;266:13783–13788. [PubMed: 1649829]

35.

Snijder HJ, Ubarretxena-Belandia I, Blaauw M. et al. Structural evidence for dimerization-regulated activation of an integral membrane phospholipase. Nature. 1999;401:717–721. [PubMed: 10537112]

36.

Jia W, Zoeiby AE, Petruzziello TN. et al. Lipid trafficking controls endotoxin acylation in outer membranes of Escherichia coli. J Biol Chem. 2004;279:44966–44975. [PubMed: 15319435]

37.

Pluckthun A, Dennis EA. Acyl and phosphoryl migration in lysophospholipids: Importance in phospholipid synthesis and phospholipase specificity. Biochemistry. 1982;21:1743–1750. [PubMed: 7082643]

38.

Anliker B, Chun J. Lysophospholipid G protein-coupled receptors. J Biol Chem. 2004;279:20555–20558. [PubMed: 15023998]

39.

Prescott SM, Zimmerman GA, Stafforini DM. et al. Platelet-activating factor and related lipid mediators. Annu Rev Biochem. 2000;69:419–445. [PubMed: 10966465]

40.

Morais Cabral JH, Atkins GL, Sanchez LM. et al. Arachidonic acid binds to apolipoprotein D: Implications for the protein's function. FEBS Lett. 1995;366:53–56. [PubMed: 7789516]

41.

Handa N, Terada T, Doi-Katayama Y. et al. Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8. Protein Sci. 2005;14:1004–1010. [PMC free article: PMC2253440] [PubMed: 15741337]

42.

Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. [PMC free article: PMC308517] [PubMed: 7984417]

43.

Page RD. TreeView: An application to display phylogenetic trees on personal computers. Comput Appl Biosci. 1996;12:357–358. [PubMed: 8902363]

44.

Altschul SF, Gish W, Miller W. et al. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. [PubMed: 2231712]

45.

Nielsen H, Brunak S, von Heijne G. Machine learning approaches for the prediction of signal peptides and other protein sorting signals. Protein Eng. 1999;12:3–9. [PubMed: 10065704]