A Quantitative Model of Daptomycin Binding to Lipid Bilayers - The

Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

pubs.acs.org/JPCB

A Quantitative Model of Daptomycin Binding to Lipid Bilayers Antje Pokorny,* Tala O. Khatib, and Heather Stevenson Department of Chemistry and Biochemistry, University of North Carolina Wilmington, Wilmington, North Carolina 28403, United States

J. Phys. Chem. B Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 09/25/18. For personal use only.

S Supporting Information *

ABSTRACT: Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multidrug resistant infections, including those caused by methicillinresistant S. aureus strains. Similar to many other antimicrobial peptides, daptomycin binds with preference to anionic membranes such as those typically found in prokaryotes. However, in contrast to most linear α-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions, and its activity in vivo is absolutely Ca2+-dependent. Here, we describe the early events that occur in the binding of daptomycin to lipid bilayers using a quantitative model to analyze both equilibrium and kinetic binding data. The goal of the analysis was to obtain a precise description of the early events that occur in the interaction of daptomycin with lipid and calcium ions at low daptomycin concentrations. In the course of the analysis, we also determined the rate and equilibrium constants for binding of daptomycin to lipid and Ca2+. The model used to describe the experimental data comprises a soluble daptomycin monomer that binds calcium ions in solution with low affinity, a soluble, Ca2+-bound dimer, and a 1:1 daptomycin−lipidCa complex. A strong interaction of daptomycin with Ca2+-complexed lipid, the amount of which depends on the availability of calcium ions in the bulk solution, appears central to its function.

■

INTRODUCTION Secondary metabolites from bacteria, fungi, and plants fulfill a variety of functions, ranging from communication and signaling to absorption of micronutrients and the defense against competing organisms.1 Actinomycetesmembers of a large and diverse group of Gram-positive filamentous bacteriahave provided an exceptionally rich source of secondary metabolites with antibiotic properties. Among those is daptomycin, a cyclic peptide antibiotic produced by Streptomyces roseosporus. It was the first member of an entirely new class of antibiotic substances to enter clinical use since the mid-20th century, making its discovery a notable step forward in antibiotic development. Daptomycin was approved by the US FDA in 2003 as an antibiotic for the treatment of skin and skin structure infections (SSSI) caused by a number of Grampositive pathogens and, a few years later, right-sided endocarditis and bloodstream infections (bacteremia).2,3 Daptomycin and related macrocyclic peptide antibiotics are structurally complex, presumably to evade proteolysis.4 They owe their classification as cyclized lipodepsipeptides to the fatty acyl chain that usually modifies the N-terminal residue and an ester linkage that leads to ring closure and formation of a macrolactone. Daptomycin is nonribosomally synthesized, and three of its 13 amino acids are in the D-configuration: DAsn-2, D-Ala-8, and D-Ser-11. Of the 10 residues that form the cyclized peptide domain, three are nonproteinogenic: ornithine (Orn-6), 3-methylglutamate (MeGlu-12), and kynurenine (Kyn-13) (Figure 1). In daptomycin, ring closure occurs between residues Thr-4 and Kyn-13. The latter is highly © XXXX American Chemical Society

Figure 1. Daptomycin structure (source: Public Domain, Wikimedia Commons).

fluorescent and functions as a Förster resonance energy transfer (FRET) acceptor to the N-terminal Trp.5,6 The primary target of daptomycin is thought to be the bacterial cytoplasmic membrane rather than a protein receptor. However, sublethal exposure to daptomycin leads to the development of resistance in S. aureus. Resistance generally correlates with a number of downstream effects, among which are mutations in the multiple-peptide-resistance-factor-F (mprF) operon.7,8 MprF is linked to the lysylation of the anionic membrane lipid phosphatidylglycerol (PG), the most abundant phospholipid in the cell membranes of GramReceived: August 2, 2018 Revised: September 7, 2018

A

DOI: 10.1021/acs.jpcb.8b07503 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B

Thus, the mechanism of daptomycin is known in general terms, but quantitative details regarding the binding of daptomycin to calcium ions and lipid continue to remain elusive (see also ref 23). Here, we set out to probe the initial interaction of daptomycin with PG-containing lipid vesicles starting from first-principles, through kinetic and equilibrium binding experiments. In the process, we also determined the rate and equilibrium constants for binding of daptomycin to lipid and Ca2+. We show that in the presence of physiological Ca2+ concentrations daptomycin binds to lipid vesicles containing 30 mol % PG with an on-rate constant close to the diffusion limit and desorbs with a rate constant on the order of 0.1 s−1. Moreover, we show that daptomycin only interacts with Ca2+-bound PG and that the Ca2+ dependence of daptomycin binding to lipid arises from the availability of Ca2+bound PG, which is a function of the bulk Ca2+ concentration.

positive bacteria. More recent data on the effects of daptomycin in whole cells show that daptomycin also induces lipid clustering in Bacillus subtilis.9,10 The subsequent delocalization of a series of proteins essential to cell envelope synthesis has been postulated to be responsible for cell death. Daptomycin-induced clustering of anionic lipids in the presence of physiological calcium ion concentrations also occurs in giant lipid vesicles, as we recently showed by confocal microscopy.11 The biological activity of daptomycin is absolutely dependent on the presence of Ca2+ ions, which parallels the observation that in lipid model systems binding of daptomycin to the bilayer requires Ca2+.12 In liposomes, daptomycin causes the flux of small ions, such as K+ and Na+, across the lipid bilayer, and in culture, exposure to daptomycin leads to membrane depolarization. However, why calcium ions are required for binding of daptomycin to the lipid bilayer is not well established. Daptomycin contains four acidic residues, Asp-3, Asp-7, Asp-9, and MeGlu-12. Their pKa values are 4.2, 1.3, 3.8, and 4.6, in that order.13,14 The ionized form of Asp-7 is likely stabilized by a salt bridge to the neighboring Orn-6, which would account for the unusually low pKa of Asp-7. The pKa of the ornithine side chain is >1013,14 and that of the arylaminogroup in Kyn is ∼1.5,14 Thus, at neutral pH, daptomycin carries one positive and four negative charges, giving it a net charge of −3. One prevalent idea is that binding of Ca2+ renders the anionic daptomycin a “de facto” cationic peptide at neutral pH, which binds preferentially to anionic bacterial cell membranes.15 However, it is not known exactly how many calcium ions bind to daptomycin, what their binding affinity is, or where the binding sites are. Most studies on the structure of daptomycin in solution or bound to a lipid bilayer have been restricted to daptomycin concentrations in the millimolar range due to the limited sensitivity of the experimental techniques.16−20 In NMR and CD experiments, for instance, the conformational changes induced by the addition of an equimolar amount of CaCl2 have been taken as indicative of a 1:1 daptomycin−Ca2+ binding stoichiometry.17−20 However, a conformational change induced by a 1:1 ratio of daptomycin−Ca2+ in solution does not require a 1:1 stoichiometry of binding. Moreover, the analysis of available data with respect to the binding stoichiometry are complicated by a series of factors. One, the assignment of a binding stoichiometry without knowledge of the binding constant of calcium ions to daptomycin is difficult. At the requisite high lipopeptide concentrations, only very large binding constants would ensure complete binding of Ca2+ to daptomycin. Second, at daptomycin concentrations above ∼1 mM, binding of Ca2+ is coupled to the formation of aggregates, which further complicates the determination of binding constants in aqueous solution. Third, studies of daptomycin binding to liposomes containing PG in the presence of Ca2+ have not taken into account the binding of Ca2+ to PG.21 To date, the best evidence for a 1:1 daptomycin−Ca 2+ stoichiometry stems from the X−ray structure of tsushimycin, a peptide antibiotic loosely related to daptomycin that has been crystallized in the presence of Ca2+.22 Tsushimycin is an amphomycin derivative with a cyclic decapeptide core and a single exocyclic residue, the N−terminus of which is amidated with a fatty acyl chain. It shares very little sequence similarity with daptomycin, but the two Asp residues that are involved in coordinating a single Ca2+ ion are separated by four residues, just as Asp-3 and Asp-7 are in daptomycin.

■

METHODS Chemicals. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′rac-glycerol) (POPG), and 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE) were purchased from Avanti Polar Lipids (Alabaster, AL). 1-Palmitoyl-2-oleoyl-sn-glycero3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE), POPE labeled with NBD through an amide bond to the amino group of the ethanolamine headgroup, was synthesized in our laboratory24 or purchased from Invitrogen Molecular Probes (Thermo Fisher Scientific, Waltham, MA). Lipids and probes were tested by TLC and used without further purification. Calcium chloride dihydrate and Chelex 100 resin (sodium form, 50−100 mesh) were purchased from Sigma-Aldrich (St. Louis, MO) and analytical thin layer chromatography (TLC) plates from Analtech (Newark, DE). Chlorinated solvents, ethanol, and methanol (high performance liquid chromatography, American Chemical Society grade) were purchased from Burdick & Jackson (Muskegon, MI). Daptomycin was obtained from Cubist Pharmaceuticals, Inc. (Lexington, MA). Stock solutions of daptomycin were prepared in deionized water−ethanol 1:1 and stored at −20 °C. Peptide concentrations of the stock solutions were determined precisely by measuring the absorbance at 280 nm and using a molar extinction coefficient of tryptophan of 5600 M−1cm−1. Preparation of Large Unilamellar Vesicles. Large unilamellar vesicles (LUVs) were prepared by mixing the lipids in chloroform in a round-bottom flask. For vesicles containing NBD−PE, the probes were added to the lipid in chloroform solution at a final probe concentration of 2 mol %. The solvent was rapidly evaporated using a rotary evaporator (Büchi R-3000, Flawil, Switzerland) at 60 °C. The lipid film was then placed under a high vacuum for 2 h and hydrated by the addition of buffer containing 20 mM MOPS, pH 7.5, and 100 mM KCl that had been passed through a Chelex column to remove residual calcium ions. The suspension of multilamellar vesicles was subjected to five freeze−thaw cycles and extruded 10× through two stacked polycarbonate filters of 0.1 μm pore size (Nuclepore, Whatman, Florham, NJ), using a water-jacketed high-pressure extruder (Lipex Biomembranes, Inc., Vancouver, Canada) at room temperature. Lipid concentrations were assayed by the Bartlett phosphate method.25 Kinetics of Daptomycin Binding to Lipid Vesicles. Daptomycin working solutions were prepared by diluting the B


Article

The Journal of Physical Chemistry B concentrated stock solutions in Ca2+-free buffer (20 mM MOPS, pH 7.5, 100 mM KCl). The daptomycin solution and lipid suspension were supplemented with calcium ions by adding a small aliquot from a concentrated stock solution of calcium chloride (0.1 or 1 M) to the working solution to the desired final Ca2+ concentration. The kinetics of association of daptomycin with LUVs were recorded on an Applied Photophysics SX.18MV stopped-flow fluorimeter (Leatherhead, Surrey, UK) at room temperature by rapidly mixing equal volumes of daptomycin solution and lipid suspension from the reservoir syringes. The final concentrations of the reagents in the observation chamber was thus halved with respect to those in the reservoir syringes. Fluorescence resonance energy transfer (FRET) between the intrinsic Kyn residue of daptomycin and NBD−POPE incorporated in the lipid membrane was used to monitor peptide binding and dissociation from LUVs.26 The Kyn residue was excited at 350 nm through a monochromator using a slit width of 1 mm. Energy transfer to NBD−POPE, which absorbs maximally at 463 nm, was monitored by measuring the emission of NBD− POPE (λmax 536 nm) through an OG-515 long-pass filter (Edmund Industrial Optics, Barrington, NJ). Daptomycin binding leads to an increase in fluorescence emission from the lipid acceptor NBD−POPE, which was monitored as a function of time. Titrations. For the lipid titration experiments, 5−10 mL of a 1 μM solution of daptomycin in buffer was prepared at the desired Ca2+ concentration. A 1 mL aliquot of this solution was then mixed with a concentrated suspension of LUVs, that had also been supplemented with calcium ions, to a final lipid concentration that corresponded to the end point of the titration. This daptomycin−lipid mixture was then diluted serially with the daptomycin solution in separate vials and allowed to equilibrate for a short period of time. Each sample was then measured in a Horiba Jobin−Ivon Fluorolog-3 spectrophotometer (Horiba Scientific, Edison, NJ) equipped with dual monochromators in the emission and excitation path to minimize signal distortions from light scattering at high lipid concentrations. This method has two advantages. One, it ensures that the daptomycin concentration remains constant throughout the experiment, and thus avoids having to correct the fluorescence signal for dilution caused by lipid addition. Second, since each daptomycin−lipid sample is measured individually, the signal is not influenced by the history of the sample. For instance, the samples at the higher lipid concentrations contain no contribution from the samples at low lipid concentrations in which the high daptomycin−lipid ratio can potentially lead to a irreversible distortion of the lipid matrix. Description of the Model. The model used to describe the kinetic and equilibrium data is summarized in the following reaction scheme

occurs to the outer monolayer of the entire lipid vesicle; however, we express vesicle concentrations in terms of the lipid concentration in outer monolayer, which is the more common practice.) The rate constants for the individual processes are indicated next to the reaction arrows and are used to obtain the corresponding equilibrium constants. Ko = ko1/ko2 is the binding constant of calcium ions to soluble daptomycin, Kagg = kagg/kdagg, the equilibrium constant that describes dimerization, and KL = k1/k−1 the equilibrium constant for DCa binding to Ca2+−complexed lipid, LCa. The reaction scheme above is described by the following set of differential equations: d[D] /dt = ko2[DCa ] − ko1[Ca][D] d[DCa ] /dt = k −1[DCa LCa] − k1[LCa][DCa ] + 2kdagg[(DCa )2 ] − 2kagg[DCa ]2 − ko2[DCa ] + ko1[Ca][D] d[(DCa )2 ] /dt = kagg[DCa ]2 − kdagg[(DCa )2 ] d[DCa LCa] /dt = k1[LCa][DCa ] − k −1[DCa LCa] , (1)

For the stopped-flow experiments, the initial concentrations of the three solution states of daptomycin, D, DCa, and (DCa)2, were calculated as follows. The daptomycin solution and lipid suspensions were allowed to equilibrate at room temperature in the reservoir syringes at the appropriate Ca2+ concentration. Both the lipid and daptomycin concentrations in the reservoir syringes are twice that of the final experimental concentrations. In the reservoir syringe, the initial concentration of free daptomycin is 2D(0)

1 = − (1 + [Ca 2 +]Ko 4 − 8[Ca 2 +]2 K aggKo2(2Dt ) + [Ca 2 +]2 Ko2 + 2[Ca 2 +]Ko + 1 /([Ca 2 +]2 K aggKo2))

where Dt is the total daptomycin concentration after mixing. The initial concentration of Ca2+-bound daptomycin is 2DCa(0)

1 = Ko[Ca 2 +](− (1 + [Ca 2 +]Ko 4 − 8[Ca 2 +]2 K aggKo2(2Dt ) + [Ca 2 +]2 Ko2 + 2[Ca 2 +]Ko + 1 /([Ca 2 +]2 K aggKo2)))

Finally, the initial concentration of the soluble daptomycin dimer is 2(DCa)2 (0) = K aggKo2[Ca 2 +]2 (− 1/4(1 + [Ca 2 +]Ko −

8[Ca 2 +]2 K aggKo2(2Dt ) + [Ca 2 +]2 Ko2 + 2[Ca 2 +]Ko + 1

/([Ca 2 +]2 K aggKo2)))2

At t = 0, equal volumes of lipid suspension and daptomycin solution are mixed. The total daptomycin concentration in the reaction chamber at t = 0 is then halved and equal to Dt. Similarly, the concentrations of monomerfree and Ca2+boundand dimer in solution in the reaction chamber at t = 0 are D, DCa, and (DCa)2. Since Kagg is obtained from the fit, the initial concentrations of the solution species were recalculated

where D is the daptomycin monomer; DCa, Ca2+-bound daptomycin; and (DCa)2, the daptomycin dimer, with bound Ca2+. D, DCa, and (DCa)2 are solution species. DCaLCa is the daptomycin monomer bound to Ca2+-complexed lipid, and LCa is the concentration of Ca2+ complexed lipid. (Peptide binding C


Article


we began by analyzing the kinetic data describing daptomycin binding to lipid with a very simple two-state model. When this simple model ceased to accurately describe the data, we expanded the model in a stepwise fashion to account for the complexity of the data. Additional states were chosen according to criteria detailed below. The numerical values of the parameters were additionally constrained by performing equilibrium titrations of daptomycin solutions with a suspension of lipid vesicles as a function of calcium ion concentration. In the following, the development of the model is described in more detail. In the simplest scenario, daptomycin binds to lipid vesicles in a single reversible step. In that case, the reaction can be written as

iteratively until self-consistency was achieved. The binding constant of calcium ions to soluble daptomycin, Ko, was determined by first estimating its value from the Ca2+ dependence of the slow phase in the kinetic traces (see the Results and Discussion). For the global kinetic fits, an essentially diffusion-limited on-rate of ko1 > 106 was assumed, and Ko = ko1/ko2. The value of Ko was then adjusted by calculating the fraction of lipid-bound daptomycin as a function of lipid concentration and Ko at t = ∞ and comparing the obtained values to those from the experimentally obtained equilibrium titration date. This procedure was repeated until consistency between the kinetic and equilibrium titration data sets was achieved. Only the membrane-bound state of daptomycin was assumed to contribute to the FRET signal.

■

k1

RESULTS AND DISCUSSION We measured binding of the lipopeptide daptomycin to PGcontaining lipid vesicles as a function of lipid and calcium ion concentration. Equilibrium and kinetic data were analyzed using exact models to expand the current understanding of daptomycin function. Association of daptomycin with lipid vesicles is accompanied by a large increase in the fluorescence emission from kynurenine, a nonproteinogenic amino acid located in the cyclized peptide domain of daptomycin (Figure 2). Thus,

D + L HooI DL

(2)

k −1

where D stands for daptomycin in solution, L, for lipid vesicles, and DL for lipid-bound daptomycin. Binding of daptomycin occurs independently and, if [L] and [D] are chosen such that [L] ≫ [D], the kinetics of binding follow a pseudo first-order rate law and reaction 2 simplifies to k1[L]

D HoooI DL

(3)

k −1

In that case, the amount of bound daptomycin as a function of time is described by an exponential function with an apparent rate constant, kapp, which is a function of the lipid vesicle concentration, kapp = k1[L] + k−127,28 DL(t ) = Dt

k1[L] (1 − e−kappt ) k −1 + k1[L]

(4)

where Dt is the total daptomycin concentration. The experimentally observed increase in fluorescence emission is proportional to DL(t) since it results from the increased FRET that accompanies daptomycin binding to lipid. If binding occurs as described by reaction 3, a fit of the kinetic data with an exponential function yields kapp, and the rate constants k1 and k−1 can be obtained from the slope and y-intercept of a plot of kapp against lipid concentration. To a first approximation, that is what we observed when the data were analyzed on short time scales (Figure 3). However, two subsequent observations indicated that this simple model is insufficient to quantitatively describe the experimental data. First, the kinetics of daptomycin binding to PG-containing lipid vesicles depend strongly on the calcium ion concentration in solution (Figure 4). The activity of daptomycin is known to critically depend on the presence of Ca2+.19,29,30 In principle, the Ca2+ dependence could arise from the interaction of Ca2+ with either daptomycin or lipid. Let us first explore the possibility of Ca2+ binding to daptomycin. In the absence of calcium ions, we were not able to detect binding of daptomycin to lipid, which implies that perhaps daptomycin requires complexation with calcium ions to bind to lipid bilayers. The Ca2+ dependence of binding would then arise from the larger fraction of Ca2+−bound daptomycin available at higher calcium ion concentrations. However, while a larger fraction of binding-competent daptomycin would increase the rate of binding, it would not alter the rate constants associated with binding, k1 and k−1, and, thus, kapp. Therefore, in order to understand the Ca2+

Figure 2. Lipid-bound fluorescence spectra of daptomycin. Daptomycin fluorescence spectra in the region of kynurenine emission as a function of lipid concentration. The concentration of Ca2+ was 2 mM, and that of daptomycin was 1 μM. Lipid used was a 70:30 mixture of POPC and POPG in concentrations ranging from 0 (purple trace) to 500 μM (red trace). The remaining spectra were generated by diluting a 500 μM lipid sample with a solution containing 2 mM Ca2+ and 1 μM daptomycin in 1:2 dilution steps, resulting in an exponential series of lipid concentrations (500 μM, 250 μM, 125 μM··· 0.977 μM) at constant Ca2+ and daptomycin concentrations.

daptomycin binding to the water-bilayer interface can be either monitored directly, through the fluorescence increase that accompanies association, or through fluorescence resonance energy transfer (FRET) from kynurenine to an acceptor fluorophore located in the lipid bilayer. The two methods yield equivalent data; however, the latter leads to an improved signal-to-noise ratio. We took a sequential approach to develop an accurate and quantitative model of daptomycin−lipid interactions. Briefly, D


Article


Figure 3. Kinetics of daptomycin binding to lipid vesicles as a function of the lipid concentration. (A) Experimental binding kinetics in 5 mM Ca2+ buffer for 5, 100, and 200 μM lipid (70:30 POPC−POPG). The data have been normalized. (B) Apparent rate constant from a single-exponential fit to the kinetic data, kapp, as a function of lipid concentration. The slope of the line yields k1 and the y-intercept, k−1.

Note that the possibility of daptomycin binding calcium ions in solution cannot be excluded at this point. We just showed that the Ca2+ dependence of kapp determined from kinetic binding data on short time scales yields no information on whether Ca2+ binds to daptomycin in solution. A second observation required further expansion of the model. Over longer time scales, the experimental binding kinetics deviate significantly from single exponential behavior (Figure 5A). We found this to be true over all lipid and Ca2+ concentrations tested. In the simplest terms, deviation from single exponential kinetics indicates that binding does not occur in a single equilibrium reaction such as that shown in eq 5 but points to the presence of an additional state, either preceding or following binding of daptomycin to lipid (states X or X′ in eq 8). k1[LCa]

Figure 4. Apparent rate constant of binding, kapp, depends on the bulk calcium ion concentration. The dashed line is drawn to guide the eye.

X F D HoooooI DLCa F X′ k −1

The two scenarios are predicted to influence the binding kinetics in different fashions.33 A process preceding daptomycin binding to lipid will lead to an additional phase in the binding kinetics with an amplitude that is independent of lipid concentration. A process following that of the initial daptomycin binding will also introduce an additional phase to the binding kinetics. However, the amplitude associated with that equilibrium will now depend on lipid concentration, in contrast to the first scenario, because the additional state is membrane-associated. The experimental data clearly show that at a fixed Ca2+ concentration, the amplitude of the second, slow phase is independent of lipid concentration (Figure 5B). We conclude that no additional lipid-bound states of daptomycin are required to describe the initial phases of binding. Moreover, we can also exclude other processes that are of higher order with respect to lipid concentration, such as lipid vesicle fusion, as factors contributing to the binding kinetics on the experimental time scale. Rather, the binding kinetics point to a second solution state of daptomycin. Based on the observation that no large daptomycin oligomers exist even at high Ca2+ concentrations34,35 and that tsushimycin, a related Ca2+-dependent lipopeptide, can form dimers in the presence of Ca2+,22 we modeled the additional solution state as dimers. With the inclusion of daptomycin dimerization in solution, the binding process now reads

effect on binding, we need to explore factors that directly affect k1 or k−1or both. Lipid vesicles with PG fractions between 20 and 50 mol % have been shown to specifically bind Ca2+ with uncorrected binding constants on the order of 100−1000 M−1.21,31,32 Thus, a straightforward way to account for the Ca2+ effect is to explicitly include binding of calcium ions to PG in the binding model. If Ca2+ binding to lipid is included in binding eq 3, the concentration of Ca2+-bound lipid, LCa, becomes a function of the calcium ion concentration and the binding equilibrium now reads k1[LCa]

D HoooooI DLCa

(5)

k −1

where LCa =

Kc[Ca 2 +] 1 + Kc[Ca 2 +]

[L] (6) 2+

2+

Here, Kc is the binding constant of Ca to lipid, [Ca ], the concentration of calcium ions, and [L] the total lipid concentration. We can now also define the binding constant of daptomycin binding to lipid, KL, as KL =

[DLCa] k = 1 [D][LCa] k −1

(8)

(7) E


Article


Figure 5. Kinetics of daptomycin binding to lipid (70:30 POPC− POPG) on a 5 s time scale. (A) Binding kinetics in buffer containing 5 mM CaCl2. Lipid concentration was 100 μM, and that of daptomycin was 1 μM. Experimental data, black dots. The solid blue line is the result of a one−exponential fit to the experimental data. (B) Experimental binding kinetics as a function of total lipid concentrations (see legend). kagg k1[LCa] 1 D2 HoooI D HoooooI DLCa 2 kdagg k −1

Figure 6. Kinetics of daptomycin binding to 100 μM total lipid (70:30 POPC−POPG) as a function of [Ca2+]. (A) Black trace, 0.5 mM Ca2+; red trace, 1 mM Ca2+; green trace, 2 mM Ca2+; blue trace, 5 mM Ca2+; magenta trace, 10 mM Ca2+ (see legend). (B) Dependence of the smaller apparent rate constant, k′, resulting from a two-exponential fit to the data in (A) on [Ca2+]. The daptomycin concentration was 1 μM. The dashed line was drawn to guide the eye.

(9)

At neutral pH, daptomycin carries a net charge of −3, which led us to hypothesize that dimerization may be promoted at high calcium ion concentrations due to the charge neutralization resulting from the binding of Ca2+. If that is indeed the case, the analysis of the minor slow phase should shed some light on this issue because, in the current model, the apparent rate constant that characterizes this phase, k′, is dominated by the rate constants associated with daptomycin dimerization in solution. Thus, the dependence of k′ on the Ca 2+ concentration can be taken as indicative of a Ca2+-induced oligomerization. The experimental data in Figure 6 show that this is indeed the case, from which we conclude that even at low daptomycin concentration the binding of calcium ions is coupled to daptomycin dimerization. Based on these results, we formulated a final model that includes binding of calcium ions to soluble daptomycin monomers in a 1:1 stoichiometry, Ca2+-induced dimerization in solution, and binding of the Ca2+-bound daptomycin monomers to Ca2+-complexed lipids:

The model described by reaction 10 was formulated as a set of coupled differential equations (equation array (1) in the Methods), which were solved by numerical integration. The numerical solutions were simultaneously fitted to 15 kinetic data sets, spanning total lipid concentrations ranging from 50− 200 μM and Ca2+ concentrations from 1−20 mM. The values of the fit parameters were tightly constrained by the simultaneous fits, with the exception of k−1 and Ko, the binding constant of Ca2+ to daptomycin monomers in solution, both of which were only poorly defined. That is, their numerical values could be varied over at least 2 orders of magnitude without the fit quality deteriorating significantly. To better define k−1 and Ko, we performed equilibrium titrations of daptomycin solutions with lipid vesicle suspensions containing 30 mol % PG, a composition identical to that used in the kinetic experiments. The fraction of DCaLCa at infinite time was first calculated using reaction 10. The procedure was then repeated for different [LCa], and the results F


Article


Figure 7. Equilibrium titrations of a 1 μM daptomycin solution with lipid vesicles (70:30 POPC−POPG) as a function of Ca2+ concentration. Plotted on the x-axis is the lipid concentration in the outer monolayer: (A) 0.1 mM Ca2+, (B) 1 mM Ca2+. The black circles are normalized experimental data points (two independent data sets in (A) and three independent data sets in (B)). The solid black lines are drawn through points obtained from the kinetic fit program as explained in the text, using k−1 = 0.1 s−1. The lower of the two dashed lines in each panel was obtained using k−1 = 0.15 s−1, and the upper dashed line, k−1 = 0.05 s−1.

distribution of daptomycin states as a function of the bulk Ca2+ concentration using the final model shows that at low Ca2+ concentrations, when binding to lipid is roughly half-maximal, two of the solution states, D and DCa, are populated to a significant extent (Figure 10A). Therefore, a likely explanation for the appearance of the isosbestic point in the titrations is that the daptomycin solution states have similar CD spectra. More specifically, isosbestic points occur when an observed spectrum is the sum of only two-component spectra. The presence of additional molecular species with different spectra will abolish the isosbestic point. However, isosbestic points also occur if several molecular species share the same spectrum over the wavelength range examined. For instance, we propose the existence of four molecular species. If the solution species are indistinguishable by CD, the experimental CD spectrum of daptomycin in the presence of lipid will contain only two componentsone resulting from the three solution species and the other from the membrane-bound form. The UV−vis and CD spectra of daptomycin in solution have been shown to be independent of Ca2+ (our observation and ref 17). In fact, it is the absence of a distinct Ca2+-dependent signal in the UV− vis range that prevents the determination of the binding constant of Ca2+ to daptomycin in solution by CD or UV−vis spectroscopy in the first place. Thus, we posit that the isosbestic point in the CD titrations observed by Lee et al. arises because the solution states of daptomycin, D, DCa, and (DCa)2 are indistinguishable by CD spectroscopy. Our model is not the first attempt to characterize the interaction of daptomycin with its lipid target. For example, Muraih et al.29 have proposed a model describing daptomycin−lipid interaction that bears some general resemblance to the one presented here. However, there are some important differences. For one, these authors again find no evidence for daptomycin oligomerization in solution by FRET. However, the fraction of daptomycin dimers depends critically on the daptomycin concentration. At concentrations typically used in steady-state fluorescence measurements (∼1 μM) and in the absence of lipid, the fraction of dimers in solution is only about 0.2 (Figure 10B) at physiological concentrations of Ca2+ (1−2 mM). In FRET experiments that use two populations of labeled monomers, the fraction of “hetero”-dimers that are actually able to undergo FRET is even lower, making the detection of dimers in solution virtually impossible. The same

were plotted and compared with the experimental titration data (Figure 7). The equilibrium titrations proved to be quite sensitive the numerical value of k−1, which allowed us to narrow its value to 0.10 ± 0.05 s−1 (Figure 7). Ko remains not particularly well-defined; however, to bring the kinetic and equilibrium data in agreement, we had to assume Ko = 2 × 104 M, a value that is in reasonable agreement with Ca2+ binding constants measured for a series of small dicarboxylic acids.36 To estimate the error in k1, k−1, and kdagg, the kinetic traces were next fit individually, using the parameters from the global fit as starting values and allowing k1, kagg, and kdagg to vary while keeping k−1, Kc, and Ko constant. The results are shown in Figure 8, and the numerical values of the fit parameters are listed in Table 1. Binding of daptomycin to PC−PG vesicles at high Ca2+ concentrations also provides a convenient way to estimate the daptomycin−lipid stoichiometry (Figure 9). At 20 mM Ca2+, the lipid in the outer monolayer is saturated with Ca2+. With each lipid addition, all available lipid binding sites become occupied by daptomycin, and daptomycin is removed from solution in direct proportion to the amount of lipid added until all daptomycin is bound to lipid (Figure 9). The data points thus fall on two straight lines and the stoichiometric lipid:daptomycin ratio can be extracted from the point at which the two lines intersect. Using a total daptomycin concentration of 1 μM, the point of intersection occurs at 3.2 ± 0.5 μM lipid. On the abscissa, we plotted the total lipid concentration in the outer monolayer. If we assume that Ca2+ binds only to the anionic PG in the mixture (PGCa, 30 mol % of the total lipid), the resulting daptomycin−PGCa stoichiometry is approximately 1:1. Lee et. al35 came to a similar conclusion by titrating a 40 μM daptomycin−lipid mixture with calcium ions. However, the same authors also concluded, based on the appearance of an isosbestic point in CD spectra obtained from the titration of daptomycin with PG−PC liposomes in the presence of Ca2+, that at low daptomycin concentrations, only two daptomycin states exist: free in solution and bound to lipid. Since we have provided evidence here for the existence of four daptomycin states, we sought to examineand perhaps reconcilethe differing claims. In principle, only two states may exist in amounts significant enough to contribute to the CD spectra. However, a plot of the G


Article


Figure 9. Equilibrium titrations of a 1 μM daptomycin solution with lipid vesicles (70:30 POPC−POPG) in buffer containing 20 mM CaCl2. The circles represent normalized experimental data from three independent data sets. Lipid concentration in the outer monolayer is plotted on the x-axis. The two lines are straight-line fits to either the initial data points or the final data points. The point of intersection is used to determine the lipid−daptomycin stoichiometric ratio (see text).

existence of daptomycin oligomers in the time frame of the kinetic experiments. This may simply mean that oligomers are only formed on longer time scales. However, the existence of a FRET signal alone is not sufficient evidence for oligomerization, unless the signal is corrected for the background FRET that results from the random collision of monomers in the plane of the bilayer.37−39

■

SUMMARY AND CONCLUSION In summary, we successfully described the early events that occur in the binding of daptomycin to lipid bilayers using an exact model to analyze both equilibrium and kinetic binding data. The model comprises a soluble daptomycin monomer that binds calcium ions with low affinity, a soluble, Ca2+-bound dimer, and a 1:1 daptomycin−lipidCa complex. In this process, we also determined the rate and equilibrium constants for binding of daptomycin to lipid and Ca2+. Thus, we showed that the dependence of daptomycin binding on Ca2+ is a consequence of a very favorable interaction with Ca2+complexed lipid (K ≈ 107 M−1), the amount of which depends on the availability of calcium ions in the bulk solution. Further, the affinity of daptomycin for lipid does not arise from irreversible formation of the final lipid-associated state. Rather, the magnitude of the dissociation constant (k−1 = 0.1 s−1) shows that daptomycin readily dissociates from lipid vesicles. The large daptomycin−lipid−Ca2+ domains observed in whole cells and by confocal microscopy9−11 are likely to form on time scales longer than those examined in this work. In any case, the interaction of daptomycin with Ca2+-complexed lipid appears central to its function. It also raises the possibility that

Figure 8. Final result of the kinetic fits for three sets of lipid concentrations. Black dots represent experimental data points and red lines, fit results using the parameters listed in Table. 1: (A) 50 μM total lipid, (B) 100 μM total lipid, (C) 200 μM total lipid. In each panel, Ca2+ concentrations are 1, 2, 5 , 10, and 20 mM, from right to left, with the lowest Ca2+ concentration always resulting in the slowest binding kinetics.

authors also attributed the FRET signal between two fluorescently labeled daptomycin populations in the presence of lipid to the formation of daptomycin oligomers on the membrane. In our work, we found no evidence for the

Table 1. Results from Fitting the Final Model to the Experimental Data.a k1 (M−1 s−1) (2.1 ± 0.4)×10

6

k−1 (s−1)

kagg (M−1 s−1)

0.10 ± 0.05

(5.3 ± 2.5)×10

4

kdagg (s−1)

Kc (M−1)

0.36 ± 0.16

28 ± 5

a The value of the binding constant of Ca2+ to daptomycin monomers in solution was assumed to be Ko = 2.0 × 104 M−1. Kc is the binding constant of Ca2+ to lipid. Binding of daptomycin to Ca2+−bound lipid is described by k1 and k−1 (see eq 7), KL = k1/k−1 = (2.1 ± 1.1) × 107 M−1 and Kagg = kagg/kdagg = (1.5 ± 1.0) × 105 M−1.

H


Article


Figure 10. Distribution of daptomycin states according to the final model described by eq 10. (A) Distribution of states for 200 μM total lipid as a function of the bulk Ca2+ concentration. Black line: free monomer in solution D. Red line: Ca2+-bound monomer in solution DCa. Green line, soluble daptomycin dimers (DCa)2. Blue line: lipid-bound daptomycin monomers DCaLCa. (B) Distribution of states in buffer containing 1 mM CaCl2 as a function of the total daptomycin concentration, Dt, in the absence of lipid. Black line: free monomer in solution D. Red line: Ca2+-bound monomer in solution DCa. Green line: soluble daptomycin dimers. Fractions were calculated using SageMath.40

daptomycin may compete for other surface-bound Ca2+ ions, such as those found to the bacterial cell wall,41 potentially compromising the integrity of the cell wall. In that case, the lipidation found in daptomycin and related Ca2+-dependent lipopetides would simply serve to reduce their solubility in water and drive the association with surfaces and other interfaces.

■

(5) Muangsiri, W.; Kirsch, L. E. The Kinetics of the Alkaline Degradation of Daptomycin. J. Pharm. Sci. 2001, 90, 1066−1075. (6) Fukunaga, Y.; Katsuragi, Y.; Izumi, T.; Sakiyama, F. Fluorescence Characteristics of Kynurenine and N’-Formylkynurenine. Their Use as Reporters of the Environment of Tryptophan 62 in Hen Egg-white Lysozyme. J. Biochem. 1982, 92, 129−141. (7) Friedman, L.; Alder, J. D.; Silverman, J. A. Genetic Changes that Correlate with Reduced Susceptibility to Daptomycin in Staphylococcus aureus. Antimicrob. Agents Chemother. 2006, 50, 2137− 2145. (8) Ernst, C. M.; Peschel, A. Broad-spectrum Antimicrobial Peptide Resistance by MprF-mediated Aminoacylation and Flipping of Phospholipids. Mol. Microbiol. 2011, 80, 290−299. (9) Pogliano, J.; Pogliano, N.; Silverman, J. A. Daptomycin-mediated Reorganization of Membrane Architecture Causes Mislocalization of Essential Cell Division Proteins. J. Bacteriol. 2012, 194, 4494−4504. (10) Müller, A.; Wenzel, M.; Strahl, H.; Grein, F.; Saaki, T. N.; Kohl, B.; Siersma, T.; Bandow, J. E.; Sahl, H. G.; Schneider, T. Daptomycin Inhibits Cell Envelope Synthesis by Interfering with Fluid Membrane Microdomains. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E7077− E7086. (11) Kreutzberger, M. A.; Pokorny, A.; Almeida, P. F. Daptomycin− Phosphatidylglycerol Domains in Lipid Membranes. Langmuir 2017, 33, 13669−13679. (12) Silverman, J. A.; Perlmutter, N. G.; Shapiro, H. M. Correlation of Daptomycin Bactericidal Activity and Membrane Depolarization in Staphylococcus aureus. Antimicrob. Agents Chemother. 2003, 47, 2538−2544. (13) Kirsch, L. E.; Molloy, R. M.; Debono, M.; Baker, P.; Farid, K. Z. Kinetics of the Aspartyl Transpeptidation of Daptomycin, a Novel Lipopeptide Antibiotic. Pharm. Res. 1989, 6, 387−393. (14) Qiu, J.; Yu, L.; Kirsch, L. E. Estimated pKa Values for Specific Amino Acid Residues in Daptomycin. J. Pharm. Sci. 2011, 100, 4225− 4233. (15) Bayer, A. S.; Schneider, T.; Sahl, H. G. Mechanisms of Daptomycin Resistance in Staphylococcus aureus: Role of the Cell Membrane and Cell Wall. Ann. N. Y. Acad. Sci. 2013, 1277, 139−158. (16) Rotondi, K. S.; Gierasch, L. M. A Well-defined Amphipathic Conformation for the Calcium-free Cyclic Lipopeptide Antibiotic, Daptomycin, in Aqueous Solution. Biopolymers 2005, 80, 374−85. (17) Jung, D.; Rozek, A.; Okon, M.; Hancock, R. E. Structural Transitions as Determinants of the Action of the Calcium-dependent Antibiotic Daptomycin. Chem. Biol. 2004, 11, 949−957. (18) Ball, L. J.; Goult, C. M.; Donarski, J. A.; Micklefield, J.; Ramesh, V. NMR Structure Determination and Calcium Binding Effects of Lipopeptide Antibiotic Daptomycin. Org. Biomol. Chem. 2004, 2, 1872−1878.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b07503. Distribution of daptomycin species in solution and in the presence of lipid (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel: (910) 962-4231. Fax: (910) 962-3013. E-mail:almei [email protected]. ORCID

Antje Pokorny: 0000-0001-6675-9966 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported in part by NIH Grant No. AI088567 and a UNCW SURCA grant. The SageMath computer algebra system was used throughout.40 We also thank Paulo Almeida for an introduction to the beauty of partition functions, many discussions, and the basic Fortran code used to construct the fit routines.

■

REFERENCES

(1) Demain, A. L.; Fang, A. The Natural Functions of Secondary Metabolites. Adv. Biochem. Eng. Biotechnol. 2000, 69, 1−39. (2) Strieker, M.; Marahiel, M. A. The Structural Diversity of Acidic Lipopeptide Antibiotics. ChemBioChem 2009, 10, 607−616. (3) Eisenstein, B. I.; Oleson, F. B., Jr.; Baltz, R. H. Daptomycin: From the Mountain to the Clinic, with Essential Help from Francis Tally, MD. Clin. Infect. Dis. 2010, 50, S10−S15. (4) Baltz, R. H.; Miao, V.; Wrigley, S. K. Natural Products to Drugs: Daptomycin and Related Lipopeptide Antibiotics. Nat. Prod. Rep. 2005, 22, 717−741. I


Article


(40) SageMath [S+09], the Sage Mathematics Software System (Version 7.5.1), The Sage Developers, 2017, http://www.sagemath. org (accessed Sep 7, 2018). (41) Thomas, K. J., III; Rice, C. V. Revised Model of Calcium and Magnesium Binding to the Bacterial Cell Wall. BioMetals 2014, 27, 1361−1370.

(19) Straus, S. K.; Hancock, R. E. Mode of Action of the New Antibiotic for Gram-positive Pathogens Daptomycin: Comparison with Cationic Antimicrobial Peptides and Lipopeptides. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 1215−1223. (20) Ho, S. W.; Jung, D.; Calhoun, J. R.; Lear, J. D.; Okon, M.; Scott, W. R.; Hancock, R. E.; Straus, S. K. Effect of Divalent Cations on the Structure of the Antibiotic Daptomycin. Eur. Biophys. J. 2008, 37, 421−433. (21) Macdonald, P. M.; Seelig, J. Calcium Binding to Mixed Phosphatidylglycerol-Phosphatidylcholine Bilayers as Studied by Deuterium Nuclear Magnetic Resonance. Biochemistry 1987, 26, 1231−1240. (22) Bunkóczi, G.; Vértesy, L.; Sheldrick, G. M. Structure of the Lipopeptide Antibiotic Tsushimycin. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2005, 61, 1160−1164. (23) Zhang, J.; Scott, W. R. P.; Gabel, F.; Wu, M.; Desmond, R.; Bae, J.; Zaccai, G.; Algar, W. R.; Straus, S. K. On the Quest for the Elusive Mechanism of Action of Daptomycin: Binding, Fusion, and Oligomerization. Biochim. Biophys. Acta, Proteins Proteomics 2017, 1865, 1490−1499. (24) Frazier, M. L.; Wright, J. R.; Pokorny, A.; Almeida, P. F. Investigation of Domain Formation in Sphingomyelin/Cholesterol/ POPC Mixtures by Fluorescence Resonance Energy Transfer and Monte Carlo Simulations. Biophys. J. 2007, 92, 2422−2433. (25) Bartlett, G. R. Phosphorous Assay in Column Chromatography. J. Biol. Chem. 1959, 234, 466−468. (26) Lakowicz, J. R. In Principles of Fluorescence Spectroscopy, 3rd ed.; Lakowicz, J. R., Ed.;; Springer: New York, 2006; pp 443−467. (27) Pokorny, A.; Almeida, P. F. F.; Melo, E. C. C.; Vaz, W. L. C. Kinetics of Amphiphile Association with Two-Phase Lipid Bilayer Vesicles. Biophys. J. 2000, 78, 267−280. (28) Almeida, P. F.; Pokorny, A. Binding and Permeabilization of Model Membranes by Amphipathic Peptides. Methods Mol. Biol. 2010, 618, 155−169. (29) Muraih, J. K.; Pearson, A.; Silverman, J.; Palmer, M. Oligomerization of Daptomycin on Membranes. Biochim. Biophys. Acta, Biomembr. 2011, 1808, 1154−1160. (30) Taylor, S. D.; Palmer, M. The Action Mechanism of Daptomycin. Bioorg. Med. Chem. 2016, 24, 6253−6268. (31) Cevc, G. Membrane Electrostatics. Biochim. Biophys. Acta, Rev. Biomembr. 1990, 1031, 311−382. (32) Garidel, P.; Blume, A. Interaction of Alkaline Earth Cations with the Negatively Charged Phospholipid 1,2-Dimyristoyl-snglycero-3-phosphoglycerol: A Differential Scanning and Isothermal Titration Calorimetric Study. Langmuir 1999, 15, 5526−5534. (33) Kreutzberger, A. J.; Pokorny, A. On the Origin of Multiphasic Kinetics in Peptide Binding to Phospholipid Vesicles. J. Phys. Chem. B 2012, 116, 951−957. (34) Qiu, J.; Kirsch, L. E. Evaluation of Lipopeptide (Daptomycin) Aggregation using Fluorescence, Light Scattering, and Nuclear Magnetic Resonance Spectroscopy. J. Pharm. Sci. 2014, 103, 853− 861. (35) Lee, M. T.; Hung, W. C.; Hsieh, M. H.; Chen, H.; Chang, Y. Y.; Huang, H. W. Molecular State of the Membrane-Active Antibiotic Daptomycin. Biophys. J. 2017, 113, 82−90. (36) Sari, H.; Covington, A. K. Comparative Measurements of Calcium Stability Constants of Some Dicarboxylic Acids by Titrimetry with pH Glass and Calcium Ion-Selective Electrodes. J. Chem. Eng. Data 2005, 50, 1620−1623. (37) Wolber, P. K.; Hudson, B. S. An Analytic Solution to the Förster Energy Transfer Problem in two Dimensions. Biophys. J. 1979, 28, 197−210. (38) King, C.; Sarabipour, S.; Byrne, P.; Leahy, D. J.; Hristova, K. The FRET Signatures of Noninteracting Proteins in Membranes: Simulations and Experiments. Biophys. J. 2014, 106, 1309−1317. (39) King, C.; Raicu, V.; Hristova, K. Understanding the FRET Signatures of Interacting Membrane Proteins. J. Biol. Chem. 2017, 292, 5291−5310. J


A Quantitative Model of Daptomycin Binding to Lipid Bilayers - The

Recommend Documents