Structural basis for endotoxin neutralization and anti-inflammatory activity of thrombin-derived C-terminal peptides

Thrombin-derived C-terminal peptides (TCP) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. In an effort to elucidate the structural and molecular aspects of these functions, we here employ a combination of nuclear magnetic resonance spectroscopy (NMR), ellipsometry, fluorescence spectroscopy, circular dichroism (CD) measurements, and in silico multiscale modeling to define interactions and the bound conformation of a TCP generated by neutrophil elastase, HVF18 (HVFRLKKWIQKVIDQFGE) in complex with bacterial lipopolysaccharide (LPS). In contrast to the disordered state of HVF18 in aqueous solution, its binding to LPS leads to a structural transition, wherein the N- terminus of the peptide forms a unique ß-turn whilst the C-terminus becomes helical. In silico modelling and simulations demonstrated that HVF18, as well as related peptides, target the LPS-binding site of CD14, and this interaction was experimentally supported using microscale thermophoresis. Collectively, the results demonstrate the role of structural transitions in LPS complex formation as well as in CD 14 interaction, and provide a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock. Significance Thrombin-derived C-terminal peptides (TCPs) of various sizes are present in human wounds, where they bind bacteria as well as “free” lipopolysaccharide (LPS), and thereby reduce inflammation. In this work, employing a combination of cellular, biophysical and structural studies, combined with in silico multiscale modeling, we present the molecular structure of a TCP in association with LPS, and define a previously undisclosed interaction between TCPs and CD14. Further, we show that TCPs exhibit relatively weak but specific affinities, all in the μM range, to both LPS and CD14. These novel structural insights into the function of this class of host-defense molecules will facilitate rational design of novel “dual function” anti-infectives, which target both bacteria and inflammatory signaling.


Introduction
Lipopolysaccharide (LPS) sensing by Toll-like receptor 4 (TLR4) is crucial in early responses to infection, where an uncontrolled LPS-response gives rise to excessive localized inflammation, such as that found in infected wounds, but also in severe systemic responses to infection [1]. Therefore, although sensing of LPS is important for initial host defense responses, clearance and control of this molecule is critical in order to avoid excessive inflammation and organ damage. For example, LPS triggers NF-κBmediated up-regulation of tissue factor, and thus formation of thrombin, which in turn activates coagulation and fibrin formation, thereby aiding in initial hemostasis and defense against bacterial invasion [2]. Furthermore, proteolysis of thrombin by neutrophil elastase leads to formation of aggregation prone 11-kDa C-terminal fragments which are present in wounds, and bind to and form amorphous amyloid-like aggregates with both LPS and Gram-negative bacteria, aiding in the subsequent clearance of these aggregates by phagocytosis [3]. In addition, thrombin-derived C-terminal peptides (TCP) of roughly 2 kDa, such as FYTHVFRLKKWIQKVIDQFGE and HVFRLKKWIQKVIDQFGE [4][5][6], are present in wound fluids, and have been demonstrated to exert anti-endotoxic functions in vitro and in vivo [4,7]. Such smaller peptides belong to the diverse family of host-defense peptides (HDP) [8], which includes neutrophil-derived α -defensins and the cathelicidin LL-37 [9,10], all known to exhibit immunomodulatory activities [11].
Today, there is an urgent need for novel anti-infective therapies [12,13]. However, current treatments based on antibiotics target bacteria only, and not the accompanying over-activation of immune responses. This uncontrolled stimulation may cause an overwhelming production of inflammatory cytokines leading to systemic inflammation, intravascular coagulation and organ dysfunction, such as seen in sepsis [14,15]. This is a leading cause of death in the U.S alone, with over 700,000 cases estimated every year, and with mortality rates from 28 to 60% [16,17]. Treatment concepts based on Nature's own innate defense strategies, aiming at not only targeting bacteria, but also the excessive immune response, could therefore have a significant therapeutic potential. For instance, many naturally occurring HDPs, as well as fragments of LPS-recognizing proteins, display a strong affinity for LPS, blocking downstream interaction with LPS-binding protein (LBP), thereby reducing cytokine production in vitro and in vivo [18][19][20].
Neutralization of circulating LPS by HDPs has been shown to reduce adverse LPSinduced pro-inflammatory effects in experimental animal models [4,21,22]. In this context, a prototypic TCP, GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), encompassing sequences of natural TCPs previously identified in human wounds [23], has been shown to protect against P. aeruginosa sepsis and LPS-mediated shock, mainly via reduction of systemic cytokine responses in vivo [4,24]. These observations, however, disclose neither the exact mode of action, nor whether these peptides can target other molecules apart from LPS. With this background, we set out to characterize the structural prerequisites at the molecular level which underlie the anti-endotoxic actions of such TCPs.

Results
Structural considerations and background on peptide structures. The C-terminal region of thrombin of which the sequence GKYGFYTHVFRLKKWIQKVIDQFGE is highlighted in the crystal structure (shown in Fig. 1A), comprises a flexible β-strand segment and a compact amphipathic helix. Previous studies have shown that various proteases can generate TCPs derived from this region [23]. For illustrative purposes, and to highlight the complete, albeit gradual, formation of such fragments, the generation of the 2 kDa TCP HVF18 HVFRLKKWIQKVIDQFGE, cleaved out by human neutrophil elastase [7], is depicted in Figure 1B. A similar generation of the peptide FYT21 (FYTHVFRLKKWIQKVIDQFGE) has been observed after digestion with the bacterial M4 peptidases Pseudomonas aeruginosa elastase [6,23] and Staphylococcus aureus aureolysin [23]. Incorporating these endogenous sequences, the prototypic peptide GKYGFYTHVFRLKKWIQKVIDQFGE has been further used in in vitro studies on its mode-of-action [24,25], as well as in several therapeutic in vivo studies, aimed at targeting endotoxin-mediated inflammatory responses [4,7]. The truncated peptide variant VFR12 (VFRLKKWIQKVI) has been shown to constitute a minimal LPS binding site, while not exhibiting any anti-endotoxic effects in cell models [26]. Some key physicochemical parameters of these peptides are indicated in Figure 1C.
Interactions of TCPs with LPS. Intrinsic tryptophan fluorescence was employed to monitor the interaction of the TCPs with E. coli LPS. Figure 1D shows changes in the fluorescence emission spectra of HVF18 as a function of increasing concentration of LPS. In the absence of LPS, the tryptophan residue has an emission maximum at 357 nm.
Addition of LPS results in a concentration-dependent blue shift in the emission maximum, indicating peptide binding to LPS. A similar shift in emission maximum was observed for the other TCPs, albeit differences in the extent of the blue shift were noted ( Fig. S1). The change in fluorescence emission maxima (λ max ) of the TCPs was utilized to obtain an indication of peptide binding affinities to LPS (Fig. 1E). As shown in Table   1, GKY25 displayed the highest affinity (K d =2 ± 0.29 μM) among the peptides investigated, followed by the endogenous fragments HVF18 (K d = 4.24 ± 0.90 μM), and the shorter VFR12 (K d =11.45 ± 0.45 μM). Furthermore, the localization of the tryptophan residue in the LPS complexes was determined by quenching of the tryptophan fluorescence with a neutral quencher, acrylamide. Table 1 shows the obtained Stern-Volmer quenching constants (K SV ) of the peptide in unbound and LPS-bound states.
Although many factors determine the photophysics of tryptophan, a large K SV for the peptides in their unbound state generally indicates complete exposure of the tryptophan residue to buffer, while a low K SV in the LPS-bound states implicates that exposure of the tryptophan to the solvent is restricted owing to incorporation of the tryptophan side chain into the hydrophobic milieu of LPS.
By analogy, ellipsometry results indicate that these TCPs bind substantially to LPS (  1F). Taken together, the ellipsometry and tryptophan fluorescence results indicate that TCPs exhibit a peptide length-dependent binding to LPS.

Effects of TCPs on LPS scavenging and blocking of pro-inflammatory responses.
Next, we assessed the ability of the peptides to neutralize free LPS in solution using the highly sensitive limulus amebocyte lysate assay (LAL) assay [27]. As can be seen in Figure 2A, GKY25, FYT21, and HVF18, but not VFR12, neutralized LPS in the LAL assay in the dose range studied, and a length-dependent inhibitory activity was observed, corresponding to the ellipsometry and tryptophan fluorescence data outlined above.
Likewise, using LPS-stimulated THP-1 monocytes followed by evaluation of NF-κB activation, a similar blocking of endotoxin responses was observed (Fig. 2B), in which VFR12 did not block NF-κB activation (Fig. S1D) Likewise, for VFR12 a large number of intense NOE cross-peaks involving backbone and side-chain proton resonances were observed in the presence of LPS (Fig. S4). As above, the continuity of sequential (i to i+1) NH/NH and Cα/NH NOEs was disturbed due to overlap. However, few medium-range (i to i+2) NH/NH NOEs and (i to i+2, i+3 and i+4) Cα/NH NOEs were observed ( Fig. S4B-C). Notably, long-range NOE contacts between the F2 aromatic ring protons with CαH and CβH of W7, as well as NOE contacts between the W7 NεH proton and F2 backbone and side-chain protons, were observed (Fig. S4D). In addition, the indole proton of W7 showed NOE contacts with the aliphatic side-chain proton resonances of residues I8, V11, I12, suggesting close interactions of amino acid side-chains in LPS-bound VFR12. In summary, the analyses of tr-NOESY spectra of HVF18 and VFR12 demonstrate that LPS binding induces backbone stabilization and (partial) formation of α-helical conformation.
Structure of HVF18 and VFR12 bound to LPS micelles. The 3D structure of LPSbound HVF18 was determined from a total of 145 tr-NOE-derived distance restraints and dihedral angle restraints ( Fig. 4B and Table 2). Figure 4C superposes the backbone atoms of the 20 lowest energy structures of HVF18. The structure is well-defined, with a backbone and heavy atom RMSD of 0.88 ± 0.41 and 1.73 ± 0.46 Å, respectively. In the LPS-bound state, the C-terminus of HVF18, encompassing residues Q10-G17, forms a compact helix. In contrast, the N-terminal residues, F3-L5 are in an extended state, followed by a β-turn involving residues K6-K7-W8-I9 (Fig. 4D). The presence of strong backbone HN/HN (i to i+1) NOEs, medium intensity Cα/NH (i to i+2), alongside the absence of Cα/NH (i to i+3) NOEs, confirms the formation of type II β-turn (Fig. 4A).
Furthermore, the close proximity of the K6 carbonyl oxygen and the amide proton of I9 (2.2 -2.4 Å), in all calculated structures indicate the occurrence of hydrogen-bond formation (Fig. 4E). The β-turn gives the peptide backbone a curved shape with amphipathic side-chain dispositions. The side-chains of R4/K6/K7 and Q10/K11/Q15 form two hydrophilic clusters connected by the central hydrophobic W8 residue (Fig. 4F) on the convex side, whereas the side-chains of V2/F3/I9/V12/I13/F16 form a concave hydrophobic core (Fig. 4G).
Likewise, the LPS-bound structure of VFR12 was determined using the tr-NOEs distance restraints ( Fig. S5 and Table 2). The structure is well-defined with a backbone and heavy atom RMSD of 0.84 ± 0.34 and 1.70 ± 0.23 Å, respectively ( Fig S5C). As in HVF18, the N-terminal residues (F2-L4) are extended with a β−turn (K5-I8), followed by an extremely short C-terminus helical turn I8-V11 ( Fig S5B). The formation of the helical segment is confirmed by the diagnostic medium-range HN/HN (i to i+2), Cα/NH (i to i+2, i+3 and i+4) contacts for the C-terminal residues ( Fig S5A). Consequently, the overall structure of LPS bound VFR12 is linear and the amphipathic distribution of side chains is disturbed. Although a hydrophilic surface including R3/K5/K6/Q9/K10 is formed in VFR12, the hydrophobic patch is small, involving only side-chains of F2/W7/V11 (Fig S5E-F).
Simulated assembly of TCPs with lipid A aggregates. Next, tr-NOESY derived HVF18 and VFR12, as well as modeled GKY25, were allowed to bind (at a 1:2 ratio of peptide:lipid) to lipid A aggregates comprising 60 molecules during coarse-grained (CG) molecular dynamics (MD) simulations (Fig. S6). Lipid A was chosen for computational efficiency in the simulations, as it is the core effector component of LPS [18]. Divalent Ca 2+ ions included in the simulations served to cross-link phosphate moieties between lipid A molecules, and are known to be essential for the stability of the resultant lamellar phase membranes and other biologically relevant phases [28,29]. At t=250 ns, large clusters of peptides were formed on the surface of the lipid aggregates. Eventually, all of these peptide clusters dispersed into the aggregate over the course of each 10 μ s simulation (Fig. S6A). The positively charged N-terminal residues were observed to compete with the Ca 2+ ions that cross-linked between the phosphates of lipid A, resulting in more loosely connected headgroups ( Fig S6B). This "breaking up" of the headgroupcrosslinking interactions occurred concurrently with the dispersion of the peptide clusters into the lipid aggregates. The dispersion of VFR12 was more rapid than that of HVF18 and GKY25, both of which formed more stable clusters that did not fully disperse within the 10 μ s of simulation. All three peptides, were able to interact with multiple lipid molecules simultaneously ( Fig S6B). Indeed, both GKY25 and HVF18 were most often found to interact simultaneously with five separate lipid A moieties. In contrast, the shorter VFR12 appeared to be less able to interact with multiple lipids, preferentially binding to four separate molecules corresponding to the reduced ability of VFR12 to neutralize LPS (Fig. S1D). The hydrophilic and positively charged residues at the Nterminus, K2, T7, H8* R11*, K13* and K14* of GKY25 (* also in HVF18) were found to be in contact with only the head group particles of lipid A ( Fig S7A). The aromatic residues, Y3, F5, Y6, and F10*, are interfacial residues, binding both the head and tail parts of the lipid, while the C-terminal F22* was more often in contact with the tails. The hydrophilic and negatively charged residues of the C-terminal helix were solvent exposed, and had a propensity to interact with the Ca 2+ ions after they had been displaced from their sites crosslinking lipid phosphates. Furthermore, high resolution all-atom (AA) molecular dynamics (MD) simulations of VFR12 and HVF18 served to validate the coarse-grained (CG) simulations ( Fig 5A). These additional simulations revealed a similar mode of binding to lipid A moieties as observed in the CG simulations, with the N-terminal positively charged residues serving to make contacts with headgroups, and the hydrophobic residues of the C-terminal helix making contacts with the lipid interfacial region and tails atoms (Fig. 5B). Taken together, the combined CG and AA MD simulations correlate with the structural and in vitro effect studies, providing a detailed description of TCP-LPS interactions as well as regions directly contacting LPS that is reflected in the LPS neutralizing capabilities of the different TCPs.
In silico analysis and microscale thermophoresis studies of LPS and TCP interaction with CD14. CD14 is a well-known pattern recognition receptor that plays a prominent role in sensitizing cells to LPS, and transferring it to the TLR4 signaling complex [30].
Considering the ability of TCPs to bind LPS and block TLR4 dimerization at cell surfaces [6,24], in silico analyses were performed to investigate possible TCP-CD14 interactions. Mutational studies suggest that charged surfaces at the amino-terminus of CD14 may be used to "capture" LPS, and the hydrophilic "rim" in particular to be of importance for LPS binding and cell activation [31]. In the absence of a ligand-bound structure for CD14, unbiased all-atom MD simulations were performed with the previously reported crystal structure of human CD14 [32], to which a nearby lipid A molecule spontaneously bound to the suggested binding cavity (Fig. 6A). Subsequently, the energetic properties of binding a lipid A molecule to the hydrophobic pocket were investigated ( Fig. S8) using biased all-atom MD simulations, based on an umbrella sampling (US) methodology [33]. The resultant potential of mean force (PMF) provides a measure of the lipid binding free-energy as a function of distance from the CD14 binding cavity [34]. The calculated PMF displays a smooth rise in free-energy upon extraction of lipid A from the CD14 binding cavity towards the bulk solvent phase, plateauing ~2 nm from the cavity (Fig. S8A). The absolute free-energy change between the CD14-bound and fully solvent-dissolved states was ~200 kJ mol -1 (Fig. S8B). This is similar in magnitude to the previously calculated binding free-energy change of lipid A to the structurally characterized LPS binding cavity of MD-2, the co-receptor of TLR4 [35], hence confirming that the proposed N-terminal location of CD14 is indeed a true binding site for LPS molecules.
Docking of tr-NOESY derived HVF18 to human CD14 using the ClusPro server [36] indicated the peptide to be bound to the amino terminal hydrophobic pocket of CD14 in >90% of the docked models retrieved from the server (Fig. 6). In majority of these structures, the N-terminal tail of HVF18 was buried deeply into the hydrophobic pocket of CD14, making clear contacts with rim residues W45, F49, V52, F69 and Y82 that bind LPS tails during simulations ( Fig 6A). In contrast, the C-terminal helix of the peptide bound to variety of hydrophilic rim residues, such as K71, R72, and Q81, as well as to rim residues specific for human CD14, such as the D44, S46, T85, and L89 ( Fig 6A). In all the docked poses, HVF18 would contact CD14 in a common orientation with the Cterminal residues of Q10, K11, D14, Q15, and E18 exposed to the solvent. In contrast, VFR12 had a less specific mode of interaction, binding to CD14 in a variety of orientations. In a few cases, VFR12 would lie across the entrance to the hydrophobic pocket, making contact with all the rim residues of CD14. A GKY25-bound state was inferred by modeling the N-terminal residues as a random coil, using HVF18 as an initial template. The highest quality models of the GKY25-CD14 complex, i.e., those with the lowest values of the objective energy function, would either loop the N-terminal residues into and out of the pocket (Fig S7B), or lay at the N-terminus across the entrance to the CD14 pocket, completely blocking entry.
Likewise, microscale thermophoresis (MST), a highly sensitive technique probing interactions between components in solution, validated the in silico simulations presented above. As can be seen in Figure 6D, fluorescence-labeled CD14 binds E. coli LPS, with a K d of 2.1 ± 1.4 μM, corresponding with previous reports [3]. Interestingly, both HVF18 and GKY25 showed a significant affinity to CD14 (K d of 1.2 ± 0.6 μM and 6.0 ± 0.5 μM, respectively), contrasting the lower interaction observed for VFR12 (K d of 16.8 ± 3.2 μM). Overall, these results indicate that the endogenous HVF18 and prototypic GKY25 efficiently block LPS-induced downstream signaling through direct LPS sequestration as well as high affinity antagonist binding to the amino terminal hydrophobic binding pocket of CD14, blocking LPS CD14 interactions.

Discussion
Today, there is an increasing interest in developing HDPs as immune modulators [11,37,38], and natural sequences such as the TCPs studied here are of interest for use in human therapy. In this perspective, it is important to define peptide actions at the molecular level residues critical for the anti-inflammatory activity of TCPs (Fig 6). In contrast to HVF18 and GKY25, VFR12 lacks the N-terminal hydrophobic residues as well as the C-terminal hydrophilic residues that display increased contacts with the LPS head and acyl chain.
Consequently, VFR12, though capable of still binding to LPS (via R3, K5, K6) comprises a relatively small hydrophobic patch, making it inefficient in penetrating the hydrophobic lipid phase of the aggregates in solution as well as interfering with LPS binding to the pocket of CD14 ( Fig S5).
Over the years, tr-NOESY has been employed to obtain three-dimensional structures of The heparin cofactor II-derived peptide KYE28 displays an N-terminal short helical region followed by a loop, two turns, and an extended C-terminus when bound to LPS [52]. Nevertheless, a feature of these determined structures is that they display partly well-defined and flexible structures in complex with LPS, characterized by an amphipathic fold [53]. In this perspective, it is of interest that TCPs share many of these overall characteristics, although they form a unique C-shaped structure, reminiscent of the T-shaped fold of LF11 (Fig. S9). Interestingly, the TCP-region RLKKWI contains the cationic and aromatic amino acid residues proposed as critical for lipid A interactions [54], and present in several LPS-binding proteins [55]. From an evolutionary perspective, it is notable that these residues show a high degree of conservation among various thrombin species. It is also notable that these residues are part of the region which binds to the LPS-binding pocket of CD14, whereas the less conserved residues in the Cterminal end are outside this pocket (Fig. S10A).
From a biological perspective, we have previously shown that TCPs such as GKY25 and FYT21 interact and bind directly to macrophages/monocytes in vivo, leading to inhibition of TLR4 dimerization and subsequent abrogation of pro-inflammatory cytokine production [6,24]. Although these data hinted at the possibility that TCPs may interact with targets other than LPS, the mechanistic basis for the anti-inflammatory effects of such peptides remained unclear. Using in silico docking and MST analysis, we here show that TCPs bind to the amino terminal hydrophobic LPS binding pocket of human CD14, the primary receptor of LPS with high affinity (Fig. 6). This prevents LPS transfer to TLR4 and consequently reduces macrophage activation. Secondly, the regions identified to interact with LPS overlap with the residues involved in the interaction with CD14, suggesting that TCPs can interfere with LPS-CD14 interactions, hence abrogating downstream transfer of LPS to the MD-2 complex and other effectors. It is also notable that HVF18 [6, 23], binds CD14 with an affinity similar to that obtained for LPS. In this context, it is therefore interesting that multiple variants of TCPs are generated by human and bacterial proteases, several of which are found in human wound fluids ( Fig. S10B) [23]. This observation implies that shorter TCP fragments, related to VFR12, may be exclusively binding to LPS and bacteria, whereas longer fragments like the HVF18 or FYT21 have the capacity also to neutralize LPS and inhibit TLR4 signaling pathways.
Furthermore, the fact that the major TCP fragment generated by human neutrophil elastase, HVF18, shows a higher affinity for CD14 than the longer GKY25, whereas the reciprocal applies to their interactions with LPS, illustrates that protease-mediated generation of multiple TCPs may "tune" LPS-responses during wounding and infection.
It is also interesting that the affinities between TCPs and CD14 were in the μM range,

Materials and Methods
Peptides. The peptides GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), FYT21 (FYTHVFRLKKWIQKVIDQFGE), HVF18 (HVFRLKKWIQKVIDQFGE), and VFR12 (VFRLKKWIQKVI), were synthesized by Biopeptide (San Diego, CA). We confirmed the purity (over 95%) using mass spectral analysis (MALDI-TOF Voyager, USA).  corresponds to saturation in the LPS adsorption isotherm under these conditions. Nonadsorbed LPS was removed by rinsing with Tris buffer at 5 ml/min for a period of 30 minutes, allowing the buffer to stabilize for 20 minutes. Peptide addition was subsequently performed to 0.01, 0.1, 0.5, and 1 µM, and adsorption monitored for at least one hour after each addition. All measurements were performed in at least duplicate at 25

°C.
Limulus amebocyte (LAL) assay. The ability of peptides to sequester LPS was assayed using a commercially available LAL chromogenic kit (QCL100 Cambrex, Lonza, USA), according to the manufacturer's protocol. LPS activates a proenzyme in the Limulus amebocyte lysate (LAL), which in turn catalytically splits a colorless substrate to the colored product para-nitroanilide (pNA), which is measured spectrophotometrically at 410 nm. Prior to running the assay, the peptides were dissolved at 200μM in pyrogen-free water, supplied with the kit.
NF-κB activation assay. THP1-XBlue-CD14 reporter cells (1x10 6 cells/mL) (InvivoGen, San Diego, USA) were seeded in phenol red RPMI (180,000 cells/well), supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) Antibiotic-Antimycotic solution (AAS), and allowed to adhere before they were stimulated with 100 ng/mL buffer. The molecular weight of LPS was considered to be 10 kDa, according to a previous report [61]. All spectra were recorded at 25°C from 190 to 260 nm, using a bandwidth of 1-nm, and averaged over three scans. Baseline scans were obtained using the same parameters for buffer or LPS and subtracted from the respective data scans with peptides. The final corrected averaged spectra were expressed in mean residue ellipticity. To ensure that the conformational space accurately reproduced the tr-NOESY derived structures, the NOEs were modeled in the simulations as flat-bottomed distance restraint potentials with a force constant of 1000 kJ mol -1 nm -2 . The time constant for the distance restraints running average was 10 ns. Angle restraints of 100 kJ mol -1 rad -2 were also applied to maintain the experimentally-derived backbone conformation. A lipid A aggregate was created by manually positioning ten lipid A molecules in solution using VMD [73] and allowing them to self-assemble over a timescale of 100 ns. The lipid parameters were used as described previously [74,75]. The peptides were positioned ~2.0 nm from the aggregate surface. Energy minimization, using the steepest descent algorithm, was performed for 1000 steps. Position restraints, with a force constant of 1000 kJ mol -1 nm -2 , were placed on the Cα particles of the peptides for 5 ns. Production simulations were performed for 250 ns.
CG MD simulations were performed using the MARTINI 2.2 force field [76]. DSSP was used to determine the secondary structures of the tr-NOESY derived VFR12 and HVF18 structures. The DSSP secondary structure categories served as constraints in the preparation of MARTINI protein parameters [77,78]. The additional seven residues at the N-terminus of GKY25, with respect to HVF18, were modeled as a random coil. The large lipid A aggregate was created by allowing 60 randomly placed lipid molecules to assemble over the course of a 1 μs simulation. The final frame of this simulation was used as the starting point for the creation of three further systems in which 30 GKY25, HVF18 or VFR12 peptides, respectively, were scattered randomly in the simulation cell, all at least 2 nm from the aggregate. The GKY25, HVF18 and VFR12 CG production simulations were each run for 10 μs. A simulation of the aggregate alone, with no peptides added, was also extended to 11 μs, and this served as negative control. All CG simulations were maintained at a constant temperature of 313 K using velocity rescale thermostat [72]. A system pressure of 1 bar was maintained isotropically using the Parrinello-Rahman barostat [79,80] with a coupling constant of 20 ps. Electrostatic interactions were treated using the reaction-field method [81] with a dielectric constant of 15 within a 1.1 nm cutoff, and zero beyond the cutoff. The van der Waals interactions were shifted to zero at 1.1 nm.
Peptide and LPS interference with CD14 interactions. The tr-NOESY derived structures of VFR12 and HVF18 were docked to human CD14 using the ClusPro server [82]. The structure of CD14 with GKY25 bound was modeled using Modeller v9.15 [83] with the HVF18-CD14 complex model as a template via the loopmodel function with the MD refinement set to "refine.very_slow". A total of 100 models of the GKY25-CD14 complex were generated. The structure with the lowest value of Modeller's objective function was selected for visualization in Figure 6. In order to validate the GKY25-CD14 model, GKY25 was also modeled in isolation using tr-NOESY derived structure of HVF18 as a template. One hundred models were generated, and the model with the lowest objective function was selected for further docking studies. The modeled GKY25 structure was docked to human CD14 using the ClusPro server.
Lipid A was assembled in an unbiased mode to CD14 via molecular simulations.
Initially, a single lipid A molecule was placed ~2.0 nm from the binding pocket (measured from the center of mass of the terminal acyl carbons of lipid A to the center of the cavity). Rapid spontaneous adsorption and insertion of lipid A into the CD14 Nterminal binding cavity was observed (within 10 ns). The simulation was subsequently run for over 1 μ s and the lipid molecule remained stably bound within the N-terminal pocket.
For free-energy calculations, the lipid A bound CD14 structure was extracted after 1 μ s of simulation. The protein and ligand were manually positioned so that pulling the lipid A molecule along the unit cell z-axis would correspond to the axis of the cavity mouth.
The system was then re-solvated and minimized before a 4 ns steered MD simulation was performed, pulling the lipid A molecule 4 nm from the protein using a pulling force of 1000 kJ mol -1 nm -2 . Forty windows were extracted at 0.1 nm intervals from this trajectory for subsequent umbrella sampling (US). During US, each window was sampled for 50 ns.
The lipid A sugar backbone and phosphate groups served as the reference point of the ligand, and the backbone atoms of residues 8 to 12 and 31 to 35 corresponding to the first two LRRs at the cavity entrance, were used as the reference point for CD14.

Microscale thermophoresis (MST). MST analysis was performed using a NanoTemper
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