Comparison of Experimental Approaches Used to Determine the Structure and Function of the Class D G Protein-Coupled Yeast α-Factor Receptor.

The α-factor mating pheromone receptor (Ste2p) has been studied as a model for the large medically important family of G protein-coupled receptors. Diverse yeast genetic screens and high-throughput mutagenesis of identified a large number of loss-of-function, constitutively-active, dominant-negative, and intragenic second-site suppressor mutants as well as mutations that specifically affect pheromone binding. Facile genetic manipulation of Ste2p also aided in targeted biochemical approaches, such as probing the aqueous accessibility of substituted cysteine residues in order to identify the boundaries of the seven transmembrane segments, and the use of cysteine disulfide crosslinking to identify sites of intramolecular contacts in the transmembrane helix bundle of Ste2p and sites of contacts between the monomers in a Ste2p dimer.

Oligomerization of yeast α-factor receptor detected by fluorescent energy transfer between ligands.

G-protein-coupled receptors (GPCRs) comprise a large superfamily of transmembrane receptors responsible for transducing responses to the binding of a wide variety of hormones, neurotransmitters, ions, and other small molecules. There is extensive evidence that GPCRs exist as homo-and hetero-oligomeric complexes; however, in many cases, the role of oligomerization and the extent to which it occurs at low physiological levels of receptor expression in cells remain unclear. We report here the use of flow cytometry to detect receptor-receptor interactions based on fluorescence resonance energy transfer between fluorescently labeled cell-impermeant ligands bound to yeast α-mating pheromone receptors that are members of the GPCR superfamily.

Identification of variant HIV envelope proteins with enhanced affinities for precursors to anti-gp41 broadly neutralizing antibodies.

HIV envelope protein (Env) is the sole target of broadly neutralizing antibodies (BNAbs) that are capable of neutralizing diverse strains of HIV. While BNAbs develop spontaneously in a subset of HIV-infected patients, efforts to design an envelope protein-based immunogen to elicit broadly neutralizing antibody responses have so far been unsuccessful. It is hypothesized that a primary barrier to eliciting BNAbs is the fact that HIV envelope proteins bind poorly to the germline-encoded unmutated common ancestor (UCA) precursors to BNAbs.

Display of the HIV envelope protein at the yeast cell surface for immunogen development.

As a step toward the development of variant forms of Env with enhanced immunogenic properties, we have expressed the glycoprotein in the yeast surface display system in a form that can be subjected to random mutagenesis followed by screening for forms with enhanced binding to germline antibodies. To optimize the expression and immunogenicity of the yeast-displayed Env protein, we tested different approaches for cell wall anchoring, expression of gp120 and gp140 Env from different viral strains, the effects of introducing mutations designed to stabilize Env, and the effects of procedures for altering N-linked glycosylation of Env. We find that diverse forms of HIV envelope glycoprotein can be efficiently expressed at the yeast cell surface and that gp140 forms of Env are effectively cleaved by Kex2p, the yeast furin protease homolog.

Human CaaX protease ZMPSTE24 expressed in yeast: Structure and inhibition by HIV protease inhibitors.

The function and localization of proteins and peptides containing C-terminal "CaaX" (Cys-aliphatic-aliphatic-anything) sequence motifs are modulated by post-translational attachment of isoprenyl groups to the cysteine sulfhydryl, followed by proteolytic cleavage of the aaX amino acids. The zinc metalloprotease ZMPSTE24 is one of two enzymes known to catalyze this cleavage. The only identified target of mammalian ZMPSTE24 is prelamin A, the precursor to the nuclear scaffold protein lamin A.

Structure of the SLC4 transporter Bor1p in an inward-facing conformation.

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg.

Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast.

We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities.

Deducing the symmetry of helical assemblies: Applications to membrane proteins.

Helical reconstruction represents a convenient and powerful approach for structure determination of macromolecules that assemble into helical arrays. In the case of membrane proteins, formation of tubular crystals with helical symmetry represents an attractive alternative, especially when their small size precludes the use of single-particle analysis. An essential first step for helical reconstruction is to characterize the helical symmetry.

The Crystal Structure of an Integral Membrane Fatty Acid α-Hydroxylase.

Neuronal electrical impulse propagation is facilitated by the myelin sheath, a compact membrane surrounding the axon. The myelin sheath is highly enriched in galactosylceramide (GalCer) and its sulfated derivative sulfatide. Over 50% of GalCer and sulfatide in myelin is hydroxylated by the integral membrane enzyme fatty acid 2-hydroxylase (FA2H).

A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs.

Determination of high-resolution, three-dimensional structures of transmembrane proteins (TMPs) has, in many cases, only been accomplished through the use of stabilized variant forms of the proteins being studied. For the important G protein-coupled receptor superfamily, this has most often been achieved by inserting a stable soluble protein, such as T4 lysozyme (T4L), in an internal loop of a receptor. However, creation of such fusion proteins generally results in loss of the ability of receptors to activate their cognate cytoplasmic G proteins.

Identification of destabilizing and stabilizing mutations of Ste2p, a G protein-coupled receptor in Saccharomyces cerevisiae.

The isolation of mutations affecting the stabilities of transmembrane proteins is useful for enhancing the suitability of proteins for structural characterization and identification of determinants of membrane protein stability. We have pursued a strategy for the identification of stabilized variants of the yeast α-factor receptor Ste2p. Because it was not possible to screen directly for mutations providing thermal stabilization, we first isolated a battery of destabilized temperature-sensitive variants, based on loss of signaling function and decreased levels of binding of the fluorescent ligand, and then screened for intragenic second-site suppressors of these phenotypes.

Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors.

G protein coupled receptors are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remain unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes that can be difficult to extract from X-ray structures.

Guided reconstitution of membrane protein fragments.

Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs.

Structure of the integral membrane protein CAAX protease Ste24p.

Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e.

Coordinating the impact of structural genomics on the human α-helical transmembrane proteome.

With the recent successes in determining membrane protein structures, we explore the tractability of determining representatives for the entire human membrane proteome. This proteome contains 2,925 unique integral α-helical transmembrane domain sequences that cluster into 1,201 families sharing more than 25% sequence identity. Structures of 100 optimally selected targets would increase the fraction of modelable human α-helical transmembrane domains from 26% to 58%, thus providing structure/function information not otherwise available.

Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast.

The insertion of a stable soluble protein into loops of transmembrane proteins has proved to be a successful approach for enhancing their stabilities and crystallization, and may also be useful in contexts where the inserted proteins can modulate or report on the activities of membrane proteins. While the use of T4 lysozyme to replace portions of the third intracellular loops of G protein-coupled receptors (GPCRs) has allowed determination of the structures of members of this important class of receptors, the creation of such fusion proteins generally leads to loss of signaling function of the resulting fusion protein, since the third intracellular loops of GPCRs play critical roles in their interactions with G proteins. We describe here a random screening approach allowing insertion of T4 lysozyme into diverse positions in the third loop of the yeast α-pheromone receptor, a GPCR encoded by the yeast STE2 gene.

Functional and physical interactions among Saccharomyces cerevisiae α-factor receptors.

The α-factor receptor Ste2p is a G protein-coupled receptor (GPCR) expressed on the surface of MATa haploid cells of the yeast Saccharomyces cerevisiae. Binding of α-factor to Ste2p results in activation of a heterotrimeric G protein and of the pheromone response pathway. Functional interactions between α-factor receptors, such as dominant-negative effects and recessive behavior of constitutive and hypersensitive mutant receptors, have been reported previously.

Nanochannel-based single molecule recycling.

We present a method for measuring the fluorescence from a single molecule hundreds of times without surface immobilization. The approach is based on the use of electroosmosis to repeatedly drive a single target molecule in a fused silica nanochannel through a stationary laser focus. Single molecule fluorescence detected during the transit time through the laser focus is used to repeatedly reverse the electrical potential controlling the flow direction.

Identifying functionally important conformational changes in proteins: activation of the yeast α-factor receptor Ste2p.

We have developed a procedure in which disulfide cross-links are used to identify regions of proteins that undergo functionally important intramolecular motion. The approach was applied to the identification of disulfide bonds that stabilize the active state of the yeast α-mating pheromone receptor Ste2p, a member of the superfamily of G protein-coupled receptors. Cysteine residues were introduced at random positions in targeted regions of a starting allele of Ste2p that completely lacks cysteines.

Oligomeric state of the oxalate transporter, OxlT.

OxlT, the oxalate transporter of Oxalobacter formigenes, was studied to determine its oligomeric state in solution and in the membrane. Three independent approaches were used. First, we used triple-detector (SEC-LS) size exclusion chromatography to analyze purified OxlT in detergent/lipid micelles.

Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p.

We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites.

Binding of fluorinated phenylalanine alpha-factor analogues to Ste2p: evidence for a cation-pi binding interaction between a peptide ligand and its cognate G protein-coupled receptor.

Ste2p, a G protein-coupled receptor (GPCR), binds alpha-factor, WHWLQLKPGQPMY, a tridecapeptide pheromone secreted by yeast cells. Upon alpha-factor binding, Ste2p undergoes conformational changes activating a signal transduction system through its associated heterotrimeric G protein leading to the arrest of cell growth in the G1 phase to prepare cells for mating. Previous studies have indicated that Tyr at position 13 of alpha-factor interacts with Arg58 on transmembrane one (TM1) of Ste2p.

Identification of human gene products containing Pro-Pro-x-Tyr (PY) motifs that enhance glutathione and endocytotic marker uptake in yeast.

In an attempt to identify genes involved in glutathione (GSH) transport, a human mammary gland cDNA library was screened for clones capable of complementing a defect in GSH uptake in yeast cells that lack Hgt1p, the primary yeast GSH uptake transporter. Five genes capable of rescuing growth on sulfur-deficient GSH-containing medium were identified: prostate transmembrane protein, androgen induced 1 (PMEPA1); lysosomal-associated protein transmembrane 4 alpha (LAPTM4alpha); solute carrier family 25, member 1 (SLC25A1); lipopolysaccharide-induced TNF factor (LITAF); and cysteine/tyrosine-rich-1 (CYYR1). All of these genes encode small integral membrane proteins of unknown function, although none appear to encode prototypical GSH transporters.

Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography.

To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by X-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow expression of reading frames fused to C-terminal His10 and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease.

Structural genomics of pathogenic protozoa: an overview.

The Structural Genomics of Pathogenic Protozoa (SGPP) Consortium aimed to determine crystal structures of proteins from trypanosomatid and malaria parasites in a high throughput manner. The pipeline of target selection, protein production, crystallization, and structure determination, is sketched. Special emphasis is given to a number of technology developments including domain prediction, the use of "co-crystallants," and capillary crystallization.