Validation of the mechanism of cholesterol binding by StAR using short molecular dynamics simulations

We previously proposed an original two-state cholesterol binding mechanism by StAR, in which the C-terminal α-helix of StAR gates the access of cholesterol to its binding site cavity. This cavity, which can accommodate one cholesterol molecule, was proposed to promote the reversible unfolding of the C-terminal α-helix and allow for the entry and dissociation of cholesterol. In our molecular model of the cholesterol–StAR complex, the hydrophobic moiety of cholesterol interacts with hydrophobic amino acid side-chains located in the C-terminal α-helix and at the bottom of the cavity. In this study, we present a structural in silico analysis of StAR. Molecular dynamics simulations showed that point mutations of Phe²⁶⁷, Leu²⁷¹ or Leu²⁷⁵ at the α-helix 4 increased the gyration radius (more flexibility) of the protein's structure, whereas the salt bridge double mutant E169M/R188M showed a decrease in flexibility (more compactness). Also, in the latter case, an interaction between Met¹⁶⁹ and Phe²⁶⁷ disrupted the hydrophobic cavity, rendering it impervious to ligand binding. These obtained results are in agreement with previous in vitro experiments, and provide further validation of the two-state binding mode of action.     

Cholesterol binding is a prerequisite for the activity of the steroidogenic acute regulatory protein (StAR)

Steroidogenesis depends on the delivery of cholesterol from the outer to the inner mitochondrial membrane by StAR (steroidogenic acute regulatory protein). However, the mechanism by which StAR binds to cholesterol and its importance in cholesterol transport are under debate. According to our proposed molecular model, StAR possesses a hydrophobic cavity, which can accommodate one cholesterol molecule. In the bound form, cholesterol interacts with hydrophobic side-chains located in the C-terminal alpha-helix 4, thereby favouring the folding of this helix. To verify this model experimentally, we have characterized the in vitro activity, overall structure, thermodynamic stability and cholesterol-binding affinity of StAR lacking the N-terminal 62 amino acid residues (termed N-62 StAR). This mature form is biologically active and has a well-defined tertiary structure. Addition of cholesterol to N-62 StAR led to an increase in the alpha-helical content and T degrees (melting temperature), indicating the formation of a stable complex. However, the mutation F267Q, which is located in the C-terminal helix interface lining the cholesterol-binding site, reduced the biological activity of StAR. Furthermore, the cholesterol-induced thermodynamic stability and the binding capacity of StAR were significantly diminished in the F267Q mutant. Titration of StAR with cholesterol yielded a 1:1 complex with an apparent K(D) of 3 x 10(-8). These results support our model and indicate that StAR can readily bind to cholesterol with an apparent affinity that commensurates with monomeric cholesterol solubility in water. The proper function of the C-terminal alpha-helix is essential for the binding process.     

StAR search--what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import

Cholesterol is the starting point for biosynthesis of steroids, oxysterols and bile acids, and is also an essential component of cellular membranes. The mechanisms directing the intracellular trafficking of this insoluble molecule have received attention through the discovery of the steroidogenic acute regulatory protein (StAR) and related proteins containing StAR-related lipid transfer domains. Much of our understanding of the physiology of StAR derives from studies of congenital lipoid adrenal hyperplasia, which is caused by StAR mutations. Multiple lines of evidence show that StAR moves cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. The precise mechanism by which StAR's action on the outer mitochondrial membrane stimulates the flow of cholesterol to the inner membrane remains unclear. When StAR interacts with protonated phospholipid head groups on the outer mitochondrial membrane, it undergoes a conformational change (molten globule transition) that opens and closes StAR's cholesterol-binding pocket; this conformational change is required for cholesterol binding, which is required for StAR activity. The action of StAR probably requires interaction with the peripheral benzodiazepine receptor.     

Steroidogenic acute regulatory protein binds cholesterol and modulates mitochondrial membrane sterol domain dynamics

The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K(d) values near 32 nm), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R(2/3) = 33 A) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.     

Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding

Villin-type headpiece domains are ～70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in a stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays, we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.     

Cholesterol binding does not predict activity of the steroidogenic acute regulatory protein, StAR

Steroidogenic acute regulatory protein (StAR) stimulates adrenal and gonadal steroidogenesis by increasing the influx of cholesterol into mitochondria, where it is converted to pregnenolone to initiate steroidogenesis. StAR acts on the outer mitochondrial membrane where each molecule stimulates the mitochondrial import of several hundred molecules of cholesterol, but the precise mechanism of the action of StAR remains uncertain. StAR has a sterol-binding pocket that can accommodate one molecule of cholesterol. Direct assays show that StAR can bind cholesterol with stoichiometry approaching 1:1, and several disease-causing mutants with decreased or absent activity have correspondingly decreased cholesterol binding. We show that the StAR mutant R182L, which causes severe disease and is devoid of measurable activity in transfected cells or with isolated steroidogenic mitochondria, nevertheless, can bind as much [(14)C]- or NBD-cholesterol as wild-type StAR under equilibrium conditions and can transfer cholesterol between liposomes in vitro. Similarly, the artificial mutant S195A had 46.5% of the activity of wild-type StAR but bound cholesterol indistinguishably from wild-type. Competition assays showed that the rate of binding (t((1/2)on)) for R182L was only 36% of the wild-type and the rate of dissociation (t((1/2)off)) was 57% of wild-type, whereas the t((1/2)on) and t((1/2)off) for S195A and S195D were essentially the same for wild-type. These data indicate that cholesterol binding and transfer activities are distinct from its activity to induce steroidogenesis. StAR appears to act by other mechanisms in addition to cholesterol binding.     

Binding of steroidogenic acute regulatory protein to synthetic membranes suggests an active molten globule

Steroidogenic acute regulatory protein (StAR) mediates cholesterol transport from the outer to the inner mitochondrial membrane during steroid biosynthesis. The mechanism of StAR's action is not established. To address mechanistic issues, we assessed the binding of StAR to artificial membranes by fluorescence resonance energy transfer using endogenous StAR tryptophan residues as the donor and dansyl-phosphatidylethanolamine in the bilayer as the acceptor. Mixing StAR with dansyl-labeled vesicles composed of phosphatidylcholine increased the fluorescence intensity of dansyl emission excited at 280 nm by 10-40%. This interaction was dependent on pH, with a maximum at pH 3.0-3.5 and essentially no change above pH 5. Binding experiments at different temperatures and various combinations of phosphatidylcholine, phosphatidylglycerol, cardiolipin, and cholesterol showed that binding involves an electrostatic step and one or more other steps. Although binding prefers a thermodynamically ordered bilayer, the rate-limiting step occurs either when the bilayer is in a fluid state or when there is cholesterol-induced membrane heterogeneity. Experiments with fluorescence and light scattering indicate that StAR binding promotes ordering and aggregation of anionic membranes. The inactive StAR mutant R182L had lower affinity for the membrane, and the partially active mutant L275P had intermediate affinity. Far-UV CD spectroscopy of StAR in PC membranes show more beta-structure than in aqueous buffers, and the presence of cardiolipin or cholesterol in the membrane fosters a molten globule state. Our data suggest that StAR binds to membranes in a partially unfolded molten globule state that is relevant to the activity of the protein.     

Structural requirements of tropomodulin for tropomyosin binding and actin filament capping

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.     

Role of a mutated residue at the entrance of the substrate access channel in cytochrome p450 engineered for vitamin D(3) hydroxylation activity

The cytochrome P450 enzyme engineered for enhancement of vitamin D(3) (VD(3)) hydroxylation activity, Vdh-K1, includes four mutations (T70R, V156L, E216M, and E384R) compared to the wild-type enzyme. Plausible roles for V156L, E216M, and E384R have been suggested by crystal structure analysis (Protein Data Bank 3A50 ), but the role of T70R, which is located at the entrance of the substrate access channel, remained unclear. In this study, the role of the T70R mutation was investigated by using computational approaches. Molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations were performed, and differences between R70 and T70 were compared in terms of structural change, binding free energy change (PMF), and interaction force between the enzyme and substrate. MD simulations revealed that R70 forms a salt bridge with D42 and the salt bridge affects the locations and the conformations of VD(3) in the bound state. SMD simulations revealed that the salt bridge tends to be formed strongly when VD(3) passes through the binding pocket. PMFs showed that the T70R mutation leads to energetic stabilization of enzyme-VD(3) binding in the region near the heme active site. Interestingly, these results concluded that the D42-R70 salt bridge at the entrance of the substrate access channel affects the region near the heme active site where the hydroxylation of VD(3) occurs; i.e., it is thought that the T70R mutation plays an important role in enhancing VD(3) hydroxylation activity. A significant future challenge is to compare the hydroxylation activities of R70 and T70 directly by a quantum chemical calculation, and three-dimensional coordinates of the enzyme and VD(3) obtained from MD and SMD simulations will be available for the future challenge.     

Dynamics of cholesterol exchange in the oxysterol binding protein family

The oxysterol-binding protein-related protein (ORP) family is essential to sterol transfer and sterol-dependent signal transduction in eukaryotes. The crystal structure of one ORP family member, yeast Osh4, is known in apo and sterol-bound states. In the bound state, a 29 residue N-terminal lid region covers the opening of the cholesterol-binding tunnel, preventing cholesterol exchange. Equilibrium and steered molecular dynamics (MD) simulations of Osh4 were carried out to characterize the mechanism of cholesterol exchange. While most of the structural core was stable during the simulations, the lid was partly opened in the apo equilibrium MD simulation. Helix α7, which undergoes the largest conformational change in the crystallized bound and apo states, is conformationally coupled to the opening of the lid. The movement of α7 helps create a docking site for donor or acceptor membranes in the open state. In the steered MD simulations of cholesterol dissociation, we observed complete opening of the lid covering the cholesterol-binding tunnel. Cholesterol was found to exit the binding pocket in a step-wise process involving (i) the breaking of water-mediated hydrogen bonds and van der Waals contacts within the binding pocket, (ii) opening of the lid covering the binding pocket, and (iii) breakage of transient cholesterol contacts with the rim of the pocket and hydrophobic residues on the interior face of the lid.     

Conformational flexibility,binding energy,role of salt bridge and alanine-mutagenesis for c-Abl kinase complex

Abl kinase plays a decisive role in the mechanism of the most fatal human pathogen chronic mylogenous leukemia (CML). Here, we have carried out a comprehensive study about the conformational flexibility, role of salt bridge and the protein- ligand interaction for this kinase with its well-known inhibitor, Imatinib. We have performed molecular dynamics simulations for conformational behavior, investigated the salt bridges and calculated the binding free energy of Imatinib with MM-PB/SA method for Abl kinase complex. We also explored the role of salt-bridge in the kinase complex and its effect on binding activity of inhibitors. Furthermore, to investigate the importance of those residues which form salt bridges, we mutated them by Alanine with the help of Alanine scanning program. We noticed significant variations in total free energy of Imatinib in all possible mutations. The binding free energy of ligand for kinase receptor was analyzed by molecular mechanics Poission Boltzmann surface area (MM-PB/SA) method. These results suggest that conserved glutamic acid and lysine are necessary for stability of complex.     

Structural basis for the thermostability of ferredoxin from the cyanobacterium Mastigocladus laminosus

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.     

Effects of the core lipid on the energetics of binding of ApoA-I to model lipoprotein particles of different sizes

Interaction of apolipoproteins (apo) with lipid surfaces plays crucial roles in lipoprotein metabolism and cholesterol homeostasis. To elucidate the thermodynamics of binding of apoA-I to lipid, we used lipid emulsions composed of triolein (TO) and egg phosphatidylcholine (PC) as lipoprotein models. Determination of the level of binding of wild-type (WT) apoA-I and some deletion mutants to large (120 nm diameter; LEM) and small (35 nm diameter; SEM) emulsions indicated that N-terminal (residues 44-65) and C-terminal (residues 190-243 and 223-243) deletions have large effects on lipid interaction, whereas deletion of the central region (residues 123-166) has little effect. Substitution of amino acids at either L230 or L230, L233, and Y236 with proline residues also decreases the level of binding, indicating that an alpha-helix conformation in this C-terminal region is required for efficient lipid binding. Calorimetry showed that binding of WT apoA-I to SEM generates endothermic heat (DeltaH approximately 30 kcal/mol) in contrast to the exothermic heat (ca. -85 kcal/mol) generated upon binding to LEM and egg PC small unilamellar vesicles (SUV). This exothermic heat arises from an approximately 25% increase in alpha-helix content, and it drives the binding of apoA-I to LEM and SUV. There is a similar increase in alpha-helix content of apoA-I upon binding to either SEM or SUV, but the binding of apoA-I to SEM is an entropy-driven process. These results suggest that the presence of a core triglyceride modifies the highly curved SEM surface packing and thereby the thermodynamics of apoA-I binding in a manner that compensates for the exothermic heat generated by alpha-helix formation.     

Cholesterol sensing by CD81 is important for hepatitis C virus entry

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81''s function as a molecular scaffold; these insights are relevant to CD81''s varied roles in both health and disease.     

An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.     

Latch and trigger role for R445 in DAT transport explains molecular basis of DTDS

A recent study reports on five different mutations as sources of dopamine transporter (DAT) deficiency syndrome (DTDS). One of these mutations, R445C, is believed to be located on the intracellular side of DAT distal to the primary (S1) or secondary (S2) sites to which substrate binding is understood to occur. Thus, the molecular mechanism by which the R445C mutation results in DAT transport deficiency has eluded explanation. However, the recently reported X-ray structures of the endogenous amine transporters for dDAT and hSERT revealed the presence of a putative salt bridge between R445 and E428 suggesting a possible mechanism. To evaluate whether the R445C effect is a result of a salt bridge interaction, the mutants R445E, E428R, and the double mutant E428R/R445E were generated. The single mutants R445E and E428R displayed loss of binding and transport properties of the substrate [³H]DA and inhibitor [³H]CFT at the cell surface while the double mutant E428R/R445E, although nonfunctional, restored [³H]DA and [³H]CFT binding affinity to that of WT. Structure based analyses of these results led to a model wherein R445 plays a dual role in normal DAT function. R445 acts as a component of a latch in its formation of a salt bridge with E428 which holds the primary substrate binding site (S1) in place and helps enforce the inward closed protein state. When this salt bridge is broken, R445 acts as a trigger which disrupts a local polar network and leads to the release of the N-terminus from its position inducing the inward closed state to one allowing the inward open state. In this manner, both the loss of binding and transport properties of the R445C variant are explained.     

Does cholesterol use the mitochondrial contact site as a conduit to the steroidogenic pathway?

The first and rate-limiting step of steroidogenesis is the transfer of cholesterol from the outer mitochondrial membrane to the inner membrane where it is converted to pregnenolone by cytochrome P450 side-chain cleavage (P450scc). This reaction is modulated in the gonads and adrenals by the steroidogenic acute regulatory protein (StAR), however, the mechanism used by StAR is not understood. The outer and inner mitochondrial membranes are joined at contact sites that are thought to be held in place by protein complexes that bridge the two membranes. While it is generally accepted that proteins are imported into the mitochondrion via contact sites, it is not clear whether cholesterol takes the same conduit to the inner membrane. Strategies to combat diseases caused by interrupted cholesterol transfer will rely on a full understanding of the steroidogenic mechanism. The challenge for the future is to determine whether StAR relies on the molecular architecture that spans the mitochondrial intermembrane space to deliver its cargo.     

Calpain activation by cooperative Ca2+ binding at two non-EF-hand sites

The active site residues in calpain are mis-aligned in the apo, Ca(2+)-free form. Alignment for catalysis requires binding of Ca2+ to two non-EF-hand sites, one in each of the core domains I and II. Using domain swap constructs between the protease cores of the mu and m isoforms (which have different Ca2+ requirements) and structural and biochemical characterization of site-directed mutants, we have deduced the order of Ca2+ binding and the basis of the cooperativity between the two sites. Ca2+ binds first to the partially preformed site in domain I. Knockout of this site through D106A substitution eliminates binding to this domain as shown by the crystal structure of D106A muI-II. However, at elevated Ca2+ concentrations this mutant still forms the double salt bridge that links the two Ca2+ sites and becomes nearly as active as muI-II. Elimination of the bridge in E333A muI-II has a more drastic effect on enzyme action, especially at low Ca2+ concentrations. Domain II Ca2+ binding appears essential, because Ca(2+)-coordinating side-chain mutants E302R and D333A have severely impaired muI-II activation and activity. The introduction of mutations into the whole heterodimeric enzyme that eliminate the salt bridge or Ca2+ binding to domain II produce similar phenotypes, suggesting that the protease core Ca2+ switch is crucial and cannot be overridden by Ca2+ binding to other domains.     

Modeling the structure of the StART domains of MLN64 and StAR proteins in complex with cholesterol

Steroidogenic acute regulatory protein-related lipid transfer (StART) domains are ubiquitously involved in intracellular lipid transport and metabolism and other cell-signaling events. In this work, we use a flexible docking algorithm, comparative modeling, and molecular dynamics (MD) simulations to generate plausible three-dimensional atomic models of the StART domains of human metastatic lymph node 64 (MLN64) and steroidogenic acute regulatory protein (StAR) proteins in complex with cholesterol. Our results show that cholesterol can adopt a similar conformation in the binding cavity in both cases and that the main contribution to the protein-ligand interaction energy derives from hydrophobic contacts. However, hydrogen-bonding and water-mediated interactions appear to be important in the fine-tuning of the binding affinity and the position of the ligand. To gain insights into the mechanism of binding, we carried out steered MD simulations in which cholesterol was gradually extracted from within the StAR model. These simulations indicate that a transient opening of loop Omega1 may be sufficient for uptake and release, and they also reveal a pathway of intermediate states involving residues known to be crucial for StAR activity. Based on these observations, we suggest specific mutagenesis targets for binding studies of cholesterol and its derivatives that could improve our understanding of the structural determinants for ligand binding by sterol carrier proteins.