Computational tools unravel putative sterol binding ... - ACS Publications

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Computational Biochemistry

Computational tools unravel putative sterol binding sites in the lysosomal NPC1 protein Nadia Elghobashi-Meinhardt J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00186 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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Journal of Chemical Information and Modeling

Computational tools unravel putative sterol binding sites in the lysosomal NPC1 protein Nadia Elghobashi-Meinhardt∗ Technische Universität Berlin, Department of Chemistry, 10623 Berlin, Germany E-mail: [email protected] Phone: +49-30-314-79386. Fax: +49-30-314-21122 Abstract Two proteins have been linked as the critical components in the molecular mechanisms involved in the Niemann Pick type C (NPC) disease: NPC1, a 140 kDa polytopic membrane-bound protein and the smaller (132 residues), water-soluble NPC2 protein. NPC1 is believed to act in tandem with NPC2, transferring cholesterol and other sterols out of the LE/Lys compartments. Mutations in either NPC1 or NPC2 can lead to an accumulation of cholesterol and lipids in the LE/Lys, the primary phenotype of the NPC disease, but approximately 95% of identified disease-causing mutations have been mapped to the membrane-bound NPC1 protein. Here, we investigate the full length, membrane-bound NPC1 protein computationally using a combination of molecular modeling, docking, and molecular dynamics (MD) simulations. An analysis of titratable amino acid side-chains, several buried in protein pockets, reveals several non-standard protonation states for the low-pH scenario (pH 5) that is realized in the lysosome. Together with the location of these buried amino acids, docking studies have identified putative lipid binding domains that are in close proximity to amino acids that, when mutated, are connected to NPC1 loss-of-function. Using energy analyses and MD simulations, we analyze these domains as potential cholesterol

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binding sites and propose the possibility of multiple sterol binding pockets enabling the intramolecular transport of sterol molecules to the transmembrane domain.

Introduction The Niemann Pick type C (NPC) disease is an autosomal recessive hereditary disease that is characterized by the accumulation of cholesterol and lipids in the late endosomal(LE)/lysosomal(Lys) compartments. Several disease-causing mutations in either NPC1 or NPC2, the two proteins associated with the disease, have been identified. 1 In 80% of cases, the disease phenotype is characterized by severe physiological disorders; the remainder is referred to as the "variant" biochemical phenotype. 2 The severity of the disease phenotype has been linked to specific mutations. However, the majority (95%) of these mutations have been mapped to the larger, membrane-bound NPC1 protein (140 kDa). 2

Human NPC1 (Figure 1A) consists of 1,278 amino acids that are arranged in 13 transmembrane (TM) helices and three distinct luminal domains. 3 The amino terminal domain (residues 25–264) is referred to as the N-terminal domain (NTD) (Figure 1B). The remaining two luminal domains (residues 370–621 and residues 854–1083) are referred to as the middle luminal domain (MLD)(Figure 1C) and the C-terminal domain, respectively (Figure 1E). Current models and structural data have suggested a cholesterol transfer path characterized by the following steps: 1) NPC2 binds cholesterol after receptormediated endocytosis of low-density lipoprotein, 2) carrying cholesterol, NPC2 docks onto the membrane-bound NPC1 protein, 3) cholesterol is transferred in a “hydrophobic hand-off" or sliding model to NPC1’s NTD, 4) downstream transfer within NPC1 moves cholesterol from the NTD binding site across the glycocalyx to another binding site closer to the TM domain. 4 Recently, however, it was shown that cholesterol can be transfered from full-length NPC1 to NCP1 lacking the NTD, supporting the hypothesis that two

2


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NPC1 molecules may interact for transfer. 5 This finding has raised the question whether alternative sterol binding sites exist in NPC1. One such candidate is the sterol sensing domain (SSD) within the TM domain (Figure 1D). Consisting of five TM helices (amino acids 621–797), the SSD is thought to be a structural element that is critical for the binding and subsequent downstream transfer of cholesterol out of the lysosomal membrane. 6–8 In fact, the SSD may be more critical for sterol metabolism than the NTD; in the absence of the NTD, NPC1 is still able to bind sterols. 9

3



NTD

B)

A)

NTD

cholesterol

His215 Leu176 Lys179

Leu175

MLD

Lys180 Lys182

CTD C)

MLD Asp531

His510 His441

SSD

His512 His492

Pro-rich linker Membrane

Glu406

TMD

Glycocalyx

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Arg404

o

80 A

Lumen

o

40 A

Cytosol

D)

SSD Leu684

CTD

Iso687

Val624 Glu688

Iso685

Phe1221

His1016

Pro691

Tyr628 Met631

E)

Tyr1225

His1029 Val689

Ile1061

Phe692 Tyr1229 Met1159

Figure 1: A) The various structural domains of NPC1 are colored as follows: NTD (amino acids 30–

250, red); linker (amino acids 251–259, cyan); MLD (amino acids 374–620, purple); TM helices including SSD (amino acids 260–373, 621–860, and 1084–1278, gray); CTD (amino acids 861–1083, green). The SSD (amino acids 621–797) is highlighted in orange; TM helices 1 and 2 are both colored in cyan to highlight the connectivity of the NTD and MLD domains. In the inserts, the individual NPC1 domains are depicted (orientations differ from the orientations in (A) to optimize visualization) with amino acids that were found to have non-standard protonation states at pH 5. B) the NTD shows residues involved in establishing an interface with the smaller, soluble NPC2 protein, as well as the cholesterol ligand in the binding pocket; C) the MLD contains several solvent-exposed histidine residues that are protonated. D) An up-close view of the hydrophobic pocket near Glu688 in the SSD is presented.

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Several mutations in the SSD, on the other hand, have been linked to the severe disease phenotype (infantile neurological onset) 10 and are known to abolish the transport of cholesterol out of the lysosome. 6,7,11 P691 and P692 in human and mouse NPC1 proteins, respectively, have been identified as critical for direct binding of cholesterol or for inducing a conformational change in the SSD required for sterol interaction. 11 In biochemical assays, mutants L724P and Q775P, both mapping to the SSD, lead to the classic biochemical phenotype, i.e. undetectable level of NPC1 protein in western blot assay; M631R is associated with the severe clinical phenotype in patients. 10 Therefore, the SSD is a vital site, not only for the egress of cholesterol, but also for the protein’s structural and functional integrity. Interestingly, the SSD shares homology with several other proteins involved in sterol metabolism, including HMG-CoA reductase, SREBP cleavage-activating protein (SCAP), NPC1-L1, and Patched, suggesting this domain is a key player in the binding and subsequent transport of cholesterol in NPC1. 3

Nonetheless, the mechanism with which sterol molecules may be transported across the glycocalyx to the TM domain, either from the NTD or from another protein domain, remains unknown. A "pocket brigade" model of cholesterol transport involving the SSD has recently been proposed as a mechanism with which to transfer and also regulate cholesterol concentrations in the lysosome lipid bilayer. 12 In this model, sterol ligands may be transferred sequentially from the NPC2 protein pocket, to the NPC1 NTD, and finally to the NPC1 SSD. 12 Interestingly, the authors suggest that the occupancy of the SSD pocket in NPC1 may regulate the uptake of cholesterol, not only in NPC1, but also in other integral membrane proteins. Nonetheless, the mechanism by which cholesterol travels from the NTD to the SSD is unknown. One proposal is that the NTD, connected to the TM domain via a proline-rich linker (amino acids 251–259,

249 PKPQPPPPPAP259 )(Figure

1A),

could dynamically reorient across the 80 Å distance of the glycocalyx to allow cholesterol to be transferred to the TM directly. 12 However, thus far, no evidence supports such a 5



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large-scale motion of the NTD, so this transfer mechanism remains speculative.

The molecular details of the steps involved in cholesterol processing within the lysosome have been investigated over the past decade in a range of biochemical experiments. 4–11,13,13–15 The emergence of high resolution structural data of the NPC1 protein in recent years has shed further light on the atomic details of the membrane bound protein. More recently, cryo-EM structures (4.4 Å resolution) of full-length human NPC1 and biochemical assays have elucidated the possible roles of the various domains, particularly as they may interact with the soluble NPC2 protein. 15 For example, deletion of the NTD (residues 25257) of NPC1 abolish more than 90% of cholesterol transfer from NPC2 to NPC1(NTD). 15 However, isolated from the rest of NPC1, the NTD is inactive, suggesting that the NTD requires other domains to be a viable sterol acceptor. 15 Recent evidence strongly suggests that NPC1 may be able to act in tandem with another NPC1 molecule to transfer cholesterol. 5

In addition to myriad biochemical assays of the NPC1 and NPC2 proteins, several computational studies have been carried out to analyze NPC1-NPC2 stability, 16,17 ligandbinding specificity, 18 and cholesterol transfer paths. 16,17,19 Nonetheless, the role of specific amino acids in determining functional behavior in NPC1 is still not fully understood, nor has the interplay between the various domains been characterized. Since the NPC1NPC2 protein-protein complex has thus far not been observed, this lack of structural data has prevented the community from proving the hypothesized mechanism of cholesterol transport. Details regarding the subsequent trafficking of cholesterol out of the lysosome are largely unknown and require further investigation.

In the present study, we aim to characterize further structure-function relationships in the NPC1 protein, particularly focusing on the possibility of a cholesterol transfer path 6


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within the NPC1 protein. Together with data from site-directed mutagenesis experiments, our results provide supporting evidence that alternative sterol binding sites exist. We use a combination of molecular modeling, pKa computations, and molecular dynamics (MD) simulations of the fully solvated protein in the lipid bilayer to analyze putative ligand binding sites and to investigate the structural flexibility of specific domains of NPC1. In the next section, results regarding pKa computations will be presented, followed by a discussion of putative sterol binding sites. Finally, we will analyze the results of MD simulations.

Results and discussion The constructed model for NPC1 includes rebuilt missing internal residues as well as a protonation pattern as determined through pKa computations (Figure 1A). Non-standard protonation states were found for nine histidine and three glutamate residues; no nonstandard protonation states were identified for Asp residues. The computed pKa values for amino acid side-chains with non-standard protonation states are listed in Table 1. Nine histidine residues and three glutamate residues have non-standard protonation states at pH 5. Several of the identified histidines are solvent-exposed such that a charged side-chain is electrostatically favorable for the polar aqueous solution. Three glutamate residues are located in hydrophobic pockets in the interior of the protein, so their sidechains are neutral. These buried amino acids were analyzed in their local protein environment to understand how the protein’s structure may be connected to function. Five of the 12 identified amino acids are located in the MLD. Of the three glutamate residues showing unusually high pKa values, Glu406 (>20), Glu688 (10.31), and Glu742 (>20), two (Glu688 and Glu742) are buried in the SSD; Glu406 is located in the MLD. His512 and His1170 are both protonated as they are involved in salt bridges with Asp531 and Glu1166, respectively.

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An up-close view of the NTD is shown in Figure 1B; His215, a solvent-exposed residue, is located on a flexible loop of the NTD, on the same face as the residues Leu175, Leu176, Lys179, Asp180, and Asp182. Notably, mutations of Leu175 and Leu176 abolish cholesterol binding, 4 and Lys179, Asp180, and Asp182 are predicted to establish an interface with NPC2 to allow for cholesterol transfer 8 (cholesterol binding pocket illustrated in Figure 1B).

Within the MLD, His441, His492, and His510 are solvent-exposed; His512 is involved in a salt bridge with Asp531, and Glu406 is buried in a hydrophobic pocket that is near the interface with the CTD (Figure 1C). Importantly, Glu406 is adjacent to Arg404 which is a disease-causing mutation (R404Q and R404W). 10 Under acidic conditions, this region of the MLD has been shown to interact with the soluble NPC2 protein to establish a binding interface. 14 Upon mutation to R404Q, NPC1 showed a drastic decrease in binding to the NPC2 protein. 14 Indeed, this region of the MLD has been hypothesized to be instrumental in positioning NPC2 to allow for sterol transfer, either to a binding site within NPC1 or directly into the lipid bilayer. 14

Two of the three glutamate residues with non-standard protonation states, Glu688 and Glu742, are buried in hydrophobic pockets in the SSD. An up-close view of the amino acids that comprise the hydrophobic pocket around Glu688 is shown in Figure 1D. Interestingly, several amino acids that have been identified with SSD loss-of-function or with classic biochemical/clinical phenotype are nearby. Pro691, a residue identified with SSD loss-of-function when mutated to P691S, 6 is in the direct vicinity (Pro Cγ –Glu Cδ separation ∼6.6 Å), indicating that this pocket represents an important structural feature in sterol sensing. Met631 (Met Cǫ –Glu Cδ separation ∼7.7 Å) is associated with the severe clinical phenotype. 10 The CTD only contains two histidine residues (His1016 and 8


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Table 1: Amino acids with non-standard protonation states at pH 5 Amino acid His215 His441 His492 His510 His512 His758 His1016 His1029 His1170 Glu406 Glu688 Glu742

pKa 6.27 6.50 7.14 8.86 10.00 6.64 7.03 6.47 >20.00 >20.00 10.31 >20.00

domain NTD MLD MLD MLD MLD SSD CTD CTD TM MLD SSD SSD

environment solvent-exposed solvent-exposed solvent-exposed solvent-exposed salt bridge with Asp531 solvent-exposed solvent-exposed solvent-exposed salt bridge with Glu1166 hydrophobic pocket hydrophobic pocket hydrophobic pocket

His1029) with non-standard protonation states, and these are both solvent-exposed. The severe disease-causing mutation I1061T, present in 15-20% of all known disease alleles, 20 is located on α5 helix of the CTD, across from the linker chain of residues (aa 251–259) connecting the NTD to the TMD (Figure 1E).

Next, using the model constructed at pH 5 to mimic lysosomal conditions, we explored putative ligand binding sites. Using AutoDock, 21 ten low energy NPC1 ligand binding sites were identified; these sites were clustered into five main binding domains (Figures 2A). To check the relative enthalpic stability of each ligand-protein binding interaction, an energy minimization of the cholesterol-NPC1 complex was performed for each of the five identified binding domains. For all sites, the cholesterol ligand was modeled inside the binding pocket in both an "up" and a "down" orientation. "Up" refers to an orientation in which the cholesterol 3’-hydroxyl group is pointing toward the luminal tip of the NTD and "down" refers to the hydroxyl group pointing away from the NTD toward the cytoplasm (Figure 3). For volumes that accommodate multiple binding poses, these conformations were also tested. In all binding sites, the "down" orientation shows enthalpic stabilization relative to the "up" orientation, which we will discuss next in more

9



detail.

In the NTD, binding site (1) overlaps with the sterol binding pocket that has been observed experimentally in X-ray crystallography studies to accommodate either cholesterol or the derivative 25-hydroxycholesterol. 4 X-ray crystallography experiments strongly indicate that the 3’-hydroxyl group is buried in the NTD with the sterol’s isooctyl chain pointing toward the protein surface. 4 This orientation is also assumed to be opposite to the orientation of cholesterol in NPC2, in which the 3’-hydroxyl group is exposed at the surface. Energy minimizations indicate that both sterol orientations are stable, with a small energetic gain (∆E ∼ 7 kcal/mol) for the orientation with the 3’-hydroxyl group pointing down (opposite to the experimentally determined orientation) (see Table 2 for energies).

The nearby (∼15 Å) site (2) (Figure 2B and C), located at the interface between the NTD and CTD, is directly adjacent to the "Ψ-loop" (residues 227–238) that has been identified as critical for NPC1 activity (Figure 2C). In absence of the "Ψ-loop", NPC1 loses 50% cholesterol esterification activity compared to WT. 22 This group of residues is thought to facilitate cholesterol transport, 22 perhaps by positioning the NPC2 protein for sterol transfer or by positioning another NPC1 molecule. 5 Energy minimization of site (2) shows an energetic gain (∆E ∼ −150 kcal/mol) when the sterol’s 3’-hydroxyl group points "down" toward the protein interior and the isooctyl tail points toward the solvent-exposed surface.

Site (3) is a binding domain that is positioned 3.4 Å from Glu406 (Figure 2D), an amino acid identified to have a pKa >20, and near Arg404, an amino acid linked to diseasecausing mutations (R404Q and R404W). 10 Three binding poses were investigated for site (3), which is located near the interface of the MLD and the CTD and near Glu406 10


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that is buried in a hydrophobic region(Figure 2D). In one pose, the "up" orientation is stabilized relative to the "down" position; in another, the opposite behavior is observed, and in the third, "up" and "down" show similar enthalpic stabilities. Energies averaged over the three poses indicate that the "down" orientation is stabilized relative to the "up" orientation (Table 2). If site (3) represents an intermediate sterol binding site, the availability of both energetically stable options ("up" or "down") may accommodate both sterol orientations, thereby optimizing increasing conformational entropy and lowering the free energy associated with the transfer process.

In the TM domain, two binding domains were identified (4 and 5). Site (4), located at the juncture between the MLD, CTD, and TMD, is located on the luminal side of Glu688 and is ∼7 Å from Arg404,(Figure 2D). Site (4) and shows nearly identical energies for both cholesterol orientations. This site has also been identified in previous docking studies as also being able to accommodate the cholesterol-trafficking inhibitor U18666A. 12 Site 4 shows an energetic stabilization (∆E ∼ 100 kcal/mol) for a "down" orientation. Site (5) is located in the SSD (Figure 2E), near the two protonated glutamate residues, Glu688 and Glu742, as well as Pro691, a residue identified with SSD loss-of-function when mutated to P691S, 6 and near Met631, associated with the severe disease phenotype (infantile neurological onset). 10 For site (5), the 3’-hydroxyl group pointing "down" and away from the interior of the TM domain is stabilized by more than 300 kcal/mol compared to the opposite orientation (Table 2). We examined the local protein structure near site (5) to understand the origin of this large energy difference between the two ligand orientations (inset of Figure 3). In the scenario of the cholesterol pointing "up" in the cavity of site (1), several amino acids containing hydrophobic side-chains (Phe692, Met1159, Tyr1229, Met1232) are within ∼4 Å of the sterol’s 3’-hydroxyl group. Cholesterol’s hydroxyl group points toward the sulfur of Met1159, with an O–S separation of 3.3 Å; the sterol oxygen is separated from Phe692 (Cǫ ) by approximately 3.3 Å (inset of Figure 3). In the "down" orientation, the 11



OH–group of cholesterol forms a stabilizing hydrogen bond with the peptide backbone oxygen of Gly1195. The sterol oxygen is separated from the Cγ and Cδ side-chain atoms of Leu1241 and Phe1199 by approximately 3.5 Å and 3.8 Å, respectively (inset of Figure 3).

Notably, the four putative binding domains that have been determined here are in close Table 2: NPC1-cholesterol minimum energies [kcal/mol] are reported for "up" and "down" cholesterol orientations in each of the ten identified docking sites. Binding site

Sterol orientation

Site 1

up (X-ray orientation) down up down up down up down up down

Site 2 Site 3 Site 4 Site 5

Energy [kcal/mol] -2084.39 -2091.57 -2262.35 -2414.75 -1597.11 -1841.43 -1623.77 -1716.13 -715.65 -1025.96

proximity to amino acids that, when mutated, are directly connected to loss-of-function. Furthermore, the identification of several enthalpically stable sterol binding sites could suggest that, after transfer of cholesterol from NPC2, the ligand may have several intermediate binding pockets available for residency, allowing the sterol to be transported step-wise through NPC1 until the ligand reaches the binding pocket in the SSD. The relative enthalpic stability of the sterol in the "down" orientation (compared to the "up" orientation) further suggests a directionality of sterol processing, namely insertion of the sterol’s isooctyl tail into the membrane. The availability of multiple binding sites, perhaps with more than one sterol binding pose, may be a mechanism with which transfer rates could be controlled and regulated: several simultaneously occupied sites may signal that metabolism should be slowed. It is worth pointing out that the energies presented here 12


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are enthalpic contributions and do not reflect any conformational sampling. Nonetheless, these results can be analyzed in the context of structural determinants that contribute to overall binding free energies.

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C) A)

B) cholesterol binding pocket observed in x-ray structure

(1)

(1)

"Ψ-loop"

(2)

(2)

D) 6.6 A 8.2 A

3.4 A

Glu406 (3)

Arg404

(3) 6.9 A

(4)

(5)

(4)

cholesterol transfer path?

E) (5)

3.6 A

Glu688 4.1 A

7.4 A

Pro691

Met631

6.0 A 5.7 A

Glu742 7.9 A

6.6 A

His1170

Figure 2: A) The modeled NPC1 protein is shown with the lowest energy ligand binding sites (site volume indicated with yellow colored spheres) as determined with AutoDock. 21 B) For each binding site, a cholesterol molecule (black) is modeled in the volume. C) Pocket (1) corresponds to the experimentally observed cholesterol binding pocket in the NTD 4 Binding site (2) is located at the interface between the NTD (red) and the MLD (green), and site (3) is located at the interface between the MLD (green) and CTD (purple). Site (4) is located at the interface of the MLD, CTD, and TM domain. Site (5) is located in the SSD of the TM domain. D) Closeup view of sites (3) and (4) is shown. Glu406, whose pKa is >20, is located adjacent to Arg404, an amino acid that is connected to the NPC disease phenotype (mutants R404Q and R404W 10 ) and has been shown to be critical for the binding of NPC2. 14 E) Closeup view of sites (4) and (5) is shown, highlighting the connectivity of the sterol binding sites. Glu688 (pKa ∼10) is near the isooctyl tail of the ligand in pocket (4) as well as near Pro691, an amino acid identified as critical for direct binding of cholesterol in the SSD required for sterol interaction. 11 The chain of putative sterol binding sites raises the possibility that multiple sites may facilitate transfer of sterol ligands from the NTD to the SSD. 14


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site (1)

up down

Met1232 Met1159

Tyr1229 3.2A

3.3A

site (4)

3.3A

Phe692

site (5)

Leu1241 3.5A 2.7A

3.8A

Phe1199 Gly1195 Lys822

Figure 3: Cholesterol is depicted in binding sites (1) (corresponding to the experimentally observed pocket

in the NTD), (4) and (5),. For the binding sites predicted in docking analyses, the sterol is modeled in both "up" and "down" orientations, according to the directions indicated by the arrows. "Up" refers to an orientation in which the cholesterol 3’-hydroxyl oxygen (shown in red) is pointing toward the NTD and "down" refers to the sterol oxygen pointing away from the NTD. An up-close view of the local protein structure around site (5) is shown in the inset for both "up" and "down" cholesterol poses. The amino acids (Phe692, Met1159, Tyr1229, Met1232) in the direct vicinity (within ∼4 Å of the 3’-hydroxyl oxygen) contain hydrophobic side-chains, stabilizing the "down" orientation of cholesterol.

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We next examined the structural dynamics of the NPC1 protein by carrying out 50 ns MD simulations of the protein in the fully solvated lipid bilayer. The RMSD of protein backbone atoms was checked to evaluate overall dynamical behavior of the protein (Figure 4); each domain RMSD was calculated individually to quantify the deviations arising from particular regions of the protein. The NTD, MLD, CTD and TM domain all exhibit relatively conserved backbone motion, with RMSD values between 2–3 Å. The sequence of amino acids forming cytoplasmic loops (amino acids 290–337) are characterized by the largest range of backbone motion with RMSD values exceeding 6 Å (Figure 4, black dotted line). The flexibility of this chain of amino acids may not be surprising given the limited resolution of the cryo-EM map for this region of atomic coordinates. On the luminal side of the TM domain, the linker region (amino acids 251–259) (light blue line) shows relatively conserved RMSD values compared to those of the other domains. This chain connects the NTD via the proline-rich linker sequence with the TM domain and has been proposed as a key structural feature in sterol transfer; movement of the cholesterol-bound NTD toward the TM domain would allow direct transfer of the ligand to the membrane. 12 However, our simulations show no large-scale hinging motion of the NTD.

One could speculate about the potential biological significance of the protein’s architecture based on the relative mobility of the various domains. The connection of the linker chain from the NTD to the TM domain via highly flexible cytoplasmic loops may indicate that this sequence is essential for the signaling pathway. It has been proposed that the binding of NPC2 to NPC1’s NTD may induce conformational changes in the MLD. 8 By dynamically adopting a new conformation upon ligand binding, this linker chain may be responsible for cross-talk between other structural domains.

We focused our attention next on the dynamics of His512, Glu406, and Glu584, amino acids which were identified as being protonated at pH 5 and which are in direct proxim16


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ity of the putative binding sites (4) and (5). His512, in the MLD, forms a salt bridge with the nearby residue Asp531 (sidechain) and a hydrogen bond with the backbone oxygen atom of Pro532 (Figure 5A, B). These intermolecular interactions essentially hold these loops in place at pH 5, resulting in higher structural integrity of the MLD. Glu406, whose pKa value was calculated to be >20.00 (Table 1) forms hydrogen bonds with the oxygen atom of the Glu584 sidechain and the –NH groups of the Arg404 guanidino sidechain Arg404 (Figure 6A and B, green line), connecting the sidechains of Glu406, Glu584, and Arg404. The motion of these three residues is dynamically coupled, evidenced by a joint structural transition around 25 ns (Figure 6B, green line). The sidechain of Glu406 rotates, thus sharing its proton with the two oxygen atoms on Glu584. A similar dynamical coupling of residues is observed in the SSD, particularly around Glu688 (Figure 6C and D). The protonated sidechain of Glu688 is connected via an H-bond network to the sidechains of Asp620 (solid black line) and Tyr1225 (dotted black line). The significance of these amino acids, located in direct vicinity of putative binding pockets, may be that they impart a controlled flexibility to the protein environment. An incoming sterol ligand may transiently disrupt a salt bridges and/or H-bonding network, but this disruption may be compensated then by the subsequent formation of additional hydrogen bonds to the sterol molecule. This hypothesis should be tested in future simulations.

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A)

NTD MLD CTD SSD

B)

TM linker cytoplasmic loops

6 5 RMSD [Å]

linker (amino acids 251-259)

4 3 2 1 0 0

5

10

15 20 25 30 35 time [nanoseconds]

40

45

50

cytoplasmic loops (amino acids 290-337)

Figure 4: A) The RMSD (Å) values of amino acid backbone atoms are plotted for the various NPC1

structural domains over the 45 ns simulation. The domains are colored as follows: NTD (amino acids 30– 250, red); linker (amino acids 251–259, light blue); MLD (amino acids 374–620, purple); TM helices (amino acids 260–373, 621–797 and 1084–1278, black solid line); SSD (amino acids 621–797, orange); CTD (amino acids 861–1083, green). The largest structural deviations arise from the cytoplasmic loops (black dotted line), reflected in the relatively high RMSD values of the TM domain as a whole (solid black line). B) The connectivity of the domains is highlighted, especially with the linker chain that connects the NTD to the TM domain. Time-lapse representation for amino acids 251–620 (cyan) shows the relatively high degree of protein backbone motion for the cytoplasmic loops (amino acids 290–337).

A)

B) Asp531 Pro532 His512

His512(N) −− Asp531/Pro532 separation [Å]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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His−Asp

His−Pro

9 8 7 6 5 4 3 2 0

10

20 30 time [nanoseconds]

40

50

Figure 5: A) Upclose view of His512 side-chain Nδ and Nǫ atoms, both protonated, forming strong hydrogen bonds with the nearby residues Asp531 and Pro532. B) The separation between His512 (Nδ ) and Oδ1 of Asp531 (solid line) and between His512 (Nǫ ) and backbone oxygen atom of Pro532 (dotted line) is measured over the course of the MD simulation carried out at pH 5. Only hydrogen atoms of heavy atoms involved in bonding are shown; the remaining H-atoms are omitted for clarity.

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A)

Trp583

Phe587

B)

Glu584 Arg404

sidechain separation [Å]

Ala605

Glu406−Glu584(O1) Glu406−Glu584(O2) Arg404(N)−Glu584(O) 6

Glu406

5.5 5 4.5 4 3.5 3 2.5 2 0

10

20

30

40

50

time [nanoseconds] Glu688−Asp620

C)

D)

Asp620

Glu688 Tyr1225

sidechain separation [Å]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Glu688−Tyr1225

10 9 8 7 6 5 4 3 2 0

10

20

30

40

50

time [nanoseconds]

Figure 6: A) In the MLD, a hydrogen bond network connects the sidechains of Glu406, Glu584, and Arg404. B) Rotation of the sidechain of Glu406 causes a dynamic hydrogen bond network in which the proton is shared between the two oxygen atoms on Glu584 (solid and dotted black lines); the network includes the –NH groups of Arg404 guanidino sidechain, so that these three residues are dynamically coupled (green line). C) In the SSD, the protonated sidechain of Glu688 is connected via an H-bond network to the sidechains of Asp620 (solid black line) and Tyr1225 (dotted black line). Only hydrogen atoms of heavy atoms involved in bonding are shown; the remaining H-atoms are omitted for clarity.

Conclusion Docking studies, together with pKa computations, have identified five distinct putative sterol binding sites in NPC1. In addition to the site in the NTD that has been confirmed experimentally, these four additional sites, spanning the length of the protein, may be viable docking pockets. A chain of putative sterol binding sites, as identified here, raises the possibility that multiple sites may facilitate transfer of sterol ligands from the NTD to 19



the TM domain, possibly via a binding site in the SSD. Indeed, modeling of cholesterol in various binding poses and orientations within these binding pockets indicates an overall stabilization of the ligand-bound system for orientations in which the sterol’s isooctyl tail is pointing toward the cytoplasm. Furthermore, NPC1 may be occupied by multiple ligands simultaneously, thereby providing the metabolic pathway with a mechanism to control transfer rates. Nonetheless, as the minimum energies reported here comprise only the enthalpic contribution to the binding free energy, in the future conformational sampling under thermal conditions should be carried out.

Our MD simulations reveal overall conservative structural behavior of the individual protein domains. For example, hydrogen bonding between protonated His512 with nearby peptide backbone oxygen atoms stabilizes neighboring helices in the MLD. No large-scale conformational changes are observed. Particularly the NTD, which has been hypothesized to hinge toward the TM domain to release cholesterol directly into the membrane, demonstrates no hinging motion. The cytoplasmic loops show the largest range of protein backbone motion, possibly a useful signaling mechanism for inter-domain communication. In the SSD, particularly near Glu688, the dynamical behavior of the protein is likely functionally important for transient sterol binding. Indeed, the SSD has been shown to be critical for binding of cholesterol; mutation in the SSD near Glu688 (P692S in mouse NPC1) leads to decreased cholesterol delivery to the plasma membrane and endoplasmic reticulum. 11

In this study we examined the structural dynamics of NPC1 embedded in a lipid bilayer to investigate the dynamical behavior of the apo protein. A necessary next step is to study the behavior of the cholesterol-bound protein, to compare the dynamics of the apo and ligand-bound proteins, especially in the lipid membrane. In addition to simulating fulllength NPC1 with cholesterol located in the binding pocket determined through X-ray 20


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crystallography, a thorough sampling of all four additional putative binding sites should be carried out to test the stability of each ligand-protein structure under thermal conditions. Indeed, simulations of the cholesterol-bound NPC1 system solvated in the lipid bilayer are the focus of ongoing computational analyses. These analyses should shed further light on the mechanism by which cholesterol exits the lysosomal compartment.

Funding N.E.-M. gratefully acknowledges financial support from the Volkswagen Stiftung (grant number 86 539).

Experimental Construction of model We modeled the NPC1 protein in a lipid bilayer as follows. The protein atomic coordinates were constructed from two sets of data. Residues 23 to 288 were taken from the lower resolution (4.43 Å) cryo-EM PDB structure 3JD8 15 and residues 334 to 1255 were taken from PDB structure 5U74 (3.33 Å from Ref. 22). The two structures were overlapped using Chimera ( 23 ) and the new protein coordinates were saved. Missing internal residues (289-333, 642-649, 800-813) were reconstructed using a combination of CHARMM 24 and the CHARMM-GUI. 25 Hydrogen atoms were added using H-build from CHARMM, 24 and the N- and C-termini were capped with neutral groups CH3 –CO– and methyl acetate –NH–CO–OCH3 , respectively. Based on the crystal structures, we constructed 15 disulfide bonds using CHARMM, [Cys25-Cys74, Cys31-Cys42, Cys63-Cys109, Cys75-Cys113, Cys97-Cys238, Cys100-Cys160, Cys177-Cys184, Cys227-Cys243, Cys240Cys247, Cys468-Cys479, Cys516-Cys533, Cys909-Cys914, Cys956-Cys1011, Cys957-Cys979, Cys967-Cys976].

21



pKa computations To determine an initial protonation pattern in NPC1, pKa values of all titratable residues were evaluated by electrostatic energy computations using karlsberg+ which combines continuum electrostatics with structural relaxation of hydrogens and salt bridges. 26–28 The electrostatic energy is determined numerically by solving the linearized PoissonBoltzmann equation (LPBE) using the Adapted Poisson-Boltzmann Solver (APBS) 29 . Solving the LPBE relies on the discretization of the PB equation and determining the electrostatic potential on a grid. The grid spacing, which can affect the solution of the LPBE, is chosen to maximize the resolution across the system of interest. As the full NPC1 protein has approximate dimensions of 70 Å × 70 Å × 160 Å, the number of grid points in the {x,y,z} dimensions for each calculation was 129, 129 and 193, respectively, resulting in grid spacings of ∼0.5 Å, ∼0.5 Å, and ∼0.8 Å, in the {x,y,z} directions, respectively. After electrostatic energies are evaluated, Monte Carlo sampling is carried out to generate protonation patterns and pH adapted protein conformations. In the absence of this sampling step, a simple continuum electrostatic approach would neglect relevant side chain conformational sampling. This method has been successfully used to study e.g. charge-transport 30 and proton pumping. 31 In the present method, several protein conformations are generated, for a given pH, with the most probable protonation microstates with the corresponding H-bond pattern. With this approach, we identified the following non-standard protonated amino acid side-chains: His215, His441, His492, His510, His512, His758, His1016, His1029, His1170, Glu406, Glu688, and Glu742.

Nonetheless, the continuum electrostatics approach does have some disadvantages, including neglect of the lipid bilayer. An alternative approach to computing pKa values of proteins is the constant-pH molecular dynamics method which relies on a potential of mean force (PMF) to allow titratable residues to change their protonation states on-the-fly during the MD simulation. 32,33 The main advantage of constant pH MD simulations is 22


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that the entire system of interest (e.g. protein, solvent, lipid bilayer) is treated explicitly with pH-coupled conformational dynamics. Especially for transmembrane proteins, the inclusion of the lipid bilayer in the calculation of pKa values may be a significant factor. In the context of transmembrane proteins, constant pH MD has recently been applied successfully to understand the atomic details of proton transport. 34–37 Nonetheless, the constant pH MD approach has an increased computational expense for frequent PMF update intervals.

The protein structure was next modeled in the lipid bilayer using the CHARMM-GUI 25,38 and OPM database. 39 For this, a lipid bilayer consisting of cholesterol (10%), DOPG (10%), and POPC (80%) was constructed. The system, including Na+ (287) and Cl− (194) ions to neutralize charge, and 99,785 explicit TIP3 water molecules, 40 had a total size of 399,269 atoms and was simulated in a rectangular box of dimension 143.5 Å× 143.5 Å× 184.9 Å.

Geometry optimizations and molecular dynamics The initial geometry of the solvated NPC1-membrane complex was optimized with 1500 steps of steepest descent (SD) energy minimization, followed by 1500 adopted basis NewtonRaphson (ABNR) 24 steps to remove any close contacts. All energy minimizations and geometry optimizations used the all-atom CHARMM36 parameter set for the protein 41 and the TIP3P model for water molecules. 40

The solvated protein-membrane complex was simulated with molecular dynamics (MD) at 310 K according to the following protocol: 1) equilibration MD with Langevin dynamics (time step of 1 fs) for 50 ps followed by CPT dynamics (time step 2 fs) for 350 ps; 2) production MD with CPT dynamics (time step 2 fs) for 50 ns. To simulate a continuous system, periodic boundary conditions were applied. Electrostatic interactions were summed with the Particle Mesh Ewald method 42 (grid spacing ∼ 1.4 Å; fftx 150, ffty 150, 23



fftz 192). A nonbonded cutoff of 16.0 Å was used, and Heuristic testing was performed at each energy call to evaluate whether the non-bonded pair list should be updated.

Docking Docking analyses of NPC1 were carried out with AutoDock4.2.6 with 126 grid points in each x, y, and z directions and a grid spacing of 1 Å. 21,43 The grid center was placed at the center of the NPC1 protein (coordinates x=123.6 Å, y=124.5 Å, z=105.5 Å). Overlap of the cholesterol ligand with the docking sites was carried out manually using VMD. 44 The coordinates of the re-positioned cholesterol were saved and the reconstructed ligandprotein complex was energy minimized in CHARMM. 24 For this energy minimization, all amino acids within a radius of 20 Å of the newly positioned cholesterol were geometry optimized while the remainder of the protein was held fixed; 500 SD steps were followed by ABNR minimization until an energy gradient of 10−3 kcal/mol/Å was obtained.

Acknowledgement N.E.-M thanks J. Dragelj and E.-W. Knapp for technical discussions.

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Graphical TOC Entry cholesterol transfer path in NPC1?

(1)

(1)

(2) (2)

(3) (3)

(4) (4)

(5) (5)

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Computational tools unravel putative sterol binding ... - ACS Publications

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