Search and Subvert: Minimalist Bacterial Phosphatidylinositol

Review Cite This: Chem. Rev. 2018, 118, 8435−8473

pubs.acs.org/CR

Search and Subvert: Minimalist Bacterial PhosphatidylinositolSpecific Phospholipase C Enzymes Mary F. Roberts,∥ Hanif M. Khan,†,§ Rebecca Goldstein,∥ Nathalie Reuter,*,‡,§ and Anne Gershenson*,⊥

Chem. Rev. 2018.118:8435-8473. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 10/03/18. For personal use only.

†

Department of Biological Sciences, ‡Department of Chemistry and, §Computational Biology Unit, University of Bergen, 5020 Bergen, Norway ∥ Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, United States ⊥ Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States ABSTRACT: Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes from Gram-positive bacteria are secreted virulence factors that aid in downregulating host immunity. These PI-PLCs are minimalist peripheral membrane enzymes with a distorted (βα)8 TIM barrel fold offering a conserved and stable scaffold for the conserved catalytic amino acids while membrane recognition is achieved mostly through variable loops. Decades of experimental and computational research on these enzymes have revealed the subtle interplay between molecular mechanisms of catalysis and membrane binding, leading to a semiquantitative model for how they find, bind, and cleave their respective substrates on host cell membranes. Variations in sequence and structure of their membrane binding sites may correlate with how enzymes from different Gram-positive bacteria search for their particular targets on the membrane. Detailed molecular characterization of protein−lipid interactions have been aided by cutting-edge methods ranging from 31P field-cycling NMR relaxometry to monitor protein-induced changes in phospholipid dynamics to molecular dynamics simulations to elucidate the roles of electrostatic and cation−π interactions in lipid binding to single molecule fluorescence measurements of dynamic interactions between PI-PLCs and vesicles. This toolkit is readily applicable to other peripheral membrane proteins including orthologues in Gram-negative bacteria and more recently discovered eukaryotic minimalist PI-PLCs.

CONTENTS 1. INTRODUCTION 2. STRUCTURAL OVERVIEW OF BACTERIAL PI-PLC ENZYMES 2.1. PI-PLC X-ray Crystal Structures and Sequence Comparisons 2.2. Differences in PI-PLC Charge Distributions 2.3. Variations in the Interfacial Binding Site (IBS) 2.3.1. Helix B and the β7−αG Rim Loop 2.3.2. S. aureus Modulation of Helix B and Rim Loop Conformations 2.4. Structural Differences between PI-PLC Enzymes from Gram-Positive and Gram-Negative Bacteria 3. PI-PLC MEMBRANE BINDING AND INTERACTIONS WITH PHOSPHOLIPIDS 3.1. Methods To Study Membrane Binding and Interactions with Phospholipids 3.1.1. Experimental Methods Used To Monitor Protein−Lipid Interactions 3.1.2. Computational Methods To Study Membrane Binding 3.2. Interfacial Binding Sites 3.2.1. Electrostatic Contributions: From Weak to Strong

© 2018 American Chemical Society

3.2.2. Protruding Structural Elements 3.2.3. Other Interesting Features 3.3. Differences in Lipid Specificity for the Three Enzymes from Gram-Positive Bacteria 3.4. Choline−Tyrosine Cation−π Interactions in Bacillus PI-PLC 3.4.1. X-ray Crystallography with Choline or Soluble Phosphatidylcholine (PC) 3.4.2. Using Fluorinated Aromatic Groups To Characterize the Roles of Aromatic Residues in the IBS 3.4.3. High Resolution NMR Relaxometry To Probe the IBS From the Perspective of the Phospholipids 3.5. Recognition of Membrane Curvature or Lipid Packing Defects? 4. ENZYMATIC ACTIVITY OF BACTERIAL PI-PLCs 4.1. PI-PLC Catalytic Mechanism 4.1.1. PI-PLCs from Gram-Positive Bacteria 4.1.2. Mammalian PI-PLCs Are Multidomain Ca2+-dependent Enzymes

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Received: March 31, 2018 Published: August 27, 2018 8435

DOI: 10.1021/acs.chemrev.8b00208 Chem. Rev. 2018, 118, 8435−8473

Chemical Reviews 4.1.3. PI-PLCs from Gram-Negative Bacteria: Str. antibioticus and Pseudomonas sp. 62186 4.1.4. DNF2: A Plant PI-PLC 4.2. Assay Systems for PI-PLC Enzymes from Gram-Positive Bacteria 4.2.1. Soluble Substrates: cIP and diC4PI 4.2.2. Phospholipid Vesicles 4.3. Substrate Specificity and Effects of Membrane Fluidity 4.3.1. PI and Substrate Constraints for PI-PLC Enzymes 4.3.2. GPI-Anchor Cleavage 4.4. Oligomerization of PI-PLCs on Membranes? 4.4.1. B. thuringiensis PI-PLC 4.4.2. S. aureus PI-PLC 4.4.3. L. monocytogenes PI-PLC 4.5. Interfacial Activation 4.6. PI-PLC Is an Allosteric Enzyme: Interfacial Allostery 4.6.1. Initial Experimental Evidence That PC Is an Allosteric Activator 4.6.2. Possible Conformations and Dynamics of Allosterically Activated Bacillus sp. PIPLC 4.7. Surface Dilution Inhibition: Interpretation on a Molecular Level? 4.7.1. NMR Evidence for Increased Protein−PC Interactions at High XPC 4.7.2. A Model To Explain Surface Dilution Inhibition at the Molecular Level 5. KINETICS OF PI-PLC/MEMBRANE INTERACTIONS 5.1. Getting to the Surface and Finding Substrates: Some Practical Considerations 5.2. Membrane Residence Times 5.3. A Model of Activity on Cell Membranes 6. MINIMALIST PI-PLCs AND INNATE IMMUNITY 6.1. L. monocytogenes PI-PLC and Autophagy 6.2. Subverting Innate Immunity by GPI-Anchor Cleavage 7. CONCLUSIONS AND PERSPECTIVES Author Information Corresponding Authors ORCID Notes Biographies Acknowledgments References

Review

have adapted to recognize surfaces in what can be very different environments with respect to pH, temperature, surface composition, and substrate distribution on the surface, to give just a few examples. Such adaptations can also affect how the enzyme activity is regulated. Adaptation to diverse environments is not unique to peripheral membrane enzymes from bacteria. Eukaryotic peripheral membrane enzymes also face diverse environments including the extracellular milieu and intracellular organelle-dependent differences in pH and membrane composition. Here we focus on the bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), also referred to as 1-phosphatidylinositol phosphodiesterase or phosphatidylinositol diacylglycerol lyase (EC 4.6.1.13), family associated with bacterial virulence.6,7 Phospholipase C’s (PLCs) are important enzymes that both propagate and regulate signaling. Eukaryotic PI-PLCs are mainly calcium-dependent multidomain proteins usually consisting of pleckstrin homology (PH) and C2 domains, that help target the protein to the membrane, EF hands domains, and the (βα)8 barrel X and Y domains which are responsible for catalytic activity.8 In contrast, PI-PLCs secreted by Grampositive and Gram-negative bacteria are minimalist enzymes encapsulating membrane binding and enzyme activity in the (βα)8 barrel itself.6,9 The first of these minimalist PI-PLCs, as well as nonspecific PLC and sphingomyelinase activities, was identified in the culture supernatants of the Gram-positive bacteria Bacillus cereus in 1965.10 Such fractionated bacterial extracts with PI-cleaving activities were early tools in exploring the asymmetric distribution of phospholipids in eukaryotic cell plasma membranes.11,12 Early work by Michell and Allan13 showed that the soluble product of PI cleavage by the Bacillus enzyme preparation was a cyclic inositol rather than the linear inositols generated by mammalian PI-PLCs (Figure 1A). Both the B. cereus and Staphylococcus aureus PI-PLCs were shown to release alkaline phosphatase from tissues14,15 catalyzing investigations of how alkaline phosphatase and other proteins are attached to the membrane leading to the discovery and characterization of GPI anchors.16 More mechanistic studies of the bacterial PIPLCs began to appear in the 1990s spurred by the X-ray crystal structure of B. cereus PI-PLC (Figures 2 and 3).17 The best characterized PI-PLCs from Gram-positive bacteria have been associated with the downregulation of innate immunity. PI-PLCs from the extracellular pathogenic bacteria Bacillus sp. and S. aureus cleave glycosylphosphatidylinositol (GPI)-anchored proteins off the extracellular surface of eukaryotic cells (Figure 1).6 Because many immune receptors are GPI-anchored proteins, this activity may downregulate eukaryotic innate immunity by interfering with proper signaling. While the PI-PLC from the intracellular bacterial pathogen Listeria monocytogenes does not cleave GPI-anchored proteins, it does cleave PI18,19 (Figure 1), and this activity downregulates autophagy, an intracellular arm of the innate immune system.7,20 With the advent of whole genome sequencing, members of this minimalist family have also been identified in higher organisms including three human homologues, PI-PLC Xdomain containing protein (PLC-XD) 1−3.21 A plant specific PLC-XD called DNF2 has also been identified in the model legume Medicago truncatula, and DNF2 mutants are defective in nitrogen fixation due to the death of rhizobia bacteria in the nitrogen-fixing root nodules.22−24 The plant findings suggest that, even in higher organisms, these minimalist enzymes may help to regulate innate immunity and, perhaps, assist in the

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1. INTRODUCTION Bacteria secrete enzymes for many purposes including acquisition of building blocks in nutrient-limited environments,1 defense against other bacteria to improve their competitiveness,2−4 and attack and modification of host cells to promote bacterial growth and infection.5 When the substrate of the secreted enzyme is incorporated into a heterogeneous surface such as a cell membrane, the enzyme has a complicated task. It must first recognize and bind the appropriate surface, find its substrate, catalyze the reaction, and then move on to find the next surface bound substrate. The diversity of bacterial lifestyles and environmental niches means that homologous bacterial enzymes that share a similar structure and catalytic mechanism 8436


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Figure 2. Snapshot of the structure and lipid binding of B. thuringiensis PI-PLC from all-atom molecular dynamics (MD) simulations.34 The Xbox and variable region of the βα barrel are in shades of blue and orange, respectively. The active site is shown by pink sticks for important catalytic residues His32, Asp33, Arg69, His82, and Asp274, and the IBS is in green with hydrophobic, Ile43 and Trp residues 47 and 242, and the cationic Lys44 residue that are all important for membrane binding as balls and sticks.

characterization of this fascinating and important PLC family. Years of work have provided a detailed catalytic mechanism for these enzymes.6 The thermodynamics of interactions between PI-PLCs from Gram-positive bacteria, particularly those from Bacillus sp., and a variety of lipid membranes have been characterized in detail.25−31 Structural insight into membrane insertion mechanisms and the relative importance of electrostatic and hydrophobic interactions have been characterized in atomistic detail using both structural and computational methods.29,32−34 The transient kinetics of protein−membrane interactions have been quantified for B. thuringiensis PIPLC,35,36 and a novel nuclear magnetic resonance (NMR) method, high resolution 31P NMR relaxometry, has provided data on how protein binding alters lipid dynamics.37,38 These experiments and computations have elucidated how these bacterial PI-PLCs bind to membranes, modulate binding to adapt to diverse environments, cleave substrates, and search for substrates on the cell membrane. The methods adapted and developed to study these PI-PLCs, as well as the detailed mechanisms for enzyme catalysis, membrane recognition, and binding, provide tools, binding motifs, and insights that are generally applicable to peripheral membrane enzymes. This review is organized as follows: Section 2 highlights the (βα)8 barrel structure of these enzymes, the poor barrel closure of the PI-PLCs from Gram-positive bacteria, and how the interfacial binding site (IBS) is mainly concentrated on loops at one end of the barrel. Section 3 discusses how this diversity translates into different modes of membrane recognition ranging from electrostatics-driven to a much lower dependence on electrostatic interactions and the presence, in some PI-PLCs, of phosphatidylcholine (PC) specific recognition mediated by choline cation−Tyr π interactions at the IBS. In section 4 catalytic mechanisms are reviewed and compared to their eukaryotic counterparts. The kinetics of PI-PLC interactions with the membrane are the focus of section 5, which considers

Figure 1. Reaction and substrates of PI-PLCs from Gram-positive bacteria. (A) The reaction catalyzed by bacterial PI-PLC enzymes occurs in two discrete steps: (1) an intramolecular phosphotransferase reaction to generate diacylglycerol (DAG) and cyclic inositol phosphate (cIP), followed by (2) hydrolysis of cIP to inositol-1phosphate (I1P). (B) Secreted bacterial PI-PLC enzymes target the phosphodiesterase bonds of GPI-anchored proteins and/or PI.

toleration of the microbiome. Thus, a detailed understanding of the similarities and differences in the structures and mechanisms of this divergent enzyme family could aid in understanding one method for targeting eukaryotic innate immunity for both health and disease. The ease with which the bacterial PI-PLCs can be overexpressed and purified, their stability, minimalist architecture (Figures 2 and 3), and sequence diversity (Figure 4) all facilitate detailed molecular and, in some cases, atomistic 8437


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Figure 3. Eukaryotic and bacterial PI-PLC enzymes. (A) Domains in eukaryotic and bacterial PI-PLC enzymes exemplified by human PLC-β2 and Bacillus sp. enzymes. The numbers indicate start and end positions for each domain (negative numbers are for the signal peptide sequence in Bacillus sp.). (B) Comparison of the secondary structure elements in B. cereus (Bc) and L. monocytogenes (Lm) PI-PLC. Strands and helices forming the βα barrel are colored in shades of blue (X-box) and orange (variable region). Structural elements only present in L. monocytogenes PI-PLC are shown in green. (C) X-ray crystal structure of B. cereus PI-PLC (PDB 1gym) showing a bound myo-inositol in the active site.45 Colors as in (B).

domains of the much larger (80−110 kDa) mammalian PI-PLC enzymes (Figure 3)42 but lacks the variable XY linker which serves as a regulatory element that can modulate enzymatic activity.8,43,44 For example, a structure-based sequence alignment of B. cereus PI-PLC17 to the X/Y domains of human PLCβ239 reveals 14% identity. To date, 34 X-ray crystal structures of bacterial PI-PLCs have been deposited in the Protein Data Bank (PDB), many of which contain a bound myo-inositol in the active site (Table 1). Helix and strand notation throughout this review uses the B. cereus PIPLC structure numbering (PDB 1gym, Figure 3B).45 Figure 3 shows the secondary structure connectivity and the crystal structure for the B. cereus PI-PLC crystallized with glucosamineα(1→6)-myo-inositol.45 The strand lengths and distribution of helices in the bacterial PI-PLCs are different from those of most other members of the (βα)8 barrel superfamily53 leading to a distorted barrel with only six alpha helices (β8α6). One of the eight strands (β5, also called Vb) is antiparallel to the other ones, and absent in the L. monocytogenes enzyme.52 The active site of the bacterial PI-PLCs resides in the open cleft in the center of the incomplete β8α6 barrel and is well conserved across all structures. The active site contains two catalytic histidine residues (His32 and His82 in the B. cereus PI-PLC structure) as well as several polar and charged amino acids, which play a role in substrate binding (Figure 2).17 PI-PLCs from Gram-positive bacteria are not Ca2+ dependent and instead contain Arg69 in the active site.54 In contrast, the Gram-negative bacterial PI-PLC have acidic side chains that can chelate Ca2+, and these active sites are very similar to those found in mammalian PI-PLCs.9 PI-PLC structures from the Gram-positive extracellular pathogens Bacillus sp. (PDB 1gym, 1t6m) and S. aureus obtained

possible relationships between membrane residence times and membrane search strategies. PI-PLCs from pathogenic bacteria are associated with downregulating innate immunity thus increasing bacterial virulence, and section 6 discusses what is known about these proposed physiological effects and recent research indicating that similar minimalist PI-PLCs are found in eukaryotes and, at least in plants, may also help to regulate innate immunity.

2. STRUCTURAL OVERVIEW OF BACTERIAL PI-PLC ENZYMES The small bacterial PI-PLCs share a basic (βα)8 barrel framework, but the overall charge distribution and amino acid composition of the IBS provide different membrane binding specificities while variation in the active site modulates activity toward GPI-anchored proteins. The similarities and differences in the structures are discussed below to set the stage for addressing membrane binding and enzymatic activity. 2.1. PI-PLC X-ray Crystal Structures and Sequence Comparisons

Mammalian PI-PLCs are divided into six classes and are organized into several distinct domains: a pleckstrin homology or PH domain; a domain comprised of four EF hands; a catalytic (βα)8 barrel domain, which is interrupted by a highly variable XY linker domain inserted between the two halves of the (βα)8 barrel; and a C2 domain.8,39 Additionally, a C-terminal coiled coil domain (CT) is necessary for homodimerization and interaction with Gα proteins in the case of PLCβ enzymes.40,41 Bacterial PI-PLCs are comprised of a single domain between 30 and 35 kDa which bears structural similarity to the catalytic X−Y 8438


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more cationic rim loop, with an additional region of cationic residues (Lys133, Lys114, and Arg166) directly adjacent to the active site. The surface potentials of the L. monocytogenes and Streptomyces antibioticus PI-PLCs, however, break this trend. The surface potential of the L. monocytogenes PI-PLC structure is quite unique. This structure shows a large (420 Å2) region of cationic charge located on the underside of the molecule, opposite the membrane interface. The Str. antibioticus structure also shows one distinctive area of intense charge, a large anionic patch comprised of the entirety of the active site cleft as well as much of the rim loop. This potential is caused by a strip of 10 Glu and Asp residues bisecting the membrane interface side of the enzyme. The strip is abutted on two sides by highly cationic regions, comprised of multiple Lys or Arg residues. These marked differences in surface electrostatic potentials likely reflect adaptation to specific environmental niches. 2.3. Variations in the Interfacial Binding Site (IBS)

2.3.1. Helix B and the β7−αG Rim Loop. In the PI-PLCs from Gram-positive bacteria, two structural elements are particularly important for membrane recognition and binding: short helix B, poised at the surface above the active site, and the “rim loop” that connects β-strand 7 to α-helix G.26,48,59,60 Consistent with its role in membrane binding, in all of the PIPLC structures from Gram-positive bacteria helix B contains one Trp residue and at least one Lys. The other key membrane binding element, the rim loop, has a variable length and contains a rather exposed aromatic residue: Trp242 in the Bacillus enzymes, Phe249 in S. aureus PI-PLC, and Phe237 in L. monocytogenes PI-PLC. The orientation of this aromatic side chain varies in the three enzymes, suggesting that the loop has significant mobility. The L. monocytogenes PI-PLC structure (2plc) shows some other structural variations: helix B is oriented ∼90° from its position in the other three structures, and there are additional differences in the loop connecting β7 to αG. Another striking difference is the absence of the Vb β-strand in Listeria; Vb in the Bacillus enzyme has been suggested to be involved in interactions with GPI anchors.19,45 2.3.2. S. aureus Modulation of Helix B and Rim Loop Conformations. Two particular features only exhibited by the S. aureus PI-PLC are (i) a large pH-dependent conformational change of the rim loop and (ii) a bound anion in a cationic pocket on the barrel rim composed of side chains His86 and Lys42, in helix B, and the amides of Lys38 and Asp39 which appears in all X-ray crystal structures except that of the S. aureus dimer where residues in helix B form the dimer interface.28,50 Under slightly basic conditions, the S. aureus PI-PLC structure closely follows the conformation of other bacterial PI-PLCs. However, when crystallized under acidic conditions, a section of the rim loop consisting of 11 residues (241S-V-A-S-G-G-S-A-FN-S251) shows a maximum backbone displacement of 9.4 Å between the acidic and basic forms (Figure 6). The conformational switch controlling this displacement is a pH-dependent cation−π interaction between His258 and Phe249. At pH 4.6, the cationic nitrogen on His258 is positioned 3.5 Å from the πsystem of Phe249, which itself is 3.5 Å from that of Tyr212 in a face−edge interaction. This π−cation interaction holds Phe249, and thus the rest of the mobile loop, in a position where Phe249, the lone aromatic residue in the mobile loop, is unable to partition into membranes. At pH 7.5, His258 will be deprotonated. With the cation no longer present, the mobile loop reverts to the more extended loop position, 10 Å away, where His258 is 11.7 Å away from the π system of Phe249.

Figure 4. Comparison of PI-PLC primary sequences from B. cereus (Bc) (Uniprot P14262), S. aureus (Sa) (P45723), and L. monocytogenes (Lm) (P34024). Secondary structure elements are shown above the sequences and are labeled as in Figure 3. Strictly conserved amino acids are highlighted in red. The first 31, 11, and 35 amino acids corresponding to signal peptides were excluded from the alignment. The alignment was generated using ClustalΟ55 and labeled using ESPript.56

at pH 7.5 (PDB 3v18) show the same general architecture and excellent structural similarity with most differences concentrated in the surface loops. The similarity between the two Bacillus PIPLCs is not surprising since they differ by only eight residues, and even the R70D mutation in the B. thuringiensis structure (PDB 1t6m), which introduces a metal binding site,47 does not disrupt the structural similarity. The overall root-mean-square deviation (rmsd) between the B. cereus structure (PDB 1gym) and the S. aureus structure (PDB 3v18) is 1.8 Å.50 The S. aureus enzyme sequences are more divergent with 40% identity to the Bacillus sequences. The third set of PI-PLC structures from Gram-positive bacteria come from L. monocytogenes, which unlike Bacillus and Staphylococcus is an intracellular pathogen. As might be expected, the L. monocytogenes structure, PDB 2plc,52 is more divergent with 26.5% identity and an overall rmsd of 6.5 Å relative to the B. cereus structure (PDB 1gym). 2.2. Differences in PI-PLC Charge Distributions

The electrostatic surface potentials can be important for membrane−protein interactions, and the potentials of the bacterial PI-PLCs vary depending on their species of origin (Figure 5). The Bacillus PI-PLCs are an example of a “typical” PI-PLC in terms of surface potential. They have relatively evenly distributed regions of positive and negative charge, with more intensely charged regions situated in the active site pocket, as well as around the rim of the βα barrel. The S. aureus PI-PLC structure shows surface potentials similar to that of the two Bacillus structures. However, S. aureus PI-PLC shows a generally 8439


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Table 1. List of Available X-ray Crystal Structures and Their Organism of Origina Gram-Positive Bacteria PDB ID

res (Å)

1ptd 1ptg 1gym 2ptd 3ptd 4ptd 5ptd 6ptd 7ptd

2.6 2.6 2.2 2.0 2.2 2.3 2.7 2.6 2.6

1t6m 3ea1 3ea2 3ea3 2or2

2.11 1.75 1.95 1.78 1.84

3v16 3v18 4f2b 4f2t 3v1h 4i9t 4i8y 4i90 4i9j 4i9mf 4rv3 4s3g

2.05 2.34 2.16 2.3 1.9 2.0 2.1 1.65 1.85 2.2 2.0 2.5

1aod 2plc

2.6 2.0

PDB ID

mutationb

ligand(s)

B. cereus (P14262) − − − myo-Ins − glucosamine-(α1→6)-D-myo-Ins D198E − D274S − D274N − H32A − H32L − R163K − B. thuringiensisc (P08954) R70D two Ca2+ per monomer Y247S/Y251S Zn2+ Y247S/Y251S Zn2+, myo-Ins Y246S/Y247S/Y248S/Y251S Mn2+ W47A/W242A − S. aureuse (P45723) − myo-Ins, Cl−, pH 4.6 − 2-propanol, SO42−, pH 7.5 − myo-Ins Y253S acetate H258Y myo-Ins, acetate H258Y myo-Ins, SO42−, triethylene glycol N254Y/H258Y acetate, Cl− N254Y/H258Y acetate, Cl−, 2 choline N254Y/H258Y acetate, diC4PC N254Y/H258Y HEPES, SO42− H258X (X = Phe-F5)g myo-Ins, acetate F249X (X = Phe-F5)g myo-Ins, acetate L. monocytogenes (P34024) − myo-inositol − − Gram-Negative Bacteria

res (Å)

3h4w 3h4x

1.5 1.23

5fyoi 5fypi 5fyri

1.5 1.17 1.45

mutationb

− − −

ligand(s) Str. antibioticus (B3A043) glycerol, ethanol, Cl− glycerol, ethanol, Cl− Pseudomonas sp. 62186 (A0A1S4NYD4) Ca2+ Ca2+ myo-Ins, Ca2+

oligomeric state

ref

monomer monomer monomer monomer monomer monomer monomer monomer monomer

17 17 45 46 46 46 46 46 46

monomer dimerd dimerd dimerd dimer

47 48 48 48 49

monomer monomer dimer monomer monomer monomer monomer monomer monomer monomer monomer monomer

50 50 28 28 50 33 33 33 33 33 51 51

monomer monomer

52 52

oligomeric state

ref

monomer monomer

released 2010h released 2010h

monomer monomer monomer

9 9 9

a

Uniprot Sequence IDs are in parentheses. bMutations are those introduced into the recombinant sequence, which may have a His tag or other handle introduced for ease of purification. cAll B. thuringiensis recombinant PI-PLC proteins have an N-terminal Met. dA crystallographic dimer with the active sites on opposite sides of the dimer as opposed to structures where the dimer positions both active sites so that they can contact the lipid interface. eAll recombinant S. aureus PI-PLC enzymes, have a C-terminal hexa-histidine tag. fsimilar to 4f2u.28 gX, the mutated residue, is a pentafluorophenylalanine introduced to weaken phosphatidylcholine (PC) cation−π interactions but strengthen hydrophobic partitioning of a phenylalanine side chain (see section 3.4.2). hNo associated publication. i5fyo and 5fyp are from two different crystal forms. 5fyo, apo enzyme, space group P4322, small unit cell; 5fyp and 5fyr, space group P43212, large unit cell.

Similarly, His258 is 4.6 Å from Tyr212. S. aureus PI-PLC is more active toward PI at acidic pH, but when PC is present the optimum shifts to more basic pH values. This pH- and PCdependent regulation of activity likely ensures that the protein has high activity toward GPI anchors in PC or sphingomyelin containing eukaryotic membranes.

and the Gram-positive bacterial PI-PLC structures are much more striking.9 The imperfect barrel is preserved in Str. antibioticus PI-PLC (PDB 3h4x), but it has eight helices unlike the Gram-positive structures which have only six. The exterior helices are shifted, with the greatest differences between αF and αG, which are rotated an angle of 54° from the homologous helices in the Bacillus structures. Furthermore, despite its importance for membrane binding in the PI-PLCs from Grampositive bacteria, helix B is not observed in the Str. antibioticus structure (Figure 7). The Pseudomonas sp. and Str. antibioticus

2.4. Structural Differences between PI-PLC Enzymes from Gram-Positive and Gram-Negative Bacteria

As elucidated by Moroz and co-workers, the differences between the two Gram-negative PI-PLC structures (PDB 3h4x and 5fyr) 8440


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Figure 6. pH dependent rim loop conformations and anion binding near helix B for S. aureus PI-PLC. The rim loop of S. aureus PI-PLC crystallized under acidic conditions (green) and basic conditions (blue). The red circle highlights the rim loop conformational change between the two structures with side chains involved in the cation−π latch (His258, Tyr249 and Tyr212) shown as sticks. A chloride anion (spacefill) is bound in the anion binding pocket partially formed by residues in helix B. myo-Inositol in the active site is shown in spacefilling representation (red), and the chloride ion is depicted as a green sphere. Reproduced from ref 50. Copyright 2012 American Chemical Society.

Figure 5. Electrostatic potentials for the bacterial PI-PLC enzymes calculated using APBS (version 1.4).57 The PI-PLCs from the Grampositive bacteria, B. thuringiensis (Bt), S. aureus (Sa), and L. monocytogenes (Lm) are shown in the upper row with helix B highlighted by the black circles. The PI-PLCs from the Gram-negative bacteria Str. antibioticus (Sab) and Pseudomonas sp. (Pssp) are shown in the lower row. The orientations of the PI-PLCs were generated using structural superpositions, and thus the apparent differences in width between the structures of the PI-PLCs from Gram-positive and Gramnegative bacteria reflect a real difference in the respective potential isocontours when viewed from the same orientation. The isocontours at +1kBT/e and −1kBT/e are shown in blue and red, respectively. The images were prepared with VMD.58

different than those of eukaryotes.61,62 To our knowledge, there is no evidence that the bacterial PI-PLC enzymes discussed in this review attack other bacteria. Even within eukaryotes, these secreted bacterial enzymes may encounter very different environments. For example, S. aureus often colonizes human skin, which is a low pH environment,63 and while Bacillus sp. and S. aureus release PI-PLC into extracellular environments, L. monocytogenes is an intracellular pathogen. Despite being homologous and structurally similar, this diversity of environments and target membranes is likely to be reflected in the lipid specificities and other membrane binding preferences of each PI-PLC. In the classical view of interactions between peripheral proteins and membranes, nonspecific electrostatic interactions drive the protein to the membrane followed by intercalation of hydrophobic amino acids into the bilayer and specific lipid recognition.64,65 We begin this section by describing methods for measuring and simulating protein−membrane interactions. We then discuss the specific interactions between PI-PLCs from Gram-positive bacteria and membranes, beginning with electrostatic interactions and detailing how interactions between PIPLC enzymes and membranes both agree with and deviate from the classical view of membrane binding.

PI-PLC structures (PDB 5fyr and 3h4x, respectively) have a more regular barrel than the Gram-positive structures, closer to the structure found in most other (βα)8 families. This change in barrel regularity arises in part from the addition of two short antiparallel strands: β2b between β2 and β3 and β8 close to the C-terminus as well as a different distribution of α helices. The Str. antibioticus and Pseudomonas sp. PI-PLC sequences are 27% identical, and the structures of the X domains are very similar, with an rmsd of 1.60 Å reported by Moroz and co-workers9 compared to an rmsd of more than 2.0 Å between the Pseudomonas sp. X domain and those of PI-PLCs from Grampositive bacteria. As noted previously, the structures of Str. antibioticus and Pseudomonas sp. PI-PLCs have a bound Ca2+ in the active site unlike the Ca2+ independent PI-PLCs from Grampositive bacteria. Thus, based on this small sample, the details of membrane binding and the catalytic mechanism likely differ between PI-PLC enzymes from Gram-positive and Gramnegative bacteria.

3. PI-PLC MEMBRANE BINDING AND INTERACTIONS WITH PHOSPHOLIPIDS The PI-PLC enzymes secreted by Gram-positive bacteria are known to interact with eukaryotic membranes. While these secreted bacterial enzymes could theoretically also interact with bacterial membranes, these membranes have lipid compositions that vary enormously between bacterial species and are very

3.1. Methods To Study Membrane Binding and Interactions with Phospholipids

3.1.1. Experimental Methods Used To Monitor Protein−Lipid Interactions. The membrane affinities of PIPLCs from Gram-positive bacteria have been investigated with many different methods and model membranes. Besides providing a detailed understanding of the thermodynamics 8441


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proteins. However, identification of the membrane binding site from sequence or even 3D structure is quite challenging. Indeed, and unlike transmembrane proteins, the membrane binding sites of peripheral proteins do not consist of well-defined secondary structure elements or hydrophobic regions easily identifiable from the primary sequence. Molecular simulations fill in a gap in the available toolbox and have proven reliable, particularly in combination with in vitro experiments,114 for a number of peripheral proteins including PI-PLCs.29,34 Molecular dynamics (MD) simulations are costly in terms of required computational power, which is proportional to the time scale simulated and the size of the system in number of particles. At the time of writing all-atom molecular dynamics (aaMD) simulations of a peripheral protein bound at, or close to, a lipid bilayer can be performed for hundreds of nanoseconds to a few microseconds. Simulations of peripheral proteins bound to phospholipid bilayers provide a detailed inventory of protein− lipid interactions, including the depth of insertion of the IBS residues in phospholipid bilayers. With sufficient computational power, aaMD simulations can be used to model the actual anchoring of the protein to a phospholipid bilayer,115 though this may be limited to a starting orientation of the protein that is fairly close to its final bilayer-bound orientation, due to limited sampling. Currently simulations of proteins bound to bilayers contain a few hundred thousand atoms in total and cover time scales from hundreds of nanoseconds up to microseconds, requiring high-performance computers. Of particular interest is a phospholipid bilayer mimetic with increased lipid mobility (highly mobile membrane mimetic, HMMM) that allows broader sampling than classical all-atom phospholipid bilayers.116−118 The use of enhanced sampling methods enables estimation of free energies of transfer119 or calculation of the contribution of individual amino acids to membrane affinity. The use of coarse-grained force fields reduces the number of particles to be simulated and thus increases the sampling and the size of systems that can be simulated but misses the atomic level of detail and thus specific protein−lipid interactions. Moreover, the Martini force field,120 one of the most used coarse-grained force fields for protein−membrane interactions, uses an elastic network to maintain protein secondary structure. Therefore, the Martini force field is not able to model conformational changes upon membrane binding even if they are as small as loop folding or extension from the protein core.121,122 Implicit membrane models represent the membrane interior/ core as a hydrophobic slab. One such model, the IMM1-GC model, adds planes of smeared charges on either side to model charged membrane surfaces.123 Such models are particularly powerful for estimating the energetic cost of embedding a protein in the membrane even though they have limitations as they do not account for lipid packing defects or heterogeneity in local lipid composition. IMM1-GC provides an estimation of the desolvation contribution and of the binding energy at a low computational cost. This increased sampling capability compared to all-atom membrane models makes such models extremely useful to discriminate between alternative membrane binding sites on a protein and thereby to predict membrane binding sites of peripheral proteins. Protein orientation with respect to the membrane model can in turn be used to initiate all-atom MD simulations.

Figure 7. Bacterial PI-PLC structural comparisons. Superposition of the structure of Pseudomonas sp. PI-PLC with myo-inositol and Ca2+ bound (PDB 5fyr,9 light orange) and (A) a PI-PLC structure from the Gram-positive bacterium B. cereus with myo-inositol bound (PDB 1ptg,17 light purple), or (B) the PI-PLC structure from another Gramnegative bacterium, Str. antibioticus (PDB 3h4w, green).

and kinetics of their membrane-binding mechanism, this wealth of data also nicely exemplifies techniques and assays that can be used to study the binding of peripheral membrane proteins to membranes, their advantages, and limitations. We summarize the most relevant methods in Table 2. We note that this list of methods is not all inclusive and a number of newer methods have been reviewed by Gavin and co-workers.66 In addition, two methods for investigating binding preferences for particular membrane curvatures/lipid packing densities (membrane tension), examining binding to membrane tubules pulled from giant unilamellar vesicles (GUVs)67 and binding to wavy patterned membranes,68 are not included in Table 2. 3.1.2. Computational Methods To Study Membrane Binding. In addition to the experimental methods listed in Table 2, computational methods can provide information directly relevant and useful to a better understanding of how peripheral membrane proteins bind to target membranes. Peripheral proteins exist in both a membrane-bound and a soluble form and are thus more easily amenable to threedimensional (3D) structure resolution than transmembrane

3.2. Interfacial Binding Sites

A widely acknowledged model for the membrane association mechanism of peripheral proteins consists of (i) an electrostati8442


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Table 2. Experimental Methods Used To Measure Protein Binding to Lipid Membranes, and Except As Noted All Methods Require Labeled,a Purified Protein

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Table 2. continued

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Table 2. continued

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Table 2. continued

a References cited in this table: 25, 26, 27, 36, 37, 38, 66, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113.

hydrophobic spikes124,125 or protruding loops.126 A close look at the X-ray structures of the bacterial PI-PLCs (see section 2) reveals such structural features. 3.2.1. Electrostatic Contributions: From Weak to Strong. Association of peripheral proteins with host membranes is often dominated by electrostatic forces due to the presence of clusters of basic residues in the IBS.127 B. thuringiensis PI-PLC does not contain an obvious cluster of basic residues, either in sequential or in spatial proximity, and its electrostatic surface potential is not strongly positive at the IBS (Figure 5). Using continuum electrostatics, Khan et al. showed that the electrostatic partition free energy of B. thuringiensis PIPLC is ∼ −0.25 kcal/mol,34 considerably lower than values calculated for other peripheral proteins (−3 to −5 kcal/mol).127 For small unilamellar vesicles (SUVs) with a mole fraction of PC

cally driven approach most often followed by (ii) the intercalation of hydrophobic side chains into the lipid bilayer.64 The first step is characterized by long-range nonspecific electrostatic forces between the negatively charged membrane and clusters of basic amino acids on the protein surface, bringing the protein into a binding-competent orientation relative to the lipid bilayer. We show below that the strength of these electrostatic forces varies from weak to strong in these bacterial PI-PLCs. The second step consists of the insertion of hydrophobic groups into the hydrophobic core of the membrane. The prototypical interfacial binding site (IBS) is thus described as containing patches of basic amino acids (lysines and arginines) and hydrophobic amino acids such as those with aromatic or aliphatic groups, or covalent lipid anchors. Membrane binding sites have been described as 8447


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(XPC) of 0.8, mutation of Lys44 increases the apparent Kd for SUVs 50−60 times or a loss of 2.4 kcal/mol in comparison to the wild-type enzyme. In contrast, mutation of other cationic residues in or near the IBS (Lys38, Arg71, and Lys279) only changes the affinity by ∼10-fold (Figure 8). If the effect of

acids to protrude from the protein globule. This short amphipathic helix, residues Pro42 through Gly48 in the Bacillus proteins (Table 3 and Figure 9), has several hydrophobic Table 3. Alignment of Primary Sequences around Helix B for the Enzymes from Gram-Positive Bacteriaa species B. cereus S. aureus L. monocytogenes

helix B sequence KLQN..PIKQVWGMTQ51 36 TLKD..PVKSVWAKTQ49 51 NGDITWTLTKPLAQTQ66 38

Amino acids in α helical segments are highlighted in bold (see also Figures 2, 3, and 9). a

Figure 9. Helix B and the rim loop (β7−αG) form hydrophobic protrusions from the protein cores. (A) Structural comparison from a superimposition of B. cereus (Bc, green, 1gym), S. aureus (Sa, blue, 3v18) and L. monocytogenes (Lm, pink, 2plc) PI-PLC X-ray crystal structures. (B) Same as (A) but from a different angle and without the Lm structure. The side chains of Trp47 (Bc) and Trp45 (Sa) from helix B, and Trp242 (Bc) and Phe249 (Sa) on the rim loop are shown with sticks. Figure 8. Effects of cationic residues on binding. (A) Affinity of B. thuringiensis PI-PLC wild type and cationic amino acid variants for SUVs. Apparent Kds were obtained using FCS and plotted as a function of XPC. (B) Electrostatic free-energy profiles of wild type and mutants, calculated using a continuum electrostatics model with a partially anionic membrane (XPC = 0.8) and a 0.1 M salt concentration. Adapted with permission from ref 34. Copyright 2016 Elsevier.

residues, most notably Trp47, but also a key cationic one, Lys44. Mutagenesis of helix B residues in the Bacillus enzyme has shown that most play important roles in membrane binding.60 Pro42 likely orients the helix in the right position for interactions with the membrane.27,60 As discussed above, Lys44 is the only basic amino acid and is important for binding to negatively charged surfaces (Figure 8).27 The side chains of Ile43, Val45, and Trp47 all point in the same direction and away from that of the Lys44 side chain, as expected for an amphipathic helix. Trp47 is likely to partition at the membrane interface given the nature of its side chain,128−130 and its mutation to Ala has a dramatic effect on the affinity for both anionic and neutral SUVs, increasing the apparent Kd 50−70-fold.60 The Kd for the Bacillus enzyme with the W47A mutation could not be rescued by placing Trp at residue 43, 45, or 49 in helix B.131 MD simulations of B. thuringiensis PI-PLC positioned at the surface of mixed anionic/ zwitterionic phospholipid bilayers show that Pro42 and Ile43 are buried below the lipid phosphate groups while Val46 and Trp47 tend to lie within the headgroup interfacial region.29,34 Table 3 shows a comparison of the primary sequences of the β1−αC loop which contains helix B, and the structures are compared in Figure 9. Bacillus and Staphylococcus proteins share a high sequence identity along the whole β1−αC loop and in helix B, suggesting a similar role for the amphipathic helix in both enzymes. In the Listeria enzyme this loop is two amino acids longer, and it does not align well to the two other sequences. However, the helix in L. monocytogenes PI-PLC still has a strong hydrophobic character with Ile54 and Leu58 and a tryptophan (Trp56). In the two structures available for L. monocytogenes PI-PLC, helix B is positioned perpendicular to that of Bacillus and Staphylococcus enzymes (Figure 9).

mutating Lys44 was solely due to electrostatic interactions between the positively charged lysine and anionic lipids, then the Kd difference between wild type and Lys44Ala at 0.8XPC should be the same as the Kd difference for wild type between 0.8XPC and 1.0XPC. However, the affinity of the wild-type enzyme for pure PC SUVs is only about 4 times that for SUVs with XPC = 0.8, demonstrating that Lys44 contributes to more than just electrostatic partitioning. Indeed, MD simulations with varying bilayer compositions reveal that the large contribution of Lys44 to binding affinity originates from a combination of long- and short-range interactions with the lipids, including electrostatics, hydrogen bonds with the headgroups, and hydrophobic contacts with the tails. Judging from the surface electrostatic potentials of the other bacterial enzymes (Figure 5), particularly their values at the postulated interfacial binding site, the magnitude of electrostatic interactions with negatively charged membranes will vary from one enzyme to the other as illustrated by the preference of Str. antibioticus and L. monocytogenes PI-PLC for negatively charged lipids (section 3.3). 3.2.2. Protruding Structural Elements. Helix B, a key structural element of many bacterial PI-PLCs (see section 2.3.1), provides a structural scaffold for hydrophobic amino 8448


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In addition to helix B, the α7−βG loop, also called the rim loop, contributes strongly to the affinity of the Bacillus enzymes for phospholipid vesicles. In particular, mutation of Trp242 to alanine dramatically reduces affinity of the protein for PC membranes.59 In B. thuringiensis PI-PLC the affinity decreases 37-fold for the Trp47Ala mutation (helix B) and 98-fold for the Trp242Ala rim loop mutation. The double mutation (Trp47Ala/Trp242Ala) virtually abolishes affinity of the enzyme for pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles. Unlike what was observed when trying to compensate for the loss of Trp47 in the Trp47Ala mutant, reintroducing a Trp into Trp242Ala as residue 236, 238, or 243 could reduce the apparent Kd for PC 10−20-fold.131 Such behavior is consistent with a flexible and dynamic conformation of this rim loop but a stable and much less dynamic structure for short helix B in solution. MD simulations show that Trp242 is buried deeper than Trp47 in the phospholipid bilayer, together with the neighboring amino acids of the rim loop. In these simulations, helix B and the rim loop are the most deeply anchored segments of the Bacillus structure. Like helix B, the length and primary sequence of the rim loop are very similar in Bacillus and Staphylococcus PI-PLCs, but the Listeria enzyme has a shorter loop with a high hydrophobic character and a primary sequence that is difficult to align to the two other enzymes from Gram-positive bacteria (see Figure 4). These results reiterate the importance of helix B and the rim loop in membrane binding, suggest that these regions have similar features in PI-PLCs from the extracellular Bacillus sp. and S. aureus pathogens, but diverge in the PI-PLC from the intracellular pathogen L. monocytogenes. 3.2.3. Other Interesting Features. In general, there are a few more interesting observations on the IBS of these enzymes. First, prolines are present in several of the membrane-interacting segments (see Figure 4 for primary sequences) possibly to maintain the orientation of turns or short helices which would be highly mobile and flexible otherwise. Prolines, which act as helix breakers in helical transmembrane segments, cause helix kinks that have been suggested to play roles in helix packing and to form hinges necessary for conformational changes in transport proteins.132 Similarly, MD simulations of B. cereus PI-PLC in solution suggest that Pro254, conserved in Bacillus sp. and S. aureus PI-PLCs but not L. monocytogenes (Figure 4), introduces a kink in helix G.79 Mutation of this proline alters protein conformational dynamics in the simulations, and compared to wild-type B. thuringiensis PI-PLC, the Pro254Tyr mutant has lower activity toward pure PI SUVs and tighter binding and higher activity toward XPC = 0.5 SUVs. These results suggest that changes in this helix G kink can affect PI-PLC dynamics, vesicle binding, and activity but that the effects of mutations may be hard to predict. Second, these enzymes have fairly different amino acid compositions of their membrane binding interfaces which are positioned in highly variable regions of the primary sequence. The overall composition reflects a high hydrophobic character with the presence of residues such as Trp, Phe, Tyr, Leu, and Ile. Trp and Tyr are known as interfacial residues in transmembrane proteins while Phe, Leu, and Ile are located in the hydrophobic region of the membrane.129,133,134 A similar dichotomy between depth of insertion of polar aromatics on one hand and Phe, Leu, and Ile on the other hand is also observed for the bacterial PIPLCs.29,34 Despite all having an IBS seemingly enriched in aromatic and hydrophobic groups, the amino acid positions are not conserved between enzymes from different bacteria. This

divergence could be the mark of evolution and reflect the different mechanisms by which the bacteria attack their hosts. The modified (βα)8 barrel fold with its numerous loops tethered to the barrel scaffold potentially offers a variety of possibilities for tuning membrane binding in terms of lipid specificity and orientation relative to the membrane. Third, we note the differential role of aromatic residues and in particular of tryptophans and tyrosines in the Bacillus enzymes. Trp residues seem to play the role of interfacial anchors independent of the lipid composition, while tyrosines have a role in PC specificity as explained below. 3.3. Differences in Lipid Specificity for the Three Enzymes from Gram-Positive Bacteria

The lipid specificities of the Bacillus PI-PLC enzymes have been scrutinized using a myriad of methods, while less is known for the PI-PLCs from Staphylococcus and Listeria. Based on the data available, and despite the similarities among the three enzymes from Gram-positive bacteria, they display very different lipid preferences, reflecting their different surface properties (Figure 5) and the variation in their primary sequences at the membrane binding sites (see section 3.2). Early in 1994 Volwerk et al. used tryptophan fluorescence spectroscopy to show that B. cereus PI-PLC binds to micelles and bilayers consisting of PC lipids, and they inferred that these PC lipids do not bind to the active site.35 Later on Hendrickson et al. used FRET between phospholipid analogue probes and tryptophans in the IBS and found the lipid preference to be dependent on lipid headgroups according to the following order: phosphatidylinositol (PI) > phosphatidylcholine (PC) > phosphatidylmethanol (PMe).135 Wehbi et al. performed binding experiments with nonsubstrate vesicles to understand the binding mechanism and lipid preferences in more detail.25 They used both zwitterionic (PC) and a range of anionic phospholipids (PMe, phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA)). Using low salt conditions and analysis with a Langmuir adsorption isotherm, they found that B. thuringiensis PI-PLC binds to anionic vesicles with much higher affinity than to zwitterionic vesicles. The authors estimated Kd’s of 64 ± 2 and 1.2 ± 0.1 μM for 1palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) SUVs, respectively. However, addition of physiological salt could dramatically reduce binding to anionic phospholipid SUVs. They concluded that the affinity for single component anionic vesicles is dominated by electrostatic interactions and that the affinity for the zwitterionic vesicles is governed by hydrophobic contributions. A few years later Pu et al. performed further experiments using FCS binding assays and mixed lipid vesicles, at physiological salt conditions, which shed new light on B. thuringiensis lipid specificity.38 They used binary component vesicles with PC (a kinetic activator, cf. section 4) and an anionic phospholipid (PMe, PA, PS, or PG) as substrate analogues. Vesicle binding was tightest for PC/PG vesicles, and PC/PS vesicles had the weakest interaction with this PI-PLC. It is worth mentioning that PG, PMe, and PA can act as competitive inhibitors of the enzyme.99 For all of these vesicles, the binding curves obtained are “ladle-shaped” with respect to the mole fraction of PC, XPC (Figure 10). The value of Kd reaches its minimum at XPC = 0.8 and then increases. This PC lipid preference by the Bacillus enzyme is due to the cation−π interaction between PC lipid headgroups and interfacial tyrosine residues.29 Hydrophobic 8449


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alanine. The cost in binding affinity for each mutation ranged from 1 to 2.5 kcal/mol and correlated well with the occupancy of the corresponding cation−π interaction observed in the MD simulations, from no significant involvement in cation−π interactions to cation−π interactions lasting most of the simulation time. Molecular mechanics force fields used for MD simulations do not explicitly account for the higher order nonbonded interactions present in cation−π interactions. Refinement of the force field to better model cation−π interactions significantly improved the correlation between cation−π occupancy in the simulations and experimental changes in Kd.138 MD simulations provide detailed pictures of these diverse and transient complexes that can involve one or two Tyr residues. The occupancies of these cation−π complexes, and not hydrophobicity or electrostatics, correlate directly with the variation of apparent Kd with XPC. However, what is the experimental evidence for such complexes? Mutagenesis says replacement of the Tyr with Ala reduces binding affinity, but it does not “prove” that the Tyr is not just inserting into the membrane. Experimental evidence for the existence of specific PC cation−Tyr π complexes has been supplied by three different techniques: (i) crystallography with choline or soluble PC,33 (ii) use of fluorinated aromatic groups at specific sites,51 and (iii) high resolution field-cycling NMR methodology to assess where persistent PC molecules bind with respect to different spin-labels on the protein.32 3.4.1. X-ray Crystallography with Choline or Soluble Phosphatidylcholine (PC). Since S. aureus PI-PLC binds poorly to PC interfaces,28,50 it serves as a good model for engineering a PC specific binding site. Comparisons between structures of B. thuringiensis PI-PLC and S. aureus PI-PLC focused on Tyr residues in helix G that are present in Bacillus sp. but not in S. aureus (Figures 3 and 4). Subsequent mutagenesis of two helix G residues, Asn254Tyr/His258Tyr, introduces PC specificity into the S. aureus enzyme. The mutant S. aureus PIPLC (N254Y/H258Y) binds with an apparent Kd value of 0.8 mM to XPC = 0.8 SUVs, a 40-fold lower Kd than wild-type S. aureus PI-PLC.33 X-ray crystal structures of the S. aureus PI-PLC N254Y/ H258Y variant soaked with choline (500 mM) or diC4PC (100 mM) show density in two sites that can be refined as the trimethylammonium moiety of choline (Figure 11A).33 The site 1 binding pocket is created by Tyr255, Asn254Tyr, and His258Tyr in helix G and Tyr212 in helix F and requires some rearrangement of side chains to accommodate the choline moiety. In site 2 a Trp residue, Trp287, as well as two Tyr residues Asn258Tyr and Tyr290 accommodate choline binding without significant side chain rearrangements relative to the apo form of the protein. When occupied, both binding sites show the common cation−π binding motif consisting of an aromatic (π) box coordinating the trimethylammonium cation.139−141 For this and other aromatic boxes, some of the aromatic residues are structurally helping to properly position the Tyr or Trp residues that bind the cation. S. aureus PI-PLC N254Y/H258Y has also been crystallized with diC4PC, and density associated with this phospholipid is only observed in site 2 (Figure 11B). Unlike binding to PC in a membrane where the acyl chains are sequestered in the bilayer, for such short, soluble PCs the acyl chains can interact with protein regions that have some nonpolar character. In the context of a bilayer the chains of the PC would be pointing up and well away from the protein (e.g., Figure 11C, D).

Figure 10. XPC dependence of B. thuringiensis PI-PLC binding to mixed anionic phospholipid (PG)/PC SUVs. Two independent data sets from experiments performed years apart with freshly prepared PI-PLC and SUVs are shown; at most a 3-fold difference is seen in the Kd at a given XPC. Given potential differences in the heterogeneity of the SUVs, particularly in the PC-rich region, this is good agreement.

interactions do not determine the lipid specificity, at least not for Bacillus PI-PLC.34 S. aureus often colonizes skin,63 a low pH environment, and S. aureus PI-PLC binds tightly to anionic PG-rich vesicles at acidic pH (6.5) where its rim loop is latched to helix G (see section 2.3.2 and Figure 6).28,50 In fact, S. aureus PI-PLC shows highest affinity (apparent Kd = 0.38 ± 0.14 mM) for pure PG vesicles at pH 6.5. Addition of PC to SUVs led to a monotonic decrease in affinity with increasing vesicle PC content, and binding of S. aureus PI-PLC to pure PC SUVs could not be measured.28 Thus, despite the fact that both Bacillus sp. and S. aureus PI-PLCs target the PC-rich136 outer membrane of eukaryotic cells, only the Bacillus PI-PLC specifically binds PC. The lack of quantitative binding data makes it more difficult to discuss the lipid specificity of L. monocytogenes PI-PLC. Monolayer studies by Chen et al. indicate that for this PI-PLC the tightest binding is achieved in the presence of an anionic membrane (PMe) rather than a zwitterionic (PC) one.26 The surface electrostatic potential of L. monocytogenes PI-PLC also strongly supports this idea (Figure 5). These results emphasize the plasticity of the IBS and the diverse membrane recognition and binding strategies used by these similar bacterial PI-PLCs. 3.4. Choline−Tyrosine Cation−π Interactions in Bacillus PI-PLC

Why does increasing PC content increase the binding affinity of Bacillus sp. PI-PLC for membranes? In the Bacillus enzyme, helix B and the rim loop are enriched in tyrosine residues (Figures 3 and 4), some of which have been shown to engage in cation−π interactions with the choline headgroups of PC. Recognition of PC lipids by PI-PLC through cation−π interactions was first proposed by Zhang et al.,137 who suggested tryptophan residues would be involved. However, subsequent mutagenesis replacing helix G Tyr residues with Ser showed a significant loss of enzymatic activity and impaired interactions with diC7PC micelles, suggesting that Tyr residues are critical.48 These aromatic residues were later observed in MD simulations to form cation−π interactions with PC.29 Mutating these tyrosine residues to alanine or serine can increase the Kd by a factor of up to 75 (Tyr88 and Tyr246), corresponding to a cost of about 2.5 kcal/mol in binding affinity. This value is significantly higher than that predicted by the Wimley−White hydrophobicity scale (∼1 kcal/mol). In total six tyrosines (86, 88, 204, 246, 247, 251) located at the IBS of B. thuringiensis PI-PLC were mutated to 8450


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interactions with PC.141,142 Thus, this substitution should result in a higher Kd (lower affinity) toward PC-rich bilayers if the aromatic residue is involved in a cation−π interaction. Fluorination also increases the hydrophobicity of the aromatic ring. This effect would enhance partitioning of the side chain into a lipid bilayer, lowering Kd (enhancing affinity). As a test of this approach for the PI-PLCs, Phe249 in the membrane binding rim loop of S. aureus PI-PLC (Figure 9) was replaced with Phe-F5. As expected since Phe249 should partition into the bilayer, the Kd clearly decreases (Figure 12A).51 With

Figure 11. Choline cation−tyrosine π binding sites from X-ray crystal structures and MD simulations. (A) S. aureus PI-PLC N254Y/H258Y crystallized in the absence (magenta, PDB 4i8y) and presence (cyan, PDB 4i90) of 500 mM choline. The choline in the binding sites are shown in yellow and orange for sites 1 and 2, respectively. (B) S. aureus PI-PLC N254Y/H258Y (1.85 Å, light blue, PDB 4i9j) crystallized in the presence of 100 mM diC4PC (orange sticks). For comparison, the structure of B. cereus PI-PLC in tan (PDB 1ptg) is superimposed. (A and B) Adapted with permission from ref 33. Copyright 2013 American Society for Biochemistry and Molecular Biology. (C and D) Snapshots of PC binding to B. thuringiensis PI-PLC from 500 ns all-atom MD simulations with the membrane at the top of each snapshot. Bound PC, with the choline labeled, is shown as sticks, and other phospholipids in the membrane are shown as lines. The B. thuringiensis residues that bind the PC headgroup are labeled and shown as sticks. Reproduced from ref 29. Copyright 2013 American Chemical Society.

Comparisons between choline and diC4PC bound to the S. aureus PI-PLC N254Y/H258Y structures33 and snapshots from MD simulations of B. thuringiensis bound to a PC-rich membrane29 show similarities and differences (Figure 11). Although similar sites are used, the residues involved in interactions with choline can differ as can the number and orientations of the residues and choline in the aromatic boxes. For example, in the S. aureus variant Tyr290 is part of an aromatic box but there is no equivalent Tyr in the Bacillus sp. structure (Figure 11B). In the simulations PC binding can also be modulated by hydrogen bonds between amino acids and the PC phosphate (e.g., Figure 11D). Some of the other differences likely reflect the plasticity of the aromatic box binding sites as well as the dynamics of PC binding where lipids enter and exit the binding sites even within the 500 ns MD simulations, whereas the X-ray crystal structures provide only a static view. There may also be real differences in the molecular details of how S. aureus PI-PLC N254Y/H258Y and Bacillus sp. bind to PC in a bilayer despite the fact that the S. aureus PI-PLC Tyr mutations were designed to mimic the Bacillus sp. enzymes. In any case, both the simulations and structures strongly support the roles of aromatic boxes in specific PC binding. 3.4.2. Using Fluorinated Aromatic Groups To Characterize the Roles of Aromatic Residues in the IBS. One way to differentiate formation of PC cation−tyrosine π complexes from nonspecific insertion of a hydrophobic side chain into the lipid bilayer is to site specifically replace an aromatic residue with a fluorinated counterpart: pentafluorophenylalanine (Phe-F5) for Phe and 3,5-difluorophotyrosine (Tyr-F2) for Tyr (fluorinated Trps are also available but were not used in the PI-PLC studies). The electronegative fluorines will lower the electron density of the π-system, which should reduce cation−π

Figure 12. Effects of fluorinated amino acids on binding affinities and PC specificity. Apparent Kd values for S. aureus PI-PLC variants with fluorinated amino acids binding to PG/PC SUVs as a function of XPC. (A) Phe249Phe-F5 (filled circles) compared to wild type (WT) (open circles). (B) Asn254Tyr/His258Tyr-F2 (filled squares) compared to Asn254Tyr/His258Tyr (open squares). Reproduced with permission from ref 51. Copyright 2015 American Society for Biochemistry and Molecular Biology.

this reassuring result in hand, Tyr-F2 was substituted into the PC binding site introduced by the Asn254Tyr/His258Tyr mutations. The S. aureus PI-PLC variant containing this substitution, Asn254Tyr/His258Tyr-F2, loses most of its affinity for PC as expected for a PC cation−Tyr π interaction (Figure 12B). The same approach was then used to test the roles of B. thuringiensis PI-PLC Tyr residues implicated in cation−π interactions (Figure 13).51 As expected, substitution of the control Tyr, Tyr247 for which mutation to Ala does not significantly affect Kd,29 with Tyr-F2 results in wild-type like affinities for PC-rich vesicles. In contrast, replacement of the other Tyr residues with the fluorinated analogue leads to Kd’s that are 1−2 orders of magnitude higher than the wild-type for PC-rich vesicles. These data also reveal another interesting effect. Fluorination alters the tyrosine phenol pKa significantly increasing the amount of deprotonated Tyr at pH 7.5;141,142 Tyr-F2 is ∼50−75% ionized at pH 7.5. Thus, fluorination alters the surface charge of the mutants by −0.5e to −0.75e. This change will weaken the affinity of the protein for vesicles rich in anionic lipids, e.g., PG. The direct effects of this charge alteration 8451


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14.04 T). At such low field strengths, the different 31P chemical shifts will merge together and the reduced difference in spins between excited and equilibrium states will result in a serious loss of sensitivity. The answer to this problem is to use shuttle field cycling to monitor R1.37,105,143,144 In this method the spins are excited at high field (for optimum sensitivity), and then the sample is rapidly shuttled to different heights in the bore of the magnet (thus lowering the field from 11.7 T to as low as 0.003 T, the latter with the use of an electromagnet mounted on top of the superconducting magnet dewar) for relaxation for a fixed period of time, followed by shuttling the sample back down into the probe for readout of residual relaxation at high field. The maximum transit time for shuttling the sample into the electromagnet is about 0.17 s, but much faster for fields of >0.1 T. Since R1 is very low at the higher fields, only a minimal change in magnetization occurs as the sample is moved to and from the low field. What is affected by the shuttling time to reach the very low fields (

Search and Subvert: Minimalist Bacterial Phosphatidylinositol

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