Crystallization of proteins from crude bovine rod outer segments.

Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques.

High-resolution crystal structures of the photoreceptor glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with three and four-bound NAD molecules.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and nonglycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD)4 , and bGAPDH(NAD)3 . The bGAPDH(NAD)4 was solved at 1.52 Å, the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD)4 and bGAPDH(NAD)3 models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54-56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies.

Detergents stabilize the conformation of phosphodiesterase 6.

Membrane-bound phosphodiesterase 6 (PDE6) plays an important role in visual signal transduction by regulating cGMP levels in rod photoreceptor cells. Our understanding of PDE6 catalysis and structure suffers from inadequate characterization of the α and ß subunit catalytic core, interactions of the core with two intrinsically disordered, proteolysis-prone inhibitory PDEγ (Pγ) subunits, and binding of two types of isoprenyl-binding protein δ, called PrBP/δ, to the isoprenylated C-termini of the catalytic core. Structural studies of native PDE6 have been also been hampered by the lack of a heterologous expression system for the holoenzyme. In this work, we purified PDE6 in the presence of PrBP/δ and screened for additives and detergents that selectively suppress PDE6 basal activity while sparing that of the trypsin-activated enzyme. Some detergents removed PrBP/δ from the PDE complex, separating it from the holoenzyme after PDE6 purification. Additionally, selected detergents also significantly reduced the level of dissociation of PDE6 subunits, increasing their homogeneity and stabilizing the holoenzyme by substituting for its native membrane environment.

StAR-like activity and molten globule behavior of StARD6, a male germ-line protein.

The steroidogenic acute regulatory protein (StAR) belongs to a family of 15 StAR-related lipid transfer (START) domain proteins termed StARD1-StARD15. StAR (StARD1) induces adrenal and gonadal steroidogenesis by moving cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane by an unclear process that involves conformational changes that have been characterized as a molten globule transition. We expressed, purified, and assessed the activity and cholesterol-binding behavior of StARD1 and StARD3-D7, showing that StARD6 had activity equal to StARD1, whereas StARD4, D5, and D7 had little or no activity with adrenal mitochondria in vitro. Partial proteolysis examined by mass spectrometry suggests that StARD6 has a protease-sensitive C-terminus, similar to but smaller than that of StARD1. Experiments using urea denaturation, stopped-flow kinetics and measurements of mitochondrial membrane association suggests that StARD1 and StARD6 both unfold and refold slowly with similar kinetic patterns. Isothermal titration calorimetry suggests that StARD6 interacts with mitochondrial membranes as well as or better than StARD1. Computational modeling of StARD6 suggests that it has a similar fold to StARD1, with a hydrophobic sterol-binding pocket and a unique C-terminal extension. StARD6, which is expressed only in male germ-line cells, thus exhibits biological and biophysical properties that imply a role in steroidogenesis.

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.

A pH-dependent molten globule transition is required for activity of the steroidogenic acute regulatory protein, StAR.

The steroidogenic acute regulatory protein (StAR) simulates steroid biosynthesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane. StAR acts exclusively on the OMM, and only StAR's carboxyl-terminal alpha-helix (C-helix) interacts with membranes. Biophysical studies have shown that StAR becomes a molten globule at acidic pH, but a physiologic role for this structural transition has been controversial. Molecular modeling shows that the C-helix, which forms the floor of the sterol-binding pocket, is stabilized by hydrogen bonding to adjacent loops. Molecular dynamics simulations show that protonation of the C-helix and adjacent loops facilitates opening and closing the sterol-binding pocket. Two disulfide mutants, S100C/S261C (SS) and D106C/A268C (DA), designed to limit the mobility of the C-helix but not disrupt overall conformation, were prepared in bacteria, and their correct folding and positioning of the disulfide bonds was confirmed. The SS mutant lost half, and the DA mutant lost all cholesterol binding capacity and steroidogenic activity with isolated mitochondria in vitro, but full binding and activity was restored to each mutant by disrupting the disulfide bonds with dithiothreitol. These data strongly support the model that StAR activity requires a pH-dependent molten globule transition on the OMM.

pH-dependent Interactions of the carboxyl-terminal helix of steroidogenic acute regulatory protein with synthetic membranes.

Steroidogenic acute regulatory (StAR) protein facilitates import of cholesterol into adrenal and gonadal mitochondria where cholesterol is converted to pregnenolone, initiating steroidogenesis. StAR acts exclusively on the outer mitochondrial membrane (OMM) by unknown mechanisms. To identify StAR domains involved in membrane association, we reacted N-62 StAR with small unilamellar vesicles (SUVs) composed of lipids resembling the OMM. Solvent-exposed domains were digested with trypsin, Asp-N, or pepsin at different pH levels, and StAR peptides protected from proteolysis were identified by mass spectrometry. At pH 4 SUVs completely protected residues 259-282; at pH 6.5 this region was partially digested into 254-272, 254-273, and 254-274. Computer-graphic modeling of N-62 StAR indicated these peptides correspond to the C-terminal alpha4 helix and that residues Leu(275), Thr(263), and Arg(272) in alpha4 form stabilizing interactions with Gln(128), Asp(150), and Asp(106) in adjacent loops. CD spectroscopy of a 37-mer model of alpha4 (residues 247-287) indicated a random coil in aqueous buffer, but in 40% methanol the peptide was alpha-helical and achieved maximal alpha-helicity at pH 5.0 in the presence of SUVs. Reacting the 37-mer with diethyl pyrocarbamate incorporated into SUVs increased the number of modified residues. Thus the C-terminal alpha4 helix is critically involved in the membrane association of StAR with OMM lipids. The membrane association and the alpha-helical structure of the C terminus in the presence of OMM lipids are also pH-dependent. These results further support StAR undergoing a pH-dependent change in its conformation when interacting with the acidic phospholipid head groups of a membrane.

A genetic isolate of congenital lipoid adrenal hyperplasia with atypical clinical findings.

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH, eventually destroying all adrenal and gonadal steroidogenesis. Lipoid CAH is caused by mutations in the steroidogenic acute regulatory protein (StAR), which facilitates the entry of cholesterol into mitochondria to initiate steroidogenesis. Patients with lipoid CAH typically present with a salt-losing crisis in the first 2 months of life, although presentation as late as 10 months with partial retention of StAR activity has been reported. We describe eight patients from six Saudi Arabian families who were first diagnosed at 1-14 months of age (median, 4-7 months; mean, 7 months). Five patients were 46,XY, and three were 46,XX. At presentation, all had hyponatremia, hyperkalemia, elevated ACTH, and low cortisol. Pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, testosterone, androstenedione, and dehydroepiandrosterone sulfate were all low in those patients in whom it was measured. DNA sequencing showed that one patient was homozygous for the StAR mutation M144R, and the other seven, from five apparently unrelated families, were homozygous for the StAR mutation R182H. Each mutation was recreated in a human StAR cDNA expression vector and found to be wholly inactive in a standard assay of COS-1 cells cotransfected with the cholesterol side-chain cleavage enzyme system. Thus, the loss of all assayable activity in vitro correlated poorly with the later onset of clinical symptoms in these patients. Lipoid CAH may present much later in life than previously thought.