The accelerated-cell-death11 (ACD11) protein 4NT2Marz molecule 14 helixes 8-26,32-42,43-51,52-55,56-61,61-72,77-88,96-121,127-139,140-143,144-155,156-158,160-169,171-199 The helix identifiers and residue ranges for ACD11 are H1(8-26)H2(32-42)H3(43-51)H4(52-55)H5(56-61)H6(61-72)H7(77-88)H8(96-121) H9(127-139)H10(140-143)H11(144-155)H12(156-158)H13(160-169),H14(171-199) 4NTGMarz molecule provides a genetic model for studying immune response activation and localized cellular suicide that halts pathogen spread during infection in plants. 12 helixes 4NTG.pdb 8-25,32-49,50-51,52-53,54-72,77-88,96-120,127-139,144-155,156-158,160-168,171-199 The helix identifiers and residue ranges for ACD11 are H1(8-25)H2(32-49)H3(50-51)H4(52-53)H5(54-72)H6(77-88)H7(96-120)H8(127-139) H9(144-155)H10(156-158)H11(160-168)H12(171-199) Backbone thin off ACD11 structure/function and show that ACD11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic programmed cell death. Normally low ceramide-1-phosphate C1P levels become elevated, but the relatively abundant cell death inducer, phytoceramide, rises acutely . ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. A π-helix (π-bulge) near the lipid-binding cleft distinguishes apo-ACD11 from other GLTP (Glycollipid Transfer Protein) folds. The global two-layer, alpha-helically-dominated, ‘sandwich’ topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a new GLTP-fold subfamily. Ceramide (Cer), ceramide-1-phosphate (C1P), and the long chain bases (LCB), sphingosine and sphingosine-1-phosphate (S1P), are bioactive lipids that function as messenger signals and mediators of eukaryotic processes such as cell growth, development, embryogenesis, senescence, inflammation, and programmed cell death (PCD). The dynamic balance between Cer (sphingoid base amide-linked to a fatty acyl chain) and its phosphorylated derivative, C1P, critically regulates PCD in plants and animals. PCD occurs during development, during disease symptoms associated with virulent infections, and during the hypersensitive response (HR) induced by avirulent stress effectors (Lam, 2004). Hallmarks of HR are local accumulation of reactive oxygen species, nitric oxide, and the phytohormone, salicylic acid. By inducing localized cell death triggered when resistance proteins recognize specific pathogen-derived molecules, HR potentiates defensive resistance. Backbone thin off GLTP and ACD11 off superpositioning (Figure 1B) reveals a positively-charged residue triad Lys64,Arg99,Arg103 in ACD11 replacing N52, L92, and W96 in GLTP. This explains the lack of glycolipid transfer by ACD11 and limited transfer of SM, which has a phosphocholine headgroup. One phosphate oxygen undergoes hydrogen bonding with the amide nitrogen of Gly144, while the sphingoid base amine hydrogen bonds with Asp60 (Figure 2D). The p-bulge centered at Asp60 (a2 helix) persists in the ACD11/lysoSM complex. off Asp60,His143=4NT2 4NT2MarzHOH426 off Asp67,HIS97,His143=4NTG 4NTGMarzHOH401,403,406,409,412,434 structure The p-bulge in apo-ACD11 brings Asp60,67 and His143,97 sufficiently close (2.725;2.514 Å) to form a salt bridge (Figure 1G), thus providing a potential regulatory mechanism for the ACD11 GLTP-fold. In other GLTP-folds, a water molecule often bridges the Asp and His residues (Figure 1H). In apo-ACD11, the Asp60-His143 salt-bridge created by the p-bulge appears to tightly seal the entry portal region of the hydrophobic pocket (Figure 1G). In D60A-ACD11, the p-bulge persists after binding 2:0-C1P but not 12:0-C1P, suggesting that salt-bridge disruption between Asp60 and His143 by itself is insufficient to induce the p-helix-to-a-helix conformational change needed for the ACD11/C1P complex to become ‘transfer viable’. In addition, the C1P acyl chain needs to be longer than only two carbons. This conclusion is supported by the structure of wt-ACD11 complexed with lysoSM, which has no acyl chain, but displays a bound conformation resembling that of 2:0-C1P in D60A-ACD11 (Figures 5 and S2C). LysoSM is tethered to the surface via its amine group interacting with Asp60, while a sulfate anion occupies the lipid headgroup (phosphate) binding pocket lending credence to the authenticity of the lysoSM binding site.
CERT is a cytoplasmic 70829-Da protein, and it contains two distinct functional domains as follow: the N-terminal pleckstrin homology domain (PH 100 amino acid residues 2RSG.pdb) and the C-terminal StAR-related lipid transfer domain (START 230 amino acid residues 4K8N.pdb). The middle region between the PH and START domains is not predicted to form a globular fold; however, it contains a short peptide motif with the consensus sequence EFFDAXE, named the “two phenylalanines in an acidic trait” (FFAT) motif. The FFAT motif interacts with VAMP(Vesicle Associated Membrane Protein) an ER-resident type II membrane protein, and the interaction is considered to be crucial for association of CERT with the ER membrane. The CERT PH domain is indispensable for the ER-to-Golgi ceramide transport. 2RSGMarz molecule Backbone thin off The three-dimensional structure and interaction study revealed the Golgi recognition mode of the CERT PH domain. The basic groove in the CERT PH domain plays a critical role in the Golgi recognition. Conservation of the basic groove within lipid transporters uncovers functional significance of the structural motif. Ceramide transport from the endoplasmic reticulum to the Golgi apparatus is crucial in sphingolipid biosynthesis, and the process relies on the ceramide trafficking protein CERT, which contains pleckstrin homology PH and StAR-related lipid transfer domains. The CERT PH domain specifically recognizes phosphatidylinositol 4-monophosphate PtdIns(4)P, a characteristic phosphoinositide in the Golgi membrane, and is indispensable for the endoplasmic reticulum-to-Golgi transport of ceramide by CERT. The structure revealed the presence of a characteristic basic groove near the canonical phosphatidylinositol 4-monophosphate PtdIns(4)P recognition site. An extensive interaction revealed that the basic groove coordinates the CERT PH domain for efficient phosphatidylinositol 4-monophosphate PtdIns(4)P recognition and localization in the Golgi apparatus. The distinctive binding modes reflect the functions of PH domains, as the basic groove is conserved only in the PH domains involved with the PtdIns(4)P-dependent lipid transport activity but not in those with the signal transduction activity. Sphingolipids are the major structural element in eukaryotic membranes, and they function as crucial signal mediators in a variety of important biological processes, including apoptosis, inflammation, etc.. Sphingolipid synthesis involves multiple steps of metabolic conversion accomplished by a series of enzymes localized in the ER,3 the Golgi apparatus, and the plasma membrane. Therefore, the transportation of sphingolipids from one organelle to others must be highly organized. The interorganelle trafficking of sphingolipids is mediated in either a vesicular or nonvesicular manner. In mammalian cells, a de novo synthesized ceramide in ER membranes is transported to the Golgi in a nonvesicular manner by an ER-to-Golgi specific ceramide transporter. CERT, also known as GPBPΔ26, a splicing variant of Goodpasture antigen-binding protein. The CERT of the ceramide trafficking activity in cells, indicating its essential role in sphingolipid biogenesis. The CERT PH domain consists and one C-terminal α-helix Gln106,107,108,109,110,111,112,113,114,Thr115 H1(106-115),.residue ranges for human CERT are of seven β-strands (β1,26-33;β2,40-48;β3,51-55;β4,67-70;β5,75-78;β6,85-90;β7,93-98) The beta strand identifiers and residue ranges are B1(26-33),B2(40-48),B3(51-55),B4(67-70),B5(75-78),B6(85-90),B7(93-98). The main chain trace of the CERT PH domain falls in a regular PH domain fold . The core structure forms a closely packed “ß-sandwich,” with two nearly orthogonal antiparallel ß-sheets ß1-ß4 and ß5-ß7. The C-terminal α-helix lies on the top of the ß-sandwich, and two loops connecting ß1/ß2 Thr34-39 and ß3/ß4 Lys56-66 are The loop identifiers and residue ranges are L1(34-39) and L2(56-66). H1106-115;B126-33,B240-48,B351-55,B467-70,B575-78,B685-90,B793-98; off at the “exposed” end of the ß-sandwich. The ß1/ß2 anti-parallel strands are almost twice as long as the ß3/ß4 anti-parallel strands. beta1,Arg26-Trp33;beta2,Trp40-48;beta3,Ala51-55;beta4,Gly67-70 beta1/2 off ;beta3/4 off This feature makes half of the ß1/ß2 anti-parallel ß-strands and the ß1/ß2 loop Trp33-Trp40 off protrude from the core ß-sandwich. The ß1/ß2Trp33-40, ß3/ß4Tyr55-67, and ß7/αCArg98-106 off loops of the CERT PH domain showed higher deviations, as compared with the other elements. In the two-dimensional 1H-15N HSQC spectrum, four backbone amide cross-peaks from the ß1/ß2 loop Asn35,Tyr36,Ile37,Gly39 off and one from the ß7/αC loop Asp103 were invisible. On the surface of the CERT PH domain, the basic residues are clustered in the middle of the molecule, forming a basic groove around the protruding part of the ß1/ß2 region. The basic groove stretches from the “exposed end” to the “side surface” of the ß-sandwich, and it includes seven basic residues Lys32,Arg43,Lys56,Arg66,His79,Arg85,Arg98 off . Conversely, the protruding ß1/ß2 region that is surrounded by the basic groove mainly consists of aromatic and/or hydrophobic residues, such as Trp33,Tyr36,Ile37,Trp40 off . off Crystal shows free cysteine sulfohydril group residues Cys27,65,70,84. Proposed mechanism of the PH domain-mediated CERT translocation to the Golgi apparatus. A, unbound state of the CERT PH domain. B, weakly bound CERT PH domain. The nonspecific electrostatic interaction was between the basic groove in the CERT PH domain and the surface of the phospholipid membrane. C, orientation of the CERT PH domain for high affinity interaction. The specific high affinity interaction with the PtdIns(4)P-embedded membrane is shown. VI. Binding of Anionic Phospholipids to the PH Domain of the Arf GAP ASAP1 Structures of the unliganded and dibutyryl PtdIns(4,5)P2-bound PH domain were solved. PtdIns(4,5)P2 made contact with both a canonical site (C site) and an atypical site (A site). 5C79Marzmolecule Backbone thin off dibutyryl PtdIns(4,5)P2PBU PtdIns(4,5)P2 dependence of binding to large unilamellar vesicles and GAP activity was sigmoidal, consistent with cooperative sites. In contrast, PtdIns(4,5) P2 binding to the PH domain of PLC delta1 was hyperbolic. PtdIns(4,5)P2-dependent binding to vesicles and GAP activity. Idea of cooperative phospholipid binding to the C and A sites of the PH domain of ASAP1. The mechanism underlies rapid switching between active and inactive ASAP1. PH domains are regulatory components of hundreds of human proteins involved in signaling, membrane traffic, and actin cytoskeleton remodeling (DiNitto and Lambright,2006; Lemmon,2008; Lemmon et al.,1996; Moravcevic et al.,2012). Domain is defined structurally as a sandwich of seven beta strands capped at one end by an alpha helix. A subset of PH domains binds to phosphoinositides and proteins. Several mechanisms by which ligand binding to PH domains regulates protein activity. Cooperative phospholipid binding to the C and A sites of the PH domain of ASAP1 is the mechanism underlies rapid switching between active and inactive ASAP1. major function of PH domains is to localize proteins to specific membrane regions through binding to specific phosphoinositides (Moravcevic et al.,2012). proteins containing phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3)-binding PH domains are recruited to membranes in which PtdIns(3,4,5)P3 is produced. Membrane localization may be further specified by the coincidence of two signals, which was first described for two independent domains within a single protein, each domain having distinct ligand specificities (Moravcevic et al.,2012). Coincidence detection may also be mediated by a single PH domain binding two distinct ligands (Balla, 2005), as recently described for FAPP1, which simultaneously binds phosphoinositides and Arf1-GTP (Godi et al.,2004; He et al.,2011; Liu et al., 2014) and Grp1, which simultaneously binds PtdIns(3,4,5)P3 and Arf6-GTP (DiNitto et al.,2007; Malaby et al.,2013). One possible dimeric association of the two ASAP1 PH domains (A and B) in crystal. Ribbon representation of two molecules (A in coral and B in purple) in the asymmetric unit is shown. The two different PtdIns(4,5)P2 binding sites are illustrated as A site formed at the dimer interface and C site formed between ß1/ß2 and ß3/ß4 loops. See also Figures S1, S2 and S3. 5C79ABMarzmolecule off Backbone thin off dibutyryl PtdIns(4,5)P2 threePBU3 One possible dimeric association of the two ASAP1 PH domains (A and B) in crystal. Ribbon representation of two molecules (A left and B right) in the asymmetric unit is shown. The two different PtdIns(4,5)P2 binding sites are illustrated as A site formed at the dimer interface and C site formed between ß1/ß2 and ß3/ß4 loops .. Interacting with this ligand, four come from B molecule Gln412,Lys349,Lys355,Trp357 off and are residues in the A site. PtdIns(4,5)P2 interacts with both the C site and the A site in solution. In 15N heteronuclear single-quantum coherence PtdIns(4,5)P2 resulted in chemical-shift perturbations for Lys349,Trp357 in the A site off and Lys348,Arg360,Arg378,Arg407 off in the C site, thus establishing that the interactions are not the result of crystal packing effects. Four side chains from the A site interact with the bound PtdIns(4,5)P2: Lys349,Lys355,Trp357,Gln412 off . The two lysines Lys349,Lys355 off form predicted salt bridges with phosphate groups at positions 1 and 5 of the inositol ring. The phosphate at the fourth position does not form any direct interactions with the protein. At the C site, the bound PtdIns(4,5)P2 contacts more than eight residues. Both the 4- and 5-phosphate interact with positively charged residues: phosphate-4 with Arg360,His373,Arg378,Ala374 off of the main-chain nitrogen. The 5-phosphate interacts with two positively charged side chains, Lys348,Arg407,Asp351 off with the latter stabilized by forming a salt bridge. Relevant in the crystal structure of two distinct sites mediating binding to phospholipid bilayers. VII. STARD4 Membrane Interactions and Sterol Binding The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/ß helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. STARD4 is expressed in most tissues as well has previously been shown to transfer sterol and membrane interaction sterol binding-uploader. 5BRLMarzmolecule 1-224 13-222 STARD4 Backbone thin off 1JSSMarzmolecule 1-224 24-222 STARD4 STARD4 interacts with anionic membranes through a surface-exposed basic patch. STARD4 membrane interaction as well sterol binding and release requires dynamic movement of both the omega 1 L1, L5 loops and membrane insertion of the C-terminal alpha-helix. C-terminal alpha-helix Pro198-121 H5198-221,H1GLY19-40,H2LYS 41-45, H3VAL78-87,H4GLY89-96,PRO198-221 Regions of STARD4 involved in membrane interactions. Ribbon representation of STARD4 in which positively charged K49,52,219,M206,R222 and Omega-1 loop Ala120,Gly121,Glu122,Leu123,Asp124,Asn125,Ile126,Ile127 off Cys112,113,148,169,173 not disulfide bond presnce observed off When cholesterol is bound in the pocket, the C-terminal α-helix would refold to form a stable protein that could diffuse in the cytosol to deliver cholesterol to the target organelle membrane. C-terminal a-helix to theL1, L5 loops between beta-strands 1,2&5,6 result in attenuated cholesterol binding and steroidogenic activity of STARD1. Following docking of cholesterol into the STARD3 structure or STARD1 model, cholesterol was released from the ligand binding pocket through a path created by conformational movement of the Omega-1 loop. Consistent with this model, crystallographic data from STARD4 and STARD11 have showed high B values suggesting structural flexibility off for the omega1 L1, L5 loops. Ala48,Lys49,Lys50,Val51,Lys52,Asp53,Val54,Thr55,Val56,Trp5 Thr119,Ala120,Gly121,Gln122,Leu123,Asp124,Asn125,Ile126,Ile127,Ser128,Pro129 L5(121-129). Sterols are a critical component of eukaryotic cell membranes. In mammalian cells, there is an approximately 7-fold range of cholesterol content in accounts for ~35% of the total lipids in the plasma membrane and is highly enriched in the endocytic recycling compartment (ERC). In comparison, in the endoplasmic reticulum (ER) where cholesterol is synthesized, cholesterol accounts for ~5% of the total lipids. There are several protein families that are classified as lipid transfer proteins that can transfer lipids among membranes. One such family is the steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) domain (STARD) family. X. Cholesterol interaction with the related STeroidogenic Acute Regulatory lipid-Transfer (START) Domain of StAR proteins STARD1 and STARD3-MLN64 Steroidogenic acute regulatory (StAR) protein related lipid transfer START domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response as receptors to ligand binding. Backbone thin off The fragment of STARD1-START hydrophobic pocket amphiphatic and aromatic amino acids:bound with cholesterol STARD1 3P0LMarz 1-285 64-276 Leu122,124,137,138,199,227,243,247,251,260,271,275 Val126,151,156,178,179,256,Ile154,216,245,255,256 Ala171,190,200,203,218,Gly145,201,221 Phe120,165,184,267,Trp147,241,250,His220,270,Tyr134 Phe Val Ala,Ile,Gly,Phe,His,Trp off The fragment of STARD3-STARThydrophobic pocket amphophatic and aromatic amino acids:bound with cholesterol: STARD3 1EM2Marz 1-445 230-443Cholesterol STARD3 2I93Marz 1-445 67-280CLR, Leu122,133,137,170,178,199,227,239,243,247,251,260,271,275 Val126,138,151,156,179,198,256,259,Ile154,245,256 Ala86,171,,172,174,175,218,Gly145,201,221 Phe120,165,184,267,Trp147,241,250,His220,270,Tyr134 Phe Leu Ala,Ile,Gly,Phe,His,Trp off off CLR The fragment of STARD4-START hydrophobic pocket amphophatic and aromatic amino acids:bound with cholesterol STARD4 1JSSMarz mouse 1-224 24-222 Leu93,98,102,123,124,145,185,193,210,217,221 Val54,56,79,82,162,202,Ile83,86,126,127189,197 Ala48,51,71,120,205,207,211,Gly73,89,121,149,151,164,170,187,195,220 Phe64,132,213,Trp95,171,His107,Tyr67,69,117,214 Leu,Val,Ala,Ile,Gly,Phe,His,Trp,Tyr off The fragment of STARD5-START hydrophobic pocket amphophatic and aromatic amino acids:bound with cholesterol STARD5 2R55Marz 1-213 2-213 Leu61,110,163,172,180,184,189 Val36,38,57,64,68,77,83,98,117,120,122,149,188,189,Ile56,88,89,111,130 Ala134,Gly35,48,53,59,73,74,85,147,151,157,184 Phe46,86,116,176,192,193,200,211,214,Trp40,65,79,His136,212,Tyr51,58,201 Leu,Val,Ala,Ile,Gly,Phe,His,Trp off which contains residues proposed to interact with cholesterol in a hydrophobic cavity. Studies suggest that cholesterol preferentially interacts with one side wall of this cavity. Differential cholesterol binding of the two most closely related START domain proteins STARD1 and STARD3. Inactivated STARD1 in humans lead to an impaired ability of the adrenal gland to produce steroid hormones, a potentially lethal disease known as congenital lipoid adrenal hyperplasia. Cholesterol is an essential multifunctional lipid in most eukaryotic cells. It exerts a strong influence on the physical state of the plasma membrane, forms cholesterol–sphingolipid-rich microdomains such as caveolae and lipid rafts, is necessary for the activity of several membrane proteins, and serves as the precursor for steroid hormones. Misfunctions of cholesterol transport are linked to a variety of diseases. The translocation of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis, is mediated by steroidogenic acute regulatory protein (StAR, STARD1). The START domain at the C-terminal half of STARD3 is believed to be exposed to the cytosol. In its isolated form, STARD3-START is able to promote steroidogenesis even more efficiently than intact STARD3. The STARD3 structure shows a hydrophobic tunnel that expands throughout the length of the START domain and is perfectly sized to accommodate a single cholesterol molecule. A similar structure has been reported for the cholesterol-regulated START protein 4 STARD4. STARD1-START shuttles cholesterol carried in its hydrophobic cavity between the outer and inner mitochondrial membranes. Biochemical data supported the view that STARD1 partially unfolds and forms molten globules in the low-pH 5 environment of the outer mitochondrial membrane. These intermediates were hypothesized to facilitate the cholesterol transfer of STARD1 to the mitochondrial inner membrane through a mechanism that does not involve sterol shuttling. Termini cartoon START domain structures are colored from the N-terminus (blue) to the C-terminus (red) STARD3 2I93Marz 1-445 67-280CLR, The crystal structure of human STARD3-START revealed an alpha/beta-fold “helix-grip” consisting of twisted curve antiparallel nine-stranded beta-sheet and four alpha-helices. The beta-strands in the order beta1,2,3,9,8,7,6,5,4 form a U-shaped beta-barrel closed with two helices near the N- and C-terminus The beta strand identifiers and residue ranges are B1(97-101),B2(107-113),B3(117-126),B9(237-243),B8(223-229),B7(197-203),B6(182-192),B5(164-171) and ,B4(151-160). are packed forming a cavity to the concave side of the sheetto. It form predominant hydrophobic cavity that is optimally sized to bind a single cholesterol molecule. STARD3 2I93Marz 1-445 67-280CLR, Leu122,133,137,170,178,199,227,239,243,247,251,260,271,275 ,Val126,138,151,156,179,198,256,259,Ile154,245,256 ,Ala86,171,,172,174,175,218,Gly145,201,221 ,Phe120,165,184,267,Trp147,241,250,His220,270,Tyr134 Val Ala,Ile,Gly,Phe,His,Trp off off CLR The roof of the cavity is mainly formed by the C-terminal alpha-helix. 69-91,129-139,142-147,231-233,252-278 The five alpha helix identifiers and residue ranges are H1(69-91),H2(129-139),H3(142-147),H5(252-278) and ,H4(231-233). The access of cholesterol to this cavity may be enabled by conformational changes of the alpha-helix H5 and the adjacent loops L3,L2. Ala174,175,Gly176,180,Asn177,Leu178,Val179; Gly145,Glu146,Trp147,Asn148,150,Pro149,Val151,Lys152 H5(252-278),L3(174-180),L2(145-151) Backbone thin off STARD1 crystal structure supports the homology model PDB 2I93. Superposition of the crystal structure with the lowest energy homology model yields an rmsd of 1.5 Å for 205 out of the 213 C alpha atoms. Major differences between these structures are found in the loops 191-196 and 209-215 Which are the beta-strands 7-6-5-4 including the 3,2-loops L3 connecting beta5,6 and part of the alpha3-helix. The beta strand identifiers and residue ranges are B7(197-203),B6(182-192),B5(164-171),B4(151-160),L3(174-180),L2(145-151) . H5(252-278) off Backbone thin off The side chain in START Glu169,Arg188,Thr223,Met225,Leu199,Ala200 (3P0L,2I93) as salt bridge was involved hydrogen bonde cholesterol binding, most likely with the 3beta-hydroxyl group of cholesterol to Leu199 peptide bond carbonyl group Leu199>C=O...H-O-CLR with 2,498 Ångströms distance between carbonyl oxygen and hydrogen of cholesterl 3beta-hydroxyl group. H5(252-278) off Backbone thin off conserved residues Trp96,Trp147,Arg217,Asp183,Asn148 STARD1 Trp147 is a possible gate keeper in lipid ligand loading. It is located in a helical loop region and interacts with the C-terminal helix H5253-280. Binding mechanism via local unfolding or a significant conformational change in the C-terminal helix H5 could be a family wide phenomenon. Mutation in the adjacent, highly conserved residue Asn148 has been observed in congenital lipoid adrenal hyperplasia (lipoid CAH) [15]. The C-terminal helix would undergo unfolding during ligand binding. Trp96,Asp183,Arg217 are all on the 'back' face of the beta-sheet. Trp147 is absolutely conserved across the family, and since the Trp147 side chain interacts with hydrophobic residues of the C-terminal helix in all the structures, this region is likely important for lipid access to the cavity due to flexibility and hydrophobic nature. Cholesterol Ser186,Glu169,Arg188,Leu199,His220 as key residues in cholesterol binding. These side chains will likely change conformation upon ligand binding. Hydrogen bond between the cholesterol hydroxyl and either the Arg188 side chain or the backbone carbonyl of Leu199. H5(252-278) off Backbone thin off A similar ion pair between the β–strands, namely, Glu169,Arg188. The STARD3 1EM2Marz 1-445 230-443Cholesterol 232-254,241-259,292-300,303-308,415-441 The five alpha helix identifiers and residue ranges are H1(232-254),H2(241-259),H3(292-300),H4(303-308),H5(415-441). STARD3 Ser330,334,362,422 hydroxyl forms a hydrogen bond to the cholesterol hydroxyl. H5 off Backbone thin off VIII. Bothnia dystrophy by domino-like rearrangements in cellular retinaldehyde-binding protein 3HY5 mutant R234W 3HX3 Cellular retinaldehyde-binding protein (CRALBP) is essential for mammalian vision by routing 11-cis-retinoids for the conversion of photobleached opsin molecules into photosensitive visual pigments. The arginine-to-tryptophan missense mutation in position 234 (R234W) in the human gene RLBP1 encoding CRALBP compromises visual pigment regeneration and is associated with Bothnia dystrophy-Night blindness occurs. 3HY5Marzmolecule23-306 1-317 RET TLA Backbone thin off 3HX3Marz molecule57-306 1-317RETx 1XGHMarz molecule66- 292 1-317RETteor The N-terminal alpha domain comprises helices alpha1–alpha5. H1,H2,H3,H4,H526-30,43-50,57-75,78-88,92-103 The core of the C-terminal alpha/beta/alpha domain comprises β-sheet with 1 antiparallel and 4 parallel strands, β1–β5 and ten helices H7-H16. 106-124,132-143,172-188,190-197,208-213,216-228,246-255,259-264,281-285,286-290,297-306 H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16 H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16 Helices alpha6 and alpha7 are packed against the concave(ielekta virsma, struktura) face over core of the C-terminal alpha/beta/alpha domain H6,H7 4 helices alpha9–alpha12are packed against the convex (izliekta) face of the sheet. H9,H10,H11,H12 third structural motif is formed between the aperiodic segment of the N terminus residues 23-42 H1 helix and loop 2 with helices alpha13, alpha14 of the C-terminus of CRALBP. H13,H14 The retinal-binding cavity is delimited by the convex side of the beta-sheet and the 6 adjacent helices. H7,H8,H9,H10,H11,H12 The interdomain contacts are governed by hydrophobic interactions between helix alpha4,6 of the N-terminal alpha domain and helices alpha7 and alpha8 of the C-terminal alpha/beta/alpha domain. H4,H6,H7,H8 Side-chain hydrogen bonds between Y117 of helix alpha6 and E185 of helix alpha8 and between Y124 of helix alpha6 and D225 of helix alpha10 control the orientation of the interdomain contact. H6,H8,H11 off Tyr117,Glu185,Tyr124,Asp225 Side-chain flips including F198, F235, and I238. As consequence the side-chain Cdelta methyl group of I238 is rotated into the retinal-binding cavity. This rotation is the only significant structural alteration in the retinal-binding pocket of the R234W 3HY5Mar off ; 3HX3Marz mutant off H9,H12 Phe198,Phe235,Ile238 off The hydrophobic retinal-binding pocket 26. H9,H12 off Backbone thin off Trp166,Leu177,215,220,227,258,262,263,Ala212,Phe161,173,204,207,240,247,Met223, Ile163,176,238,241,Val224,254,266,268,Pro145,244 Ribbon diagram of wild-type CRALBP bound to 11-cis-retinal. The helices of the N-terminal domain are indicated in H1,H2,H3; C-terminal helices are indicated in H14,H15,H16; the helical gate indicated in H11,H12,H13; beta-strands are indicated in yellow and key Arg234. H1,H2,H3,H11,H12,H13,H14,H15,H16 off R234 off The position of R234 is indicated cpk sphere, the 11-cis-retinal ligand is shown as cpk dots, and the cavity surface in the Retinoid-Binding Pocket is11-cis-retinal sequestered completely from bulk water solvent. The pocket volumes were calculated and rolling probe with radius of 1.0 Å. volume is 6.45×102Å3. The alpha,beta unsaturated aldehyde is stacked between the phenyl ring of Phe161 and the sulfur of Met223. H11off Backbone thin off Phe161,Met223 The carbonyl oxygen of the aldehyde serves as the hydrogen bond acceptor for the phenol group hydroxyl H-O- of Tyr180 and the acidic oxygen H-OOC- of Glu202 2.91 Å E202 & 2.697 Å Y180 hydrogen bonding distances between oxygen atoms >C=O...H-O-. H11off Backbone thin off Tyr180,Glu202 Hydrogen-bonding geometry between the Glu202 and Tyr180 donors and the trigonal planar sigma orbitals of the aldehyde than with the tetrahedral sigma orbitals of primary alcohol or carboxylic hydroxyl as 2.91 Å Glu202 and 2.697 Å Tyr180 hydrogen bonding distances between oxygen atoms >C=O...H-O-. The beta-ionone ring and the polyene chain are fixed by van der Waals interactions with the apolar side chains ,H7,H8,H11,H12 off Backbone thin off Ile163,Trp166,Phe173,Leu215,Leu220,Val224,Leu227,Ile238,Phe240,Phe247,Tyr251,Val254,Leu258 respectively. ,H7,H8,H9 off Backbone thin off Arg151,Asn190,Thr193,Gly155 As shown residue Arg151 is located at the end of beta-strand beta1 and is connected by network of hydrogen bonds 2.984 Å, 2.959 Å, and 3.367 Å to Gly155, Thr193 and Asn190 of helix alpha8 and to the carbonyl oxygen of residue Gly155 on beta 2 strand. Helix alpha6 and the adjacent helix upto alpha11 represent central building block of the CRAL-TRIO fold defining 1 wall of the 11-cis-retinal-binding cavity. H6,H7,H8,H9,H10,H11 IX. Structure of human StARD3 with lutein-binding domain A crystal structure of the lutein-binding domain of human StARD3 (StAR-related lipid-transfer protein 3; also known as MLN64) homology with StARD1 and shared cholesterol-binding character. 5i9jSTARD3Marzmolecule LUT,XAT Backbone thin off 5FCYMarzmolecule BCR StARD3 has since been recognized as a carotenoid-binding protein in the primate retina, where its biochemical function of binding lutein with specificity appears to be well suited to recruit this photoprotective molecule. The helix-grip fold constructed around a solvent-filled cavity. The beta-ionone ring characteristic of lutein pointing towards the bottom of the cavity were associated with fewer steric clashes, suggesting that steric complementarity and ligand asymmetry may play a role in discriminating lutein from the other ocular carotenoids zeaxanthin and meso-zeaxanthin, which only have β-ionone rings. X. Ergosterol-cholesterola oxysterols for phosphatidylinositol 4-phosphate PI(4)P inter membrane exchange Oxysterols are oxidized derivatives of cholesterol that by enzymatic pathways involve cytochrome P450 enzymes (e.g. CYP27A1, CYP7A) and non-enzymatic involve action of reactive oxygen and nitrogen species. Oxysterols play important roles in regulation of cholesterol biosynthesis and are intermediates in the synthetic pathway of the bile acids and steroid hormones [2]. Biological roles of oxysterols in cell development and differentiation, and cytotoxic and pro-apoptoic processes, and pro-inflammatory signaling pathway [3]. Pathogenic effects of some oxysterols (e.g. cholesterol-5,6-epoxide, 7-ß-hydroxycholesterol) have been described in various diseases such as cardiovascular diseases, osteoporosis, Alzheimer’s disease, and cancer [4]. Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although a- and ß-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, ß-elicitins (possessing 6–7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the alpha-isoforms (with only 1–3 lysine residues). Complex with ergosterol, cholesterol, oxysterols, analogs and phosphatidylinositol 4-phosphate PI(4)P. 2AIBMarzmolecule 1-98 ERG 4-16,17-20,21-32,43-53,53-66,84-97 H1,H2,H3,H4,H5,H6 1-29 the N-terminal segment 29 amino-acids is unfolded-open empty and forms a lid that blocks the sterol molecule in the pocket. 1ZHZMarz1-434 -1-434
The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/ß helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. STARD4 is expressed in most tissues as well has previously been shown to transfer sterol and membrane interaction sterol binding-uploader. 5BRLMarzmolecule 1-224 13-222 STARD4 Backbone thin off 1JSSMarzmolecule 1-224 24-222 STARD4 STARD4 interacts with anionic membranes through a surface-exposed basic patch. STARD4 membrane interaction as well sterol binding and release requires dynamic movement of both the omega 1 L1, L5 loops and membrane insertion of the C-terminal alpha-helix. C-terminal alpha-helix Pro198-121 H5198-221,H1GLY19-40,H2LYS 41-45, H3VAL78-87,H4GLY89-96,PRO198-221 Regions of STARD4 involved in membrane interactions. Ribbon representation of STARD4 in which positively charged K49,52,219,M206,R222 and Omega-1 loop Ala120,Gly121,Glu122,Leu123,Asp124,Asn125,Ile126,Ile127 off Cys112,113,148,169,173 not disulfide bond presnce observed off When cholesterol is bound in the pocket, the C-terminal α-helix would refold to form a stable protein that could diffuse in the cytosol to deliver cholesterol to the target organelle membrane. C-terminal a-helix to theL1, L5 loops between beta-strands 1,2&5,6 result in attenuated cholesterol binding and steroidogenic activity of STARD1. Following docking of cholesterol into the STARD3 structure or STARD1 model, cholesterol was released from the ligand binding pocket through a path created by conformational movement of the Omega-1 loop. Consistent with this model, crystallographic data from STARD4 and STARD11 have showed high B values suggesting structural flexibility off for the omega1 L1, L5 loops. Ala48,Lys49,Lys50,Val51,Lys52,Asp53,Val54,Thr55,Val56,Trp5 Thr119,Ala120,Gly121,Gln122,Leu123,Asp124,Asn125,Ile126,Ile127,Ser128,Pro129 L5(121-129). Sterols are a critical component of eukaryotic cell membranes. In mammalian cells, there is an approximately 7-fold range of cholesterol content in accounts for ~35% of the total lipids in the plasma membrane and is highly enriched in the endocytic recycling compartment (ERC). In comparison, in the endoplasmic reticulum (ER) where cholesterol is synthesized, cholesterol accounts for ~5% of the total lipids. There are several protein families that are classified as lipid transfer proteins that can transfer lipids among membranes. One such family is the steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) domain (STARD) family.
Steroidogenic acute regulatory (StAR) protein related lipid transfer START domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response as receptors to ligand binding. Backbone thin off The fragment of STARD1-START hydrophobic pocket amphiphatic and aromatic amino acids:bound with cholesterol STARD1 3P0LMarz 1-285 64-276 Leu122,124,137,138,199,227,243,247,251,260,271,275 Val126,151,156,178,179,256,Ile154,216,245,255,256 Ala171,190,200,203,218,Gly145,201,221 Phe120,165,184,267,Trp147,241,250,His220,270,Tyr134 Phe Val Ala,Ile,Gly,Phe,His,Trp off The fragment of STARD3-STARThydrophobic pocket amphophatic and aromatic amino acids:bound with cholesterol: STARD3 1EM2Marz 1-445 230-443Cholesterol STARD3 2I93Marz 1-445 67-280CLR, Leu122,133,137,170,178,199,227,239,243,247,251,260,271,275 Val126,138,151,156,179,198,256,259,Ile154,245,256 Ala86,171,,172,174,175,218,Gly145,201,221 Phe120,165,184,267,Trp147,241,250,His220,270,Tyr134 Phe Leu Ala,Ile,Gly,Phe,His,Trp off off CLR The fragment of STARD4-START hydrophobic pocket amphophatic and aromatic amino acids:bound with cholesterol STARD4 1JSSMarz mouse 1-224 24-222 Leu93,98,102,123,124,145,185,193,210,217,221 Val54,56,79,82,162,202,Ile83,86,126,127189,197 Ala48,51,71,120,205,207,211,Gly73,89,121,149,151,164,170,187,195,220 Phe64,132,213,Trp95,171,His107,Tyr67,69,117,214 Leu,Val,Ala,Ile,Gly,Phe,His,Trp,Tyr off The fragment of STARD5-START hydrophobic pocket amphophatic and aromatic amino acids:bound with cholesterol STARD5 2R55Marz 1-213 2-213 Leu61,110,163,172,180,184,189 Val36,38,57,64,68,77,83,98,117,120,122,149,188,189,Ile56,88,89,111,130 Ala134,Gly35,48,53,59,73,74,85,147,151,157,184 Phe46,86,116,176,192,193,200,211,214,Trp40,65,79,His136,212,Tyr51,58,201 Leu,Val,Ala,Ile,Gly,Phe,His,Trp off which contains residues proposed to interact with cholesterol in a hydrophobic cavity. Studies suggest that cholesterol preferentially interacts with one side wall of this cavity. Differential cholesterol binding of the two most closely related START domain proteins STARD1 and STARD3. Inactivated STARD1 in humans lead to an impaired ability of the adrenal gland to produce steroid hormones, a potentially lethal disease known as congenital lipoid adrenal hyperplasia. Cholesterol is an essential multifunctional lipid in most eukaryotic cells. It exerts a strong influence on the physical state of the plasma membrane, forms cholesterol–sphingolipid-rich microdomains such as caveolae and lipid rafts, is necessary for the activity of several membrane proteins, and serves as the precursor for steroid hormones. Misfunctions of cholesterol transport are linked to a variety of diseases. The translocation of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis, is mediated by steroidogenic acute regulatory protein (StAR, STARD1). The START domain at the C-terminal half of STARD3 is believed to be exposed to the cytosol. In its isolated form, STARD3-START is able to promote steroidogenesis even more efficiently than intact STARD3. The STARD3 structure shows a hydrophobic tunnel that expands throughout the length of the START domain and is perfectly sized to accommodate a single cholesterol molecule. A similar structure has been reported for the cholesterol-regulated START protein 4 STARD4. STARD1-START shuttles cholesterol carried in its hydrophobic cavity between the outer and inner mitochondrial membranes. Biochemical data supported the view that STARD1 partially unfolds and forms molten globules in the low-pH 5 environment of the outer mitochondrial membrane. These intermediates were hypothesized to facilitate the cholesterol transfer of STARD1 to the mitochondrial inner membrane through a mechanism that does not involve sterol shuttling. Termini cartoon START domain structures are colored from the N-terminus (blue) to the C-terminus (red) STARD3 2I93Marz 1-445 67-280CLR, The crystal structure of human STARD3-START revealed an alpha/beta-fold “helix-grip” consisting of twisted curve antiparallel nine-stranded beta-sheet and four alpha-helices. The beta-strands in the order beta1,2,3,9,8,7,6,5,4 form a U-shaped beta-barrel closed with two helices near the N- and C-terminus The beta strand identifiers and residue ranges are B1(97-101),B2(107-113),B3(117-126),B9(237-243),B8(223-229),B7(197-203),B6(182-192),B5(164-171) and ,B4(151-160). are packed forming a cavity to the concave side of the sheetto. It form predominant hydrophobic cavity that is optimally sized to bind a single cholesterol molecule. STARD3 2I93Marz 1-445 67-280CLR, Leu122,133,137,170,178,199,227,239,243,247,251,260,271,275 ,Val126,138,151,156,179,198,256,259,Ile154,245,256 ,Ala86,171,,172,174,175,218,Gly145,201,221 ,Phe120,165,184,267,Trp147,241,250,His220,270,Tyr134 Val Ala,Ile,Gly,Phe,His,Trp off off CLR The roof of the cavity is mainly formed by the C-terminal alpha-helix. 69-91,129-139,142-147,231-233,252-278 The five alpha helix identifiers and residue ranges are H1(69-91),H2(129-139),H3(142-147),H5(252-278) and ,H4(231-233). The access of cholesterol to this cavity may be enabled by conformational changes of the alpha-helix H5 and the adjacent loops L3,L2. Ala174,175,Gly176,180,Asn177,Leu178,Val179; Gly145,Glu146,Trp147,Asn148,150,Pro149,Val151,Lys152 H5(252-278),L3(174-180),L2(145-151) Backbone thin off STARD1 crystal structure supports the homology model PDB 2I93. Superposition of the crystal structure with the lowest energy homology model yields an rmsd of 1.5 Å for 205 out of the 213 C alpha atoms. Major differences between these structures are found in the loops 191-196 and 209-215 Which are the beta-strands 7-6-5-4 including the 3,2-loops L3 connecting beta5,6 and part of the alpha3-helix. The beta strand identifiers and residue ranges are B7(197-203),B6(182-192),B5(164-171),B4(151-160),L3(174-180),L2(145-151) . H5(252-278) off Backbone thin off The side chain in START Glu169,Arg188,Thr223,Met225,Leu199,Ala200 (3P0L,2I93) as salt bridge was involved hydrogen bonde cholesterol binding, most likely with the 3beta-hydroxyl group of cholesterol to Leu199 peptide bond carbonyl group Leu199>C=O...H-O-CLR with 2,498 Ångströms distance between carbonyl oxygen and hydrogen of cholesterl 3beta-hydroxyl group. H5(252-278) off Backbone thin off conserved residues Trp96,Trp147,Arg217,Asp183,Asn148 STARD1 Trp147 is a possible gate keeper in lipid ligand loading. It is located in a helical loop region and interacts with the C-terminal helix H5253-280. Binding mechanism via local unfolding or a significant conformational change in the C-terminal helix H5 could be a family wide phenomenon. Mutation in the adjacent, highly conserved residue Asn148 has been observed in congenital lipoid adrenal hyperplasia (lipoid CAH) [15]. The C-terminal helix would undergo unfolding during ligand binding. Trp96,Asp183,Arg217 are all on the 'back' face of the beta-sheet. Trp147 is absolutely conserved across the family, and since the Trp147 side chain interacts with hydrophobic residues of the C-terminal helix in all the structures, this region is likely important for lipid access to the cavity due to flexibility and hydrophobic nature. Cholesterol Ser186,Glu169,Arg188,Leu199,His220 as key residues in cholesterol binding. These side chains will likely change conformation upon ligand binding. Hydrogen bond between the cholesterol hydroxyl and either the Arg188 side chain or the backbone carbonyl of Leu199. H5(252-278) off Backbone thin off A similar ion pair between the β–strands, namely, Glu169,Arg188. The STARD3 1EM2Marz 1-445 230-443Cholesterol 232-254,241-259,292-300,303-308,415-441 The five alpha helix identifiers and residue ranges are H1(232-254),H2(241-259),H3(292-300),H4(303-308),H5(415-441). STARD3 Ser330,334,362,422 hydroxyl forms a hydrogen bond to the cholesterol hydroxyl. H5 off Backbone thin off VIII. Bothnia dystrophy by domino-like rearrangements in cellular retinaldehyde-binding protein 3HY5 mutant R234W 3HX3 Cellular retinaldehyde-binding protein (CRALBP) is essential for mammalian vision by routing 11-cis-retinoids for the conversion of photobleached opsin molecules into photosensitive visual pigments. The arginine-to-tryptophan missense mutation in position 234 (R234W) in the human gene RLBP1 encoding CRALBP compromises visual pigment regeneration and is associated with Bothnia dystrophy-Night blindness occurs. 3HY5Marzmolecule23-306 1-317 RET TLA Backbone thin off 3HX3Marz molecule57-306 1-317RETx 1XGHMarz molecule66- 292 1-317RETteor The N-terminal alpha domain comprises helices alpha1–alpha5. H1,H2,H3,H4,H526-30,43-50,57-75,78-88,92-103 The core of the C-terminal alpha/beta/alpha domain comprises β-sheet with 1 antiparallel and 4 parallel strands, β1–β5 and ten helices H7-H16. 106-124,132-143,172-188,190-197,208-213,216-228,246-255,259-264,281-285,286-290,297-306 H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16 H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16 Helices alpha6 and alpha7 are packed against the concave(ielekta virsma, struktura) face over core of the C-terminal alpha/beta/alpha domain H6,H7 4 helices alpha9–alpha12are packed against the convex (izliekta) face of the sheet. H9,H10,H11,H12 third structural motif is formed between the aperiodic segment of the N terminus residues 23-42 H1 helix and loop 2 with helices alpha13, alpha14 of the C-terminus of CRALBP. H13,H14 The retinal-binding cavity is delimited by the convex side of the beta-sheet and the 6 adjacent helices. H7,H8,H9,H10,H11,H12 The interdomain contacts are governed by hydrophobic interactions between helix alpha4,6 of the N-terminal alpha domain and helices alpha7 and alpha8 of the C-terminal alpha/beta/alpha domain. H4,H6,H7,H8 Side-chain hydrogen bonds between Y117 of helix alpha6 and E185 of helix alpha8 and between Y124 of helix alpha6 and D225 of helix alpha10 control the orientation of the interdomain contact. H6,H8,H11 off Tyr117,Glu185,Tyr124,Asp225 Side-chain flips including F198, F235, and I238. As consequence the side-chain Cdelta methyl group of I238 is rotated into the retinal-binding cavity. This rotation is the only significant structural alteration in the retinal-binding pocket of the R234W 3HY5Mar off ; 3HX3Marz mutant off H9,H12 Phe198,Phe235,Ile238 off The hydrophobic retinal-binding pocket 26. H9,H12 off Backbone thin off Trp166,Leu177,215,220,227,258,262,263,Ala212,Phe161,173,204,207,240,247,Met223, Ile163,176,238,241,Val224,254,266,268,Pro145,244 Ribbon diagram of wild-type CRALBP bound to 11-cis-retinal. The helices of the N-terminal domain are indicated in H1,H2,H3; C-terminal helices are indicated in H14,H15,H16; the helical gate indicated in H11,H12,H13; beta-strands are indicated in yellow and key Arg234. H1,H2,H3,H11,H12,H13,H14,H15,H16 off R234 off The position of R234 is indicated cpk sphere, the 11-cis-retinal ligand is shown as cpk dots, and the cavity surface in the Retinoid-Binding Pocket is11-cis-retinal sequestered completely from bulk water solvent. The pocket volumes were calculated and rolling probe with radius of 1.0 Å. volume is 6.45×102Å3. The alpha,beta unsaturated aldehyde is stacked between the phenyl ring of Phe161 and the sulfur of Met223. H11off Backbone thin off Phe161,Met223 The carbonyl oxygen of the aldehyde serves as the hydrogen bond acceptor for the phenol group hydroxyl H-O- of Tyr180 and the acidic oxygen H-OOC- of Glu202 2.91 Å E202 & 2.697 Å Y180 hydrogen bonding distances between oxygen atoms >C=O...H-O-. H11off Backbone thin off Tyr180,Glu202 Hydrogen-bonding geometry between the Glu202 and Tyr180 donors and the trigonal planar sigma orbitals of the aldehyde than with the tetrahedral sigma orbitals of primary alcohol or carboxylic hydroxyl as 2.91 Å Glu202 and 2.697 Å Tyr180 hydrogen bonding distances between oxygen atoms >C=O...H-O-. The beta-ionone ring and the polyene chain are fixed by van der Waals interactions with the apolar side chains ,H7,H8,H11,H12 off Backbone thin off Ile163,Trp166,Phe173,Leu215,Leu220,Val224,Leu227,Ile238,Phe240,Phe247,Tyr251,Val254,Leu258 respectively. ,H7,H8,H9 off Backbone thin off Arg151,Asn190,Thr193,Gly155 As shown residue Arg151 is located at the end of beta-strand beta1 and is connected by network of hydrogen bonds 2.984 Å, 2.959 Å, and 3.367 Å to Gly155, Thr193 and Asn190 of helix alpha8 and to the carbonyl oxygen of residue Gly155 on beta 2 strand. Helix alpha6 and the adjacent helix upto alpha11 represent central building block of the CRAL-TRIO fold defining 1 wall of the 11-cis-retinal-binding cavity. H6,H7,H8,H9,H10,H11 IX. Structure of human StARD3 with lutein-binding domain A crystal structure of the lutein-binding domain of human StARD3 (StAR-related lipid-transfer protein 3; also known as MLN64) homology with StARD1 and shared cholesterol-binding character. 5i9jSTARD3Marzmolecule LUT,XAT Backbone thin off 5FCYMarzmolecule BCR StARD3 has since been recognized as a carotenoid-binding protein in the primate retina, where its biochemical function of binding lutein with specificity appears to be well suited to recruit this photoprotective molecule. The helix-grip fold constructed around a solvent-filled cavity. The beta-ionone ring characteristic of lutein pointing towards the bottom of the cavity were associated with fewer steric clashes, suggesting that steric complementarity and ligand asymmetry may play a role in discriminating lutein from the other ocular carotenoids zeaxanthin and meso-zeaxanthin, which only have β-ionone rings. X. Ergosterol-cholesterola oxysterols for phosphatidylinositol 4-phosphate PI(4)P inter membrane exchange Oxysterols are oxidized derivatives of cholesterol that by enzymatic pathways involve cytochrome P450 enzymes (e.g. CYP27A1, CYP7A) and non-enzymatic involve action of reactive oxygen and nitrogen species. Oxysterols play important roles in regulation of cholesterol biosynthesis and are intermediates in the synthetic pathway of the bile acids and steroid hormones [2]. Biological roles of oxysterols in cell development and differentiation, and cytotoxic and pro-apoptoic processes, and pro-inflammatory signaling pathway [3]. Pathogenic effects of some oxysterols (e.g. cholesterol-5,6-epoxide, 7-ß-hydroxycholesterol) have been described in various diseases such as cardiovascular diseases, osteoporosis, Alzheimer’s disease, and cancer [4]. Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although a- and ß-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, ß-elicitins (possessing 6–7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the alpha-isoforms (with only 1–3 lysine residues). Complex with ergosterol, cholesterol, oxysterols, analogs and phosphatidylinositol 4-phosphate PI(4)P. 2AIBMarzmolecule 1-98 ERG 4-16,17-20,21-32,43-53,53-66,84-97 H1,H2,H3,H4,H5,H6 1-29 the N-terminal segment 29 amino-acids is unfolded-open empty and forms a lid that blocks the sterol molecule in the pocket. 1ZHZMarz1-434 -1-434