Does Cholesterol Contain Amino Acids

J Lipid Res. 2017 Jun; 58(six): 1044–1054.

Mutual structural features of cholesterol binding sites in crystallized soluble proteins

Received 2016 Nov 29; Revised 2017 April 12

Abstruse

Cholesterol-protein interactions are essential for the architectural organization of jail cell membranes and for lipid metabolism. While cholesterol-sensing motifs in transmembrane proteins have been identified, little is known almost cholesterol recognition by soluble proteins. Nosotros reviewed the structural characteristics of binding sites for cholesterol and cholesterol sulfate from crystallographic structures available in the Protein Data Depository financial institution. This assay unveiled central features of cholesterol-bounden sites that are present in either all or the bulk of sites: i) the cholesterol molecule is by and large positioned betwixt poly peptide domains that have an organized secondary structure; two) the cholesterol hydroxyl/sulfo group is often partnered by Asn, Gln, and/or Tyr, while the hydrophobic part of cholesterol interacts with Leu, Ile, Val, and/or Phe; iii) cholesterol hydrogen-bonding partners are often found on α-helices, while amino acids that interact with cholesterol'south hydrophobic core have a slight preference for β-strands and secondary construction-defective protein areas; iv) the steroid'due south C21 and C26 constitute the "hot spots" almost often seen for steroid-protein hydrophobic interactions; 5) mutual "cold spots" are C8–C10, C13, and C17, at which contacts with the proteins were not detected. Several common features we identified for soluble protein-steroid interaction appear evolutionarily conserved.

Keywords: lipids, receptors, sterols, Ten-ray crystallography, cholesterol binding site, cholesterol-protein interaction

Cholesterol is a fundamental structural component of membranes in animal cells (1). Indeed, cholesterol maintains the basic architecture of plasma membranes; in the full general bulky phospholipid bilayer, the insertion of cholesterol molecules introduces the lipid guild phase, which is essential for membrane organization and proper poly peptide part (2, 3). Moreover, cholesterol molecules usually adapt in vertical domains that span the whole bilayer leading to the formation of lipid "rafts," which are critical for cell signaling and protein sorting and trafficking (iv). Cholesterol as well serves as a metabolic forerunner of other physiologically relevant steroids, such every bit steroid hormones and bile acids (v, 6).

The primal structural and functional roles of cholesterol are underscored by the wide variety of defects that result from abnormal levels of cholesterol in the torso. Cholesterol availability to the central nervous arrangement is critical for myelin formation, brain maturation, and synapse formation and function (7, viii). Thus, the Smith-Lemli-Opitz syndrome, characterized past a marked reduction in tissue cholesterol, includes a broad variety of developmental abnormalities that range from physical malformations to developmental delay and mental retardation (9). In adult humans, depression total cholesterol levels positively correlate with increased mortality due to respiratory, digestive affliction, or traumatic events (x). In the infirmary setting, hypocholesterolemia is frequently seen later major surgery or during serious trauma, severe infections or prolonged hypovolemic stupor, with very low cholesterol levels being predictors of mortality in critically sick patients (eleven–xiv). In turn, excessive cholesterol levels, such equally in familial or caused hypercholesterolemia, lead to atherosclerosis and to a dramatically increased incidence of stroke and cardiovascular bloodshed (15–17). High cholesterol levels too correlate with progression of Alzheimer's disease and contribute to development of ocular disorders (eighteen, xix).

The multitude of pathophysiological conditions triggered by, or coincidental with, abnormal cholesterol levels fueled scientific studies on cholesterol modulation of protein office. This modulation includes control of prison cell motion and organization of the actin cytoskeleton (xx), regulation of interactions between bacterial or viral (such as HIV) pathogens and mammalian cells (21, 22), and modulation of receptor and ion channel function (23–26), even desperate modification of ion aqueduct'south pharmacology (27–29). While the mechanisms underlying cholesterol modulation of protein function are even so under debate, 2 major theories accept been recognized. The beginning is based on the ability of cholesterol to modify the physical backdrop of biological membranes, leading to the germination of the lipid order phase. Indeed, plasma membranes of mammalian cells contain big amounts of cholesterol (upwards to 50 mol%), which allows cholesterol to play a major role as a structural lipid (1). Thus, cholesterol may interact with membrane phospholipids and promote tight packing of the phospholipid acyl chains (so chosen "condensing effect" of cholesterol) (30–32). As a result, the presence of cholesterol increases lateral pressure within the membrane and introduces "packing stress" (33). The increment in lateral pressure upon cholesterol incorporation into the phospholipid bilayer has been proposed as a major mechanism for cholesterol to attune protein function (33–35). The 2d theory, however, proposes that cholesterol modifies protein part by direct steroid bounden to the protein target. Therefore, a large trunk of work has been dedicated to identifying protein motifs that could bind cholesterol molecules.

The chemic structure of cholesterol presents several key elements. First, the core of the molecule is formed past a tetracyclic (rings A–D) band system ( Fig. 1A ). A double bond in band B between carbon atoms 5 and 6 confers rigidity to the molecule. The hydrophobic tetracyclic band system is complemented past a rather flexible iso-octyl concatenation. Thus, the merely hydrophilic feature of cholesterol is a β-hydroxyl grouping at C3. Noteworthy, cholesterol is an asymmetric molecule: its α-face corresponds to a smooth, rather planar, surface, while the β-face is a surface with rough edges (methyl groups) (36).

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Cholesterol binding sites in crystallized proteins. A: Molecular construction of cholesterol (R: hydroxyl) and cholesterol sulfate (R: sulfate). B: Overview of crystallized cholesterol-poly peptide complexes. C: Overview of crystallized cholesterol sulfate-protein complexes. In all cases, the cholesterol molecule is in teal blueish; hydrogen atoms are not shown. In (B, C), arrows that point at β-strands flanking the cholesterol β-face are in yellowish whereas arrows that point at α-helixes flanking the cholesterol α-face up are in cerise.

In transmembrane protein segments, the cholesterol recognition amino acid consensus (CRAC) motif has been identified (37, 38). CRAC is a relatively brusque, linear motif with the sequence (Leu/Val)-X_one–5-(Tyr)-10_one–5-(Lys/Arg), where 10 represents any amino acid. Lys or Arg, or even Tyr, are expected to hydrogen bond with cholesterol; the Tyr effluvious ring construction stabilizes cholesterol's polycyclic core by "stacking" against it, while Leu and Val interact with the steroid's hydrophobic iso-octyl chain. Thus, the CRAC motif represents an oriented binding site for cholesterol, with nonpolar amino acids residing at the N terminus of the motif and a polar residuum(due south) at the C terminus. Cholesterol binding by CRAC motifs within poly peptide transmembrane areas and in juxta-membrane segments has been well-documented (39, 40). Even so, the cholesterol-sequestering power of CRAC motifs that are present in the sequence of nontransmembrane protein segments has been questioned repeatedly (38, 40, 41).

More recently, "reversed" CRAC motifs, in which amino acids announced in a sequence that is directionally contrary to that in CRACs, accept been described and termed "CARCs" (twoscore). In addition, several other cholesterol bounden areas on poly peptide transmembrane segments have been reported. In particular, a systematic NMR study utilizing Ala-scanning mutagenesis has revealed cholesterol-binding backdrop for the GXXXG motif in the C99 poly peptide (42). The geometry of cholesterol-binding to GXXXG and flanking areas remains speculative. Withal, mutations of thirteen amino acids scattered along GXXXG itself or its vicinity either totally ablate or significantly abolish cholesterol bounden (42).

A more precise picture of cholesterol binding to a transmembrane protein arises from an X-ray structure of the human β2-adrenergic receptor bound to cholesterol (43): although the binding surface area does not comprise conventional cholesterol-recognition motifs, information technology does include basic Arg, aromatic Trp, and aliphatic Leu/Val, a structural triad that is found in CRAC motifs. Thus, although CRAC (and CARC) motifs per se might actually have low predictive value for identifying cholesterol-binding sites, such motifs embody a general idea on the chemical forces that enable cholesterol binding to transmembrane sections of the proteins, due east.1000., hydrogen bonding with the cholesterol hydroxyl grouping and hydrophobic interactions with the cholesterol hydrophobic core (twoscore).

While there is some consensus on the structural basis of cholesterol interactions with transmembrane proteins, common structural features feature of cholesterol bounden to soluble proteins remain largely undetermined. Collectively, cholesterol-binding areas in soluble proteins are usually represented by poly peptide hydrophobic cavities that shield the steroid from an aqueous environment and besides enable cholesterol release to the membrane or a partner protein (38). An instance is the steroidogenic acute regulatory (StAR) poly peptide, which includes the steroidogenic astute regulatory protein-related lipid transfer (StART) domain (44). This domain results from an evolutionarily conserved sequence of >200 aminoacids that class an ensemble of α-helixes and β-sheets to adjust a diversity of lipid species. Two StAR poly peptide isoforms that are virtually specific for cholesterol (StAR-D1 and StAR-D3) do comprise CRACs, yet the role of these CRAC domains in cholesterol binding/transport is unclear. In synthesis, common protein secondary structural elements and amino acids that partner with the cholesterol molecule in soluble proteins remain largely unknown. Thus, the goal of our piece of work is to contribute to cover this gap in knowledge.

Assay OF CRYSTALLOGRAPHIC PROTEIN-CHOLESTEROL STRUCTURES

Nosotros performed searches of the Protein Information Depository financial institution (PDB) database (pdb.org) for poly peptide structures that contained cholesterol every bit a jump ligand. Our search yielded a total of nine structures, which had resolution and isothermic B-factor ranging from 1.45 to 3.two Å (all structures) and from xix.v to 76.23 Å² (vi structures), respectively. PDB files were downloaded into Molecular Operating Environs (MOE) software (Chemical Computing Group, Canada) and visualized using a built-in function in MOE. The Poly peptide Contacts algorithm in MOE was used to define ligand-bounden pockets with cutting-off distances of iv.five Å for both hydrophobic and ionic interactions. Histidine was treated equally a basic amino acrid while methionine was treated as hydrophobic. Five PDB entries describing cholesterol-poly peptide complexes were analyzed (Fig. 1B). The shape of cholesterol-binding sites can exist generally described as a pocket (invagination). In some instances, this description tin be extended to either a more secluded and rather straight tunnel (i.e., the binding site is virtually fully covered around its long axis by the poly peptide construction) or every bit a bean-like cavity. The latter suits the flexibility of the steroid lateral chain. In general, cholesterol-binding sites are solvent-attainable (45), with h2o molecules reported in the vicinity and/or within cholesterol-bounden sites in several crystal structures (46, 47). Using altitude measurement routine based on the receptor surface map in MOE, nosotros estimated averaged length of the cholesterol-binding pocket along steroid centrality being approximately 23 Å with the diameter of the pocket averaging 11–12 Å. The cardinal amino acid contact partners of the cholesterol molecule are summarized in Table i .

Tabular array 1.

Amino acid contacts for cholesterol/cholesterol sulfate in binding sites on crystallized proteins

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Beta-cryptogein belongs to the elicitin family and constitutes a pocket-size, extracellular, highly toxic protein that is secreted past pathogenic microorganisms and promotes leaf necrosis of the host institute (48). Cryptogeins have been reported to accept steroid-shuttling ability (49). The crystal structure of β-cryptogein from Phytophtora cryptogea in circuitous with cholesterol (PDB entry 1LRI) reveals cholesterol'southward position inside a lax, hydrophobic, and elongated tunnel that is virtually fully covered around the long axis by several α-helices and a pocket-sized fraction of β-strands. The cholesterol-binding tunnel is rather nonspecific, equally it tin accommodate a big variety of 3β-hydroxy sterols (49). Cholesterol is positioned inside the tunnel with its α-face up oriented toward the β-strands and its β-face facing the α-helical structure. Cholesterol binding involves hydrogen bonding with a Tyr located in the α-helical poly peptide domain and with h2o. Cholesterol tetracyclic nucleus and its iso-octyl chain are flanked past a multitude of amino acids (several Leu, Ile, Val, Met, and Phe). In improver to cholesterol, this β-cryptogein site binds ergosterol and fatty acids (50, 51).

Oxysterol bounden protein, Osh4, is a soluble cytosolic protein. Osh4 and related proteins are highly conserved from yeast to humans. The crystallographic construction of Saccharomyces cerevisiae Osh4 jump to cholesterol (PDB entry 1ZHY) is one of many that describe steroid/oxysterol bounden to this protein (46). In complex with cholesterol, Osh4 provides a hydrophobic pocket (i.eastward., invagination) flanked by a organization of β-strands at one side, and two α-helical structures at the other. Cholesterol is positioned inside the pocket with its β-face up oriented toward the β-strands and its α-face up facing α-helical structures. The 3β-hydroxyl of cholesterol forms hydrogen bonds with a Gln located in the α-helical domain and with water. The tetracyclic ring system and the iso-octyl tail of cholesterol are partnered with several Leu, Ile, Val, and Phe residues. Besides cholesterol, the site can also accommodate ergosterol, 7- and 25-hydroxycholesterols (46). The ability to arrange cholesterol derivatives oxygenated whether at the steroid nucleus or at the lateral chain documents the pocket'southward flexibility, which may be explained by its topology: it contains a wide lateral opening that very likely reduces overall rigidity.

The tick poly peptide, japanin, was first described in salivary glands of Rhipicephalus appendiculatus. It belongs to the lipocalin family unit of the hydrophobic molecule transporters. This soluble poly peptide exerts an immunomodulatory role by targeting and selectively reprogramming human dendritic cells (52). The crystal construction of japanin-cholesterol complex (PDB entry 4BOE) shows a hydrophobic bean-shaped fissure that is generally formed by β-strands, with curt α-helical segments flanking the cholesterol molecule (53). Cholesterol is positioned inside the cleft with its α-face oriented toward β-strands and its β-face up facing the α-helical structure. Cholesterol'due south 3β-hydroxyl forms a hydrogen bond with the backbone amide Due north provided past Glu. The hydrophobic partners of cholesterol tetracyclic rings and iso-octyl chain are several Leu and Val residues, with a single Trp or Phe also contributing.

Niemann-Option C1 (NPC1) protein is nowadays in lysosomal membranes and represents i of the fundamental molecules in cholesterol exit from the lysosome. NPC1 protein has 13 transmembrane helices, with a soluble N terminus protruding into the lysosome lumen (54–56). The N-concluding domain contains a cholesterol-bounden site, which is responsible for capturing cholesterol from NPC2, an NPC1 partner, for further shuttling cholesterol out of the lysosome past partitioning it into the lysosome membrane (55). The crystal of the NPC1 N-terminal domain bound to cholesterol (PDB entry 3GKI) shows cholesterol positioned inside a hydrophobic bean-shaped cleft that is flanked by α-helices at one side and β-strands at the other. Cholesterol is positioned inside the cleft with its β-face oriented toward β-strands and its α-face up facing α-helical structures. Cholesterol's 3β-hydroxyl forms hydrogen bonds with Asn and Gln located in the β-strand and α-helix, respectively. In plough, the tetracyclic region of cholesterol is tightly held by hydrophobic amino acids, which include Leu, Phe, Trp, and Met. Additional amino acids assist to grade the cholesterol-binding cleft. The binding crack is surrounding the cholesterol iso-octyl chain, however, loses its tight profile and opens into the solvent. The site enables adaptation of 25-hydroxycholesterol while prevents cholesterol derivatives with modifications at C3 (cholesterol sulfate and epicholesterol) from bounden (55).

Cytochrome P450 (P450scc or CYP11A1) is found just in vertebrates and serves as a key enzyme in steroidogenesis by metabolizing cholesterol and a wide array of other sterols and their derivatives (57). Mitochondrial CYP11A1 is bound to an inner mitochondrial membrane with the large soluble protein core protruding into the mitochondrial matrix (PDB entry 3N9Y) (47). The cholesterol molecule is buried inside a hydrophobic elongated pocket that is formed by several β-strands and α-helices that shield the steroid from the aqueous medium. Cholesterol is positioned inside the pocket with its β-face up oriented toward β-strands and its α-face facing the α-helical structure. The 3β-hydroxyl of cholesterol does not interact directly with CYP11A1 amino acids but binds to two water molecules that are part of a hydrogen-bond network formed past additional water molecules and the polar residues Tyr, Asn and Gln. Many amino acids (Leu, Ile, Val, Phe, and Trp) arrange a tightly fitted pocket to accommodate the hydrophobic role of cholesterol. The site does non discriminate betwixt cholesterol and 20- or 22-hydroxycholesterol, with their binding mode being strikingly similar to that of cholesterol (47).

The smoothened receptor (SMO) mediates signal transduction in the hedgehog pathway (58). The SMO construction includes a hepta-helical transmembrane domain and an extracellular cysteine-rich domain that are continued past the juxtamembrane linker domain (PDB entry 5L7D) (45). The cysteine-rich domain contains the cholesterol-bounden site, which contributes to SMO-mediated signaling (45). Within this site, the cholesterol molecule is positioned inside an elongated pocket between α-helices while the cholesterol iso-octyl concatenation is flanked by a β-canvas (45). Notably, the order of the cholesterol molecule (as measured by the lower B-factor) is higher than that of the protein backbone, an expected result considering that cholesterol'southward steroid nucleus is rather rigid (45). The 3β-hydroxyl of cholesterol forms a hydrogen bond with Asp, this bond existence role of a larger hydrogen bail network formed by Asp, Tyr and Trp (45). The steroid hydrogen-bonding Asp seems to be located within a protein region that lacks a divers secondary structure. In contrast, the hydrophobic partners of cholesterol preferentially reside in α-helices (Tabular array ane). With the exception of Trp, all these partners are Leu, Val, and Ile. SMO's cholesterol-binding site can also accommodate 20(S)-hydroxycholesterol (59).

ANALYSIS OF CRYSTALLOGRAPHIC STRUCTURES CONTAINING CHOLESTEROL SULFATE-Protein COMPLEXES

The assay of the few available crystal structures of cholesterol may not be enough to define common features of cholesterol sites in soluble proteins. Thus, nosotros as well studied cholesterol sulfate. This is a cholesterol derivative in which the 3β-hydroxyl is substituted by a sulfate group. Although this substitution diminishes the overall hydrophobicity of the molecule, the remaining structural features are identical to those of cholesterol. From a biological standpoint, cholesterol sulfate has been extensively recognized every bit i of the virtually important sulfonated steroids. Higher levels of cholesterol sulfate were found in the plasma of patients with liver cirrhosis and hypercholesterolemia (sixty) while atherosclerosis has been linked to cholesterol sulfate deficiency (61). Under normal physiology, cholesterol sulfate plays a critical office in platelet adhesion and keratinocyte differentiation. At the molecular level, this steroid regulates the action of serine proteases and, in a rather selective style, of protein kinase C isoforms. Several PDB entries draw cholesterol sulfate-protein complexes. We analyzed three complexes found in the PDB database, their topology beingness depicted in Fig. 1C. Averaged volume of the site was estimated at ii,745 ± 256 Å³. The key features of cholesterol sulfate binding sites are summarized in Table 1.

NPC2 is a soluble lysosomal protein that plays a major part in cholesterol intracellular trafficking. NPC2 deficiency is characterized by a life-threatening accumulation of cholesterol in lysosomes (62). The crystal construction of the NPC2 complex with cholesterol sulfate (PDB entry 2HKA) reveals that the steroid molecule is positioned inside a hydrophobic tunnel that is deeply buried between poly peptide β-strands (63). The sulfo-group of the sterol does non course hydrogen bond(south) with NPC2. This is a unique example, as polar groups at C3 of the steroid are expected to have a hydrogen-binding amino acid partner (Table ane). The cholesterol sulfo-group, however, protrudes into the aqueous medium which substitutes for polar partners normally provided by a poly peptide. Every bit in crystal structures that contained cholesterol, the cholesterol sulfate tetracyclic ring organization is partnered past Val, Phe, Leu, Ile and Trp. Interestingly, this site can also accommodate cholesterol, the latter having lower affinity when compared with cholesterol sulfate (63). As well cholesterol and cholesterol sulfate, the NPC2 site binds a wide variety of animal and plant sterols. Bounden of fatty acids, bile acids or glycosphingolipids, however, could non be observed (64). The lack of hydrogen-bonding protein partners for the steroid may contribute to the relatively lax specificity of the site toward cholesterol derivatives at C3: indeed, cholesteryl acetate and 5α-cholestan-3-one bind to the site (63). Unexpectedly, thiocholesterol, cholesteryl bromide and long concatenation cholesteryl esters cannot bind (63). Information technology has been proposed that the failure of binding studies was a consequence of the differential solubility of unlike lipid species in a given solvent, and to their differential ability to grade multimers in hydrophilic media (63).

The retinoic acid-related orphan receptor α (RORalpha) is an orphan member of the subfamily one of nuclear hormone receptors. ROR proteins serve as critical regulators of many physiological processes that occur during embryonic evolution and in adulthood, including regulation of circadian rhythms (65, 66). Cholesterol and cholesterol sulfate were proposed as RORalpha ligands, with cholesterol sulfate having an affinity for this receptor higher than that of cholesterol (67). Conceivably, the stronger hydrogen-bonding ability of cholesterol sulfate leads to its higher affinity to cholesterol-binding sites, as observed for both ROR and NPC2 (meet above). In the crystal structure of steroid-RORalpha complex (PDB entry 1S0X), cholesterol sulfate is positioned inside a bean-shaped hydrophobic crevice formed generally past α-helices, yet a few curt β-strand domains are also present. Cholesterol sulfate is positioned within the crevice with the steroid β-face oriented toward the β-strands and the α-confront facing the α-helical structure. Positioning of cholesterol sulfate inside the crack is very similar to cholesterol; however, cholesterol sulfate is pulled out a picayune toward the more hydrophilic side of the pocket. The 3-sulfo group hydrogen bonds with the backbone amide N of Tyr and Gln and with a sidechain N of Arg. The hydrophobic function of the molecule is partnered by Ile, Phe, Val, Trp, and Met.

Cytochrome P450 46A1 initiates the major pathway for cholesterol removal from the brain via conversion of cholesterol to 24(S)-hydroxycholesterol (68). The protein has a short North-terminal transmembrane region, with the protein core beingness soluble. Cytochrome P450 46A1'southward crystallographic structure in circuitous with cholesterol (PDB entry 2Q9F) shows cholesterol inside a bean-shaped protein cavity that shields the steroid from an aqueous medium by layers of α-helices and β-strands (69). Cholesterol is positioned inside the cavity with its β-face up oriented toward β-strands and its α-face facing the α-helical structure. Equally presented for RORalpha (see above), cholesterol'southward 3β-hydroxyl forms hydrogen bonds with backbone amide N atoms, the latter provided by His and Asn. The hydrophobic role of the cholesterol molecule is partnered past Leu, Ile, Phe and other amino acids (Tabular array 1). As well cholesterol, the protein site is expected to bind 7-dehydrocholesterol and desmosterol, as oxidation of these steroids by P450 46A1 has been documented (70).

COMPARISON OF STEROID-PROTEIN CONTACT MAPS FOR CHOLESTEROL VERSUS CHOLESTEROL SULFATE

Commutation of the hydroxyl at C3 with a sulfate does not disrupt the general topology of the steroid molecule. Thus, cholesterol-sulfate binding sites follow the full general layout of cholesterol's bounden sites (Fig. 1B, C) (67). The sulfate, however, carries a much larger charge than the hydroxyl, which results in differences in sterol-poly peptide interactions at ring A between the 2 steroids. Based on computationally assessed steroid-poly peptide contacts for each crystallographic construction, we created steroid-protein interaction maps for cholesterol and cholesterol sulfate. Several "hot spots," i.e., C atoms that stand for contact points with proteins in the majority of crystals, were identified ( Fig. 2 ): C7, C12, C21, and C26 for cholesterol, and C14, C18, C19, C21, C22, C24, C26, and C27 for cholesterol sulfate. The larger number of hot spots for cholesterol sulfate is probable explained by the smaller number of crystallographic structures analyzed (three for cholesterol sulfate vs. 6 for cholesterol). Despite the larger number of hot spots in the cholesterol sulfate structures, the contact maps of cholesterol and cholesterol sulfate are rather similar, with C21 and C26 constituting hot spots for both steroids. In addition, maps for both steroids testify that hydrophobic steroid-protein contacts are formed most exclusively by Leu, Val and Ile, with occasional advent of Phe or Trp. As expected, there are more diverse and dense ionic interactions at the sulfo-group of cholesterol sulfate when compared with those at the hydroxyl of cholesterol. However, the steroid-interacting amino acids Asn, Gln, and Tyr are common. Although steroid hydroxyl and sulfate groups have preference toward N atoms of the amino acids to grade hydrogen bonding, steroid C21 and C26 do non show stiff preference for a particular atom even when steroid molecules class contacts with the same amino acid (Fig. two). Finally, common cold spots were also detected: neither cholesterol nor cholesterol sulfate formed contacts with the proteins at steroid C atoms C8–C10, C13, or C17. Overall, the protein contact maps of cholesterol sulfate are similar to those of cholesterol.

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Schematic representation of the contact maps for cholesterol (A) and cholesterol sulfate (B) interactions with soluble proteins. Contact maps for each crystal structure were created using the "Poly peptide Contacts" function in MOE software with cut-off distances for the hydrophobic and ionic interactions fix at four.five Å. A total of six crystal structures for cholesterol and 3 for cholesterol sulfate with proteins were analyzed. Schemes reflect contacts that were detected in at least iv structures for cholesterol and at least ii structures for cholesterol sulfate. Amino acrid residues that form contacts at each carbon atom of the steroid are listed within boxes. The frequency of appearance is indicated in parenthesis. For instance, "C7: Leu (3)" ways that Leu formed contact with C7 on iii occasions. Within a given structure, several amino acids may form contact with the same carbon atom of the steroid. Thus, the sum of frequencies at which amino acids that appear at a particular contact betoken may exceed the full number of complexes in which a contact betwixt poly peptide and steroid was detected. Contact points (i.eastward., hot spots) that are common for poly peptide interactions with both cholesterol and cholesterol sulfate are shown in orangish; amino acid atoms that form contact with the steroid are listed; carbon atoms that stand for common cold spots (come across text) are numbered in dark-green.

COMMON STRUCTURAL FEATURES OF CHOLESTEROL/CHOLESTEROL SULFATE BINDING SITES IN SOLUBLE PROTEINS

Superposition of cholesterol and cholesterol sulfate bound to crystallized proteins based on the iii hot spots identified in Fig. 2 showed the clustering of steroid molecules into three conformational groups, which differed in the rotation bending of the steroid nucleus along the C3–C17 axis ( Fig. 3A ). Although the rotation of the molecule may reach 90°, data all the same show a fairly consequent conformational profile of steroid ligands bound to the crystallized proteins.

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Common design of cholesterol-binding sites in soluble proteins. A: Superposition of cholesterol and cholesterol sulfate bound to crystallized proteins is based on the three hot spots identified in Fig. 2 (east.g., oxygen at steroid C3, and carbon atoms C21 and C26). B: Proposed design for the cholesterol-binding site in crystallized soluble proteins, which includes mutual structural features that ascertain cholesterol and cholesterol sulfate binding. Spheres in light gray emphasize the cutting-off distance at which steroid-binding amino acids where adamant.

Based on the visualization of the crystal structures of soluble proteins in complexes with cholesterol/cholesterol sulfate, we were able to find the steroid bounden site's full general features, which were present in the majority of the structures ( Table 2 ). Kickoff, the steroid molecule is e'er positioned within a protein cavity/pocket to minimize the exposure of the hydrophobic steroid molecule to the aqueous environment (Fig. 3B). This finding is key to understanding steroid interaction with soluble proteins. While maintenance of a big hydrophobic cavity in an aqueous environment would be energetically unfavorable, it is believable that some rearrangement of poly peptide structure occurs in presence of steroid ligand. Indeed, the evidence favors the "induced fit" mode of cholesterol and cholesterol sulfate bounden to soluble proteins. In particular, rearrangement of protein domains upon cholesterol binding has been reported for β-cryptogein and SMO protein (45, 51). In NPC2, the sterol-all-around hydrophobic cleft is pocket-sized in the absence of cholesterol. However, pregnant reorientation of several amino acid side bondage is observed upon sterol binding, with the entire site existence thus molded around the hydrocarbon portion of the sterol to enable efficient binding (63). The malleability of these sites may account for their adaptation of various ligands, such as steroid derivatives, and fifty-fifty fatty acids (50). However, malleability is limited, and accommodation of ligands other than that of steroid family is not ever possible (63).

Tabular array 2.

Frequency of appearance of the proposed common structural features amidst cholesterol/cholesterol sulfate bounden sites on crystallized soluble proteins

Feature of Steroid-Binding Poly peptide Site	PDB Entry									Frequency of Advent
Feature of Steroid-Binding Poly peptide Site	1LRI	1ZHY	4BOE	3GKI	3N9Y	5L7D	2HKA	1S0X	2Q9F	Frequency of Advent
Protein pocket	Yes	Yep	Yep	Aye	Yes	Yeah	Yes	Aye	Yeah	ix/9
Avoidance of secondary structure-lacking areas	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yep	Yes	ix/9
Steroid α-confront facing an α-helix		Yes		Yeah	Yes	Yes		Yes	Yes	half dozen/9
Steroid β-confront facing a β-strand		Yes		Yep	Yeah		Yes	Yes	Yeah	vi/9
Hydrogen-bonding with Asn, Gln, or Tyr	Yes	Yes		Yes				Yes	Yes	v/9
Hydrogen bonding amino acrid is on an α-helix	Yes	Yes		Yes				Yes	Yes	5/9
Hydrophobic interactions with Leu, Ile, Val, or Phe	Yes	Yes	Aye	Yes	Yes	Yes	Yes	Yep	Yes	9/9
Steroid-poly peptide contacts at C21 and C26	Yes	Yes	Yes				Yep	Yes		5/ix
Hydrogen bonding with O at C3 moiety	Yep	Yes	Yes	Yes		Yep		Yes	Yes	7/9
Lack of contacts with the protein at steroid C8–C10, C13, and C17	Yes	Yep	Yeah	Yes	Yes	Yes	Yes	Yep	Aye	nine/9

2d, the steroid-all-around pocket is frequently formed by ordered domains (whether α-helices or β-strands). Thus, the steroid avoids binding to protein areas that lack a secondary structure. Secondary structure-lacking areas take a loftier degree of flexibility (71). Thus, it is conceivable that these areas: i) are unable to provide a lasting, concerted group of amino acids to capture and retain the spring steroid; and/or 2) nowadays the risk of "exposing" the steroid molecule to the aqueous solvent. Interestingly, in the bulk of structures (with the exception of NPC2 and smoothened proteins) the steroid is positioned inside the hydrophobic protein pocket that is flanked by α-helices on one side and β-strands on the other (Fig. 3B). Interestingly, the crude β-face of the steroid is preferentially facing β-strands while the smoother α-face prefers α-helices. The basis of this phenomenon remains unclear. Withal, information technology has to be taken into account that polar hydroxyl or sulfate groups are oriented toward the β-face of the steroid. Having the β-face of cholesterol oriented toward β-strands may help to avoid electrostatic repulsion between the steroid polar group and the dipole of the α-helix. Consistent with this, the more polar sulfo group (in cholesterol sulfate) ever faces a β-strand structure while the less polar hydroxyl (in cholesterol) faces this construction in virtually cases, yet is still able to face α-helices (e.chiliad., β-cryptogenin and japanin).

Tertiary, Tyr, Gln, Asn, Glu, Arg, and His were all identified equally possible partners for accommodating steroid polar groups at C3 (Table 1). Even so, only five out of 9 protein-steroid complexes included hydrogen bonding, whether directly with Asn, Gln and Tyr or via coordination through water molecules. Asn, Gln and Tyr have polar side chains and share neutrality on the scale of side chain acidity/basicity. Remarkably, only 3 other amino acids (Cys, Ser and Thr) meet this combination. These amino acids differ from Asn, Gln, and Tyr by having a much higher hydropathy index, i.east., a parameter indicative of the prevalence of hydrophobic versus hydrophilic properties of the amino acids (72). Thus, the scale of hydropathy indexes [Cys (2.five) > Thr (−0.7) > Ser (−0.eight) > Tyr (−1.iii) Gln (−three.5) = Asn (−3.5)] inversely reflects the frequency at which these amino acids have been reported to interact with the steroid polar grouping at C3 in crystallized soluble proteins: Cys, Thr and Ser are never seen; Tyr is occasionally reported; Asn and Gln are often reported. Overall, it appears that the physicochemical properties of amino acid partners of the steroid group at C3 are similar to those of the cholesterol molecule as a whole: neutral and polar. Remarkably, when either a ligand (in the example of cholesterol sulfate) or a receptor (in the case of Glu serving equally a hydrogen bail partner in japanin) becomes acidic, the ligand hydrogen bonds with the backbone amide N atoms rather than with the side concatenation of the amino acid. Yet, Asn and Gln are also institute amongst these hydrogen-bonding amino acids (e.chiliad., RORalpha and CYP46A1). Interestingly, in 5 out of nine structures, amino acids that provide hydrogen bonding at the moiety of C3 are constitute on the α-helical domains (Table 1). Although the polar α-face of cholesterol points away from the α-helices, the latter are still able to provide amino acids that satisfy geometric criteria (distance and angle) for hydrogen bonding with cholesterol.

Fourth, Ile, Val, Leu, and Phe are consistently institute partners of the hydrophobic part of steroid molecules (Table 1). This pattern is not surprising, considering that the four amino acids are at the acme of the hydropathy scale, with hydropathy indexes of 4.5, 4.2, iii.8, and 2.8, respectively (72). In dissimilarity to the hydrogen bond partners, there is no pattern regarding the location of hydrophobic residues that collaborate with the hydrophobic core of the steroid: α-helices, β-strands, and even secondary structure-lacking protein areas provide hydrophobic amino acids with similar frequency of appearance. Overall, at that place is a slight predominance of β-strands combined with secondary construction-defective areas (Table 1).

Finally, we detected both "hot" and "common cold" spots for proteins to contact the steroid: C21 and C26 found the well-nigh ofttimes hot spots for steroid-protein hydrophobic interactions while O atoms at the C3 moiety often provide bonding partners for steroid-poly peptide hydrogen bonding (Fig. 3B). In plow, mutual cold spots are presented past the steroid C8–C10, C13, and C17, at which contacts with the protein were not detected. The rather lax blueprint of cholesterol-binding sites, with simply one hot spot for possible hydrogen bonding complemented by hydrophobic interactions inside a malleable protein pocket, allows bounden of a diverse group of cholesterol derivatives into the sites within soluble proteins.

CHOLESTEROL/CHOLESTEROL SULFATE Binding SITES IN SOLUBLE PROTEINS: Further CONSIDERATIONS

A strategy similar to that followed here to place steroid binding sites in soluble proteins (computational modeling based on crystallographic data) has likewise been successful in identifying ion channel protein bounden past ethanol and related n-alkanols. Ethanol is a promiscuous, depression-analogousness ligand that interacts with both soluble and membrane proteins at aqueous concentrations in the millimolar range (73, 74). With these characteristics, many conventional methodologies, such every bit radioligand bounden or spectroscopy, are of fiddling apply to identify ethanol-binding sites. However, computational visualization and analysis of four crystal structures of booze-recognition proteins that were bachelor at the time unveiled critical common features to alcohol-sensing sites in proteins (75). These common features were used by united states as a template for the discovery of an ethanol-sensing site in the calcium/voltage-gated potassium channel of big conductance (BK), an ionotropic receptor that controls numerous physiological functions and constitutes a major target of alcohol actions in the torso (74, 76).

From a ligand perspective, cholesterol offers challenges similar to those posed by ethanol. Although the cholesterol molecule is more than complex, cholesterol modulation of poly peptide part is, as ethanol's, rather promiscuous: hundreds of cholesterol-sensing proteins that participate in cholesterol modulation of prison cell biology have been discovered (40, 77). Moreover, for some of these proteins, cholesterol effective concentrations are in the millimolar range (78, 79), this analogousness being like to that of ethanol. As found for ethanol, cholesterol-bounden sites are expected to reside in nontransmembrane regions of the protein (76, eighty). A first attempt to identify a cholesterol-sensing motif succeeded when the CRAC motif was advanced. This motif includes amino acids that are common amidst previously known cholesterol-sensing proteins (81). Moreover, upwardly to day, CRAC motifs are widely used equally a fast-screen approach in the search for putative cholesterol-sensing regions within transmembrane proteins. Our current analysis reveals that, in contrast to the rather brusk, linear CRAC motifs, cholesterol-binding sites in soluble proteins are generally large structures, with circuitous 3D organization that requires the associates of several structural elements (α-helices and β-strands), leading to the formation of cholesterol-bounden cavities/tunnels. This complexity for cholesterol-peptide interaction in soluble proteins is somewhat anticipated, as the steroid molecule has to be separated from direct contact with the aqueous medium by a hydrophobic protein shield.

A more detailed comparison of cholesterol-binding sites in soluble proteins with CRACs reveals farther differences: our study identified residues in soluble proteins that conform a "signature" theme: Asn, Gln and Tyr form ionic/hydrogen bonds with the sterol in 5 out of ix crystal structures while Leu, Ile, Val and Phe found the bulk of amino acid partners for the hydrophobic steroid nucleus and iso-octyl chain in all the crystal structures evaluated. Overall, our profiling of amino acids shows little resemblance (if whatsoever) with the CRAC "signature" motif, which e'er contains a central Tyr, in addition to Arg and Lys (39). The only overlapping amino acids between cholesterol-bounden sites in soluble proteins and cholesterol-binding sites in protein transmembrane segments (i.e., CRACs) are aliphatic Leu and Val. Every bit mentioned above, these amino acids have one of the highest hydropathy indexes (72). Thus, it is conceivable that they represent a "must take" hydrophobic element inside sites that bind such a lipophilic molecule equally cholesterol.

Finally, we found no correlation between CRAC number/distribution in soluble proteins and their ability to bind cholesterol. For instance, β-cryptogein does not accept a CRAC motif whereas the CYP11A1 protein sequence contains six CRACs. This consequence buttresses the thought that the predictive value of CRAC domains for the presence of cholesterol-bounden sites in soluble proteins has to exist taken with caution. This conclusion is in understanding with previously reported difficulties in using the CRAC motif sequence as a predictor of cholesterol-binding ability by membrane proteins themselves and proteins in general (38, 40, 41). It has been shown that the genome of Streptococcus agalactiae (GenBank accretion number NC 004368) encodes 2,094 proteins (41). The bulk of these proteins have no relation to cholesterol homeostasis, yet it has been estimated that the CRAC motif appears as frequently as every 112 aminoaacids (41). Similar examples were shown for proteomes of Staphylococcus aureus and Escherichia coli (41). Therefore, the mere occurrence of CRAC domains is non indicative of a cholesterol-binding site. Furthermore, we also showed that lack of CRAC motifs does not preclude soluble proteins from binding cholesterol (Table ane). Whether our newly identified common structural features of cholesterol-binding sites in soluble proteins (Table 2) concord predictive value remains to be established.

CONCLUSIONS

We identified common structural features of cholesterol/cholesterol sulfate binding sites in soluble proteins. The proteins under analysis cover a large evolutionary span (from fungi to Human sapiens), a wide assortment of functions (cytotoxicity, cholesterol-shuttling, catalysis, etc.), and exhibit varied topology (cytosolic, extracellular, lysosomal, etc.). Thus, the overall structural design of cholesterol-binding sites in soluble proteins is highly conserved. The common structural features herein identified tin can be used as a tool to narrow downwards the all-encompassing pool of putative cholesterol-bounden sites that ordinarily results from computational analysis of protein structures. Thus, our findings should facilitate the discovery of cholesterol-sensing areas and a rational for drug design to target pathological conditions related to disruption of cholesterol homeostasis.

Footnotes

Abbreviations:

CRAC: cholesterol recognition amino acrid consensus
CYP11A1: cytochrome P450 (P450scc)
MOE: Molecular Operating Surroundings
NPC1: Niemann-Selection C1
PDB: Protein Data Bank
RORalpha: retinoic acid-related orphan receptor α SMO, smoothened receptor
StAR: steroidogenic acute regulatory
Offset: steroidogenic acute regulatory protein-related lipid transfer

This work was supported by National Institutes of Health Grants R01 AA023764; (A.N.B.) and R01 HL104631 (A.Chiliad.D.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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