Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.

Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.

Heterodimer formation and activity in the human enzyme galactose-1-phosphate uridylyltransferase.

One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity. This question is of particular interest for the human enzyme galactose-l-phosphate uridylyl-transferase (GALT), impairment of which results in the inherited metabolic disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes. Furthermore, the broad range of phenotypic severity observed in these patients raises the possibility that allelic combination, not just allelic constitution, may play some role in determining outcome. In the work described herein, we have selected two distinct naturally occurring null mutations of GALT, Q188R and R333W, and asked the questions (i) what are the impacts of these mutations on subunit assembly, and (ii) if heterodimers do form, are they active? To answer these questions, we have established a yeast system for the coexpression of epitope-tagged alleles of human GALT and investigated both the extent of specific GALT subunit interactions and the activity of defined heterodimer pools. We have found that both homodimers and heterodimers do form involving each of the mutant subunits tested and that both heterodimer pools retain substantial enzymatic activity. These results are significant not only in terms of their implications for furthering our understanding of galactosemia and GALT holoenzyme structure-function relationships but also because the system described may serve as a model for similar studies of other complexes composed of multiple subunits.

Functional expression of mouse Mdr1 in an outer membrane permeability mutant of Escherichia coli.

Functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in Escherichia coli is providing an appropriate system for structure/function studies and might provide an invaluable tool to screen potential P-gp substrates and inhibitors. The major problem encountered in such studies, however, is the impermeability of the outer membrane of Gram-negative bacteria, which protects microorganisms against the cytotoxic effects of many lipophilic cancer drugs and blocks accessibility of P-gp reversal agents. In the present study we have constructed, by mutagenesis, a "leaky" (containing a permeable outer membrane) strain of E. coli, which is significantly more susceptible to the toxic effect of known P-gp substrates and cytotoxic agents. Expression of mouse Mdr1 in the mutant confers cross-resistance to daunomycin, quinidine, chloroquine, rhodamine 6G, and puromycin. Most importantly, reserpine and doxorubicin completely abolish Mdr1-mediated rhodamine resistance. The results provide strong support for previous observations, suggesting that Mdr1 can be expressed functionally in E. coli and indicate that the leaky mutant will be useful for further structure/function studies of the heterologously expressed eukaryotic drug efflux protein.

Novel human DNA alkyltransferases obtained by random substitution and genetic selection in bacteria.

DNA repair alkyltransferases protect organisms against the cytotoxic, mutagenic, and carcinogenic effects of alkylating agents by transferring alkyl adducts from DNA to an active cysteine on the protein, thereby restoring the native DNA structure. We used random sequence substitutions to gain structure-function information about the human O6-methylguanine-DNA methyltransferase (EC 2.1.1.63), as well as to create active mutants. Twelve codons surrounding but not including the active cysteine were replaced by a random nucleotide sequence, and the resulting random library was selected for the ability to provide alkyltransferase-deficient Escherichia coli with resistance to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Few amino acid changes were tolerated in this evolutionarily conserved region of the protein. One mutation, a valine to phenylalanine change at codon 139 (V139F), was found in 70% of the selected mutants; in fact, this mutant was selected much more frequently than the wild type. V139F provided alkyltransferase-deficient bacteria with greater protection than the wild-type protein against both the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine, increasing the D37 over 4-fold and reducing the mutagenesis rate 2.7-5.5-fold. This mutant human alkyltransferase, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of alkylation-based chemotherapeutic regimens.

Extending the chemistry that supports genetic information transfer in vivo: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA, and methylphosphonate DNA.

DNA and RNA are the polynucleotides known to carry genetic information in life. Chemical variants of DNA and RNA backbones have been used in structure-function and biosynthesis studies in vitro, and in antisense pharmacology, where their properties of nuclease resistance and enhanced cellular uptake are important. This study addressed the question of whether the base(s) attached to artificial backbones encodes genetic information that can be transferred in vivo. Oligonucleotides containing chemical variants of DNA or RNA were used as primers for site-specific mutagenesis of bacteriophage f1. Progeny phage were scored both genetically and physically for the inheritance of information originally encoded by bases attached to the nonstandard backbones. Four artificial backbone chemistries were tested: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA and methylphosphonate DNA. All four were found capable of faithful information transfer from their attached bases when one or three artificial positions were flanked by normal DNA. Among oligonucleotides composed entirely of nonstandard backbones, only phosphorothioate DNA supported genetic information transfer in vivo.

Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

The molecular role of the common gamma c subunit in signal transduction reveals functional asymmetry within multimeric cytokine receptor complexes.

The specific signal transduction function of the gamma c subunit in the interleukin (IL) 2, IL-4, IL-7, IL-9, and IL-15 receptor complexes remains undefined. The present structure-function analyses demonstrated that the entire cytoplasmic tail of gamma c could be functionally replaced in the IL-2 receptor (IL-2R) signaling complex by a severely truncated erythropoietin receptor cytoplasmic domain lacking tyrosine residues. Heterodimerization of IL-2R beta with either gamma c or the truncated erythropoietin receptor chain led to an array of specific signals normally derived from the native IL-2R despite the substitution of Janus kinase JAK2 for JAK3 in the receptor complex. These findings thus suggest a model in which the gamma c subunit serves as a common and generic "trigger" chain by providing a nonspecific Janus kinase for signaling program initiation, while signal specificity is determined by the unique "driver" subunit in each of the gamma c- containing receptor complexes. Furthermore, these results may have important functional implications for the asymmetric design of many cytokine receptor complexes and the evolutionary design of receptor subfamilies that share common trigger or driver subunits.

Identification of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a combinatorial chemical library.

Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a "synthetic peptide combinatorial library." We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an IC50 of 2 microM. The peptide is active on IN proteins from other retroviruses such as HIV-2, feline immunodeficiency virus, and Moloney murine leukemia virus, supporting the notion that a conserved region of IN is targeted. The hexapeptide was also tested in the disintegration reaction. This phosphoryl-transfer reaction can be carried out by the catalytic core of IN alone, and the peptide HCKFWW was found to inhibit this reaction, suggesting that the hexapeptide acts at or near the catalytic site of IN. Identification of an IN hexapeptide inhibitor provides proof of concept for the approach, and, moreover, this peptide may be useful for structure-function analysis of IN.

Structural and functional analysis of pp70S6k.

The pp70/85-kDa S6 kinases, collectively referred to as pp70S6k, are thought to participate in transit through the G1 phase of the cell cycle. pp70S6k regulates the phosphorylation of the 40S ribosomal protein S6 and the transcription factor CREM tau. pp70S6k is regulated by serine/threonine phosphorylation, and although 1-phosphatidylinositol 3-kinase and phospholipase C have been implicated as upstream regulators, the mechanism of activation and identity of the upstream pp70S6k kinases remain unknown. To improve our understanding of how this mitogen-stimulated protein kinase is regulated by growth factors and the immunosuppressant rapamycin, we have initiated a structure/function analysis of pp70S6k. Our results indicate that both the N and C termini participate in the complex regulation of pp70S6k activity.

Expression studies of catalytic antibodies.

We have examined the positive influence of human constant regions on the folding and bacterial expression of active soluble mouse immunoglobulin variable domains derived from a number of catalytic antibodies. Expression yields of eight hybridoma- and myeloma-derived chimeric Fab fragments are compared in both shake flasks and high density fermentations. In addition the usefulness of this system for the generation of in vivo expression libraries is examined by constructing and expressing combinations of heavy and light chain variable regions that were not selected as a pair during an immune response. A mutagenesis study of one of the recombinant catalytic Fab fragments reveals that single amino acid substitutions can have dramatic effects on the expression yield. This system should be generally applicable to the production of Fab fragments of catalytic and other hybridoma-derived antibodies for crystallographic and structure-function studies.

Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts.

The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK was replaced with Met (RII beta-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RII beta, RII beta-P, and RII beta-K, and the effects on PKA isozyme distribution and transformation properties were analyzed. DT cells contained PKA-I and PKA-II isozymes in a 1:2 ratio. Over-expression of wild-type or mutant RII beta resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RII beta cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RII beta overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RII beta. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RII beta-P or RII beta-K cells failed to respond to 8-Cl-cAMP. These data suggest that autophosphorylation and nuclear location signal sequences are integral parts of the growth regulatory mechanism of RII beta.

D1 cap region involved in the receptor recognition and neural cell survival activity of human ciliary neurotrophic factor.

Human ciliary neurotrophic factor (hCNTF), which promotes the cell survival and differentiation of motor and other neurons, is a protein belonging structurally to the alpha-helical cytokine family. hCNTF was subjected to three-dimensional structure modeling and site-directed mutagenesis to analyze its structure-function relationship. The replacement of Lys-155 with any other amino acid residue resulted in abolishment of neural cell survival activity, and some of the Glu-153 mutant proteins had 5- to 10-fold higher biological activity. The D1 cap region (around the boundary between the CD loop and helix D) of hCNTF, including both Glu-153 and Lys-155, was shown to play a key role in the biological activity of hCNTF as one of the putative receptor-recognition sites. In this article, the D1 cap region of the 4-helix-bundle proteins is proposed to be important in receptor recognition and biological activity common to alpha-helical cytokine proteins reactive with gp130, a component protein of the receptors.

High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli.

The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N omega-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

Identification of NAB1, a repressor of NGFI-A- and Krox20-mediated transcription.

NGFI-A (also called Egr1, Zif268, or Krox24) and the closely related proteins Krox20, NGFI-C, and Egr3 are zinc-finger transcription factors encoded by immediate-early genes which are induced by a wide variety of extracellular stimuli. NGFI-A has been implicated in cell proliferation, macrophage differentiation, synaptic activation, and long-term potentiation, whereas Krox20 is critical for proper hindbrain segmentation and peripheral nerve myelination. In previous work, a structure/function analysis of NGFI-A revealed a 34-aa inhibitory domain that was hypothesized to be the target of a cellular factor that represses NGFI-A transcriptional activity. Using the yeast two-hybrid system, we have isolated a cDNA clone which encodes a protein that interacts with this inhibitory domain and inhibits the ability of NGFI-A to activate transcription. This NGFI-A-binding protein, NAB1, is a 570-aa nuclear protein that bears no obvious sequence homology to known proteins. NAB1 also represses Krox20 activity, but it does not influence Egr3 or NGFI-G, thus providing a mechanism for the differential regulation of this family of immediate-early transcription factors.

Residues in the pathway through a membrane transporter.

The structure of solute transporters is understood largely from analysis of their amino acid sequences, and more direct information is greatly needed. Here we report work that applies cysteine scanning mutagenesis to describe structure-function relations in UhpT, a bacterial membrane transporter. By using an impermeant SH-reactive agent to probe single-cysteine variants, we show that UhpT transmembrane segment 7 spans the membrane as an alpha-helix and that the central portion of this helix is exposed to both membrane surfaces, forming part of the translocation pathway through this transporter.