Chemical specificity and physical properties of the lipid bilayer in the regulation of protein kinase C by anionic phospholipids: evidence for the lack of a specific binding site for phosphatidylserine.

The association of protein kinase C (PKC) with membranes was found not to be specific for phosphatidyl-L-serine (PS). In particular, a synthetic phospholipid, dansyl-phosphatidylethanolamine, proved to be fully functional in the association of PKC with lipid bilayers and in mediating the interaction of this enzyme with diacylglycerol. Dansyl-phosphatidylethanolamine was also able to activate the enzyme in a Ca2+-dependent fashion. Differences in the ability to bind and activate PKC observed for an array of anionic lipids were not larger than alterations caused by changes in acyl chain composition. Thus, although different lipids interact to different extents with PKC, there are no specific binding sites for the PS headgroup on the enzyme. We found that lipids with a greater tendency to form inverted phases increased the binding of PKC to bilayers. However, these changes in lipid structure cannot be considered separately from the miscibility of lipid components in the membrane. For pairs of lipids with similar acyl chains, the dependence on PS concentration is sigmoidal, while for dissimilar acyl chains there is much less dependence of binding on PS concentration. The results can be explained in terms of differences in the lateral distribution of components in the membrane.

Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements.

The Drosophila CF2II protein, which contains zinc fingers of the Cys2His2 type and recognizes an A+T-rich sequence, behaves in cell culture as an activator of a reporter chloramphenicol acetyltransferase gene. This activity depends on C-terminal but not N-terminal zinc fingers, as does in vitro DNA binding. By site-specific mutagenesis and binding site selection, we define the critical amino acid-base interactions. Mutations of single amino acid residues at the leading edge of the recognition helix are rarely neutral: many result in a slight change in affinity for the ideal DNA target site; some cause major loss of affinity; and others change specificity for as many as two bases in the target site. Compared to zinc fingers that recognize G+C-rich DNA, CF2II fingers appear to bind to A+T-rich DNA in a generally similar manner, but with additional flexibility and amino acid-base interactions. The results illustrate how zinc fingers may be evolving to recognize an unusually diverse set of DNA sequences.

Mutagenic specificity of solar UV light in nucleotide excision repair-deficient rodent cells.

To investigate the role of nucleotide excision repair (NER) in the cellular processing of carcinogenic DNA photoproducts induced by defined, environmentally relevant portions of the solar wavelength spectrum, we have determined the mutagenic specificity of simulated sunlight (310-1100 nm), UVA (350-400 nm), and UVB (290-320 nm), as well as of the "nonsolar" model mutagen 254-nm UVC, at the adenine phosphoribosyltransferase (aprt) locus in NER-deficient (ERCC1) Chinese hamster ovary (CHO) cells. The frequency distributions of mutational classes induced by UVB and by simulated sunlight in repair-deficient CHO cells were virtually identical, each showing a marked increase in tandem CC-->TT transitions relative to NER-proficient cells. A striking increase in CC-->TT events was also previously documented for mutated p53 tumor-suppressor genes from nonmelanoma tumors of NER-deficient, skin cancer-prone xeroderma pigmentosum patients, compared to normal individuals. The data therefore indicate that the aprt gene in NER-deficient cultured rodent cells irradiated with artificial solar light generates the same distinctive "fingerprint" for sunlight mutagenesis as the p53 locus in NER-deficient humans exposed to natural sunlight in vivo. Moreover, in strong contrast to the situation for repair-component CHO cells, where a significant role for UVA was previously noted, the mutagenic specificity of simulated sunlight in NER-deficient CHO cells and of natural sunlight in humans afflicted with xeroderma pigmentosum can be entirely accounted for by the UVB portion of the solar wavelength spectrum.

Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity.

The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr). The isolated amino-terminal domain (EIN) of E. coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr. An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein. A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield. Intact E. coli EI is effective in phosphotransfer from PEP to HPr from E. coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum. Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E. coli or M. capricolum requires the intermediacy of HPr. The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E. coli, B. subtilis, and M. capricolum as well as to EIAglc from E. coli. These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr.

Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.

Identification of a receptor/G-protein contact site critical for signaling specificity and G-protein activation.

Each G protein-coupled receptor recognizes only a distinct subset of the many structurally closely related G proteins expressed within a cell. How this selectively is achieved at a molecular level is not well understood, particularly since no specific point-to-point contact sites between a receptor and its cognate G protein(s) have been identified. In this study, we demonstrate that a 4-aa epitope on the m2 muscarinic acetylcholine receptor, a prototypical Gi/o-coupled receptor, can specifically recognize the C-terminal 5 aa of alpha subunits of the Gi/o protein family. The m2 receptor residues involved in this interaction are predicted to be located on one side of an alpha-helical receptor region present at the junction between the third intracellular loop and the sixth transmembrane domain. Coexpression studies with hybrid m2/m3 muscarinic receptors and mutant G-protein alpha q subunits showed that the receptor/G-protein contact site identified in this study is essential for coupling specificity and G-protein activation.

A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.

The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of kcat/Km). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences--strict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.

The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library.

Type I and II receptors for the transforming growth factor beta (TGF-beta) are transmembrane serine/threonine kinases that are essential for TGF-beta signaling. However, little is known about their in vivo substrates or signal transduction pathways. To determine the substrate specificity of these kinases, we developed combinatorial peptide libraries synthesized on a hydrophilic matrix that is easily accessible to proteins in aqueous solutions. When we subjected these libraries to phosphorylation by the cAMP-dependent protein kinase, we obtained the optimal peptide sequence RRXS (I/L/V), in perfect agreement with the substrate sequence deduced from mutagenesis and crystal structure analyses. By using the same libraries, we showed that the optimal substrate peptide for both the type I and II TGF-beta receptors was KKKKKK(S/T)XXX. Since the two kinases are thought to play different roles in intracellular signal transduction, it was a surprise to find that they have almost identical substrate specificity. Our method is direct, sensitive, and simple and provides information about the kinase specificity for all the amino acid residues at each position.

Phage display selection of ligand residues important for Src homology 3 domain binding specificity.

The Src homology 3 (SH3) domain is a 50-aa modular unit present in many cellular proteins involved in intracellular signal transduction. It functions to direct protein-protein interactions through the recognition of proline-rich motifs on associated proteins. SH3 domains are important regulatory elements that have been demonstrated to specify distinct regulatory pathways important for cell growth, migration, differentiation, and responses to the external milieu. By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequence and to bind to SH3 domains in one of two pseudosymmetrical orientations, class I and class II. The class I sites have the consensus sequence ZP(L/P)PP psi P whereas the class II consensus is PP psi PPZ (where psi is a hydrophobic residue and Z is a SH3 domain-specific residue). We previously showed by M13 phage display that the Src, Fyn, Lyn, and phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPP psi P. These results failed to explain the specificity for cellular proteins displayed by SH3 domains in cells. In the current study, class I and class II core ligand sequences were displayed on the surface of bacteriophage M13 with five random residues placed either N- or C-terminal of core ligand residues. These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and PI3K SH3 domains. By this approach, additional ligand residue preferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides. The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and PI3K SH3 domains showed distinct binding specificities. These results indicate that residues that flank the core binding sequences shared by many SH3 domains are important determinants of SH3 binding affinity and selectivity.

Fine specificity of the antibody response to myelin basic protein in the central nervous system in multiple sclerosis: the minimal B-cell epitope and a model of its features.

T cells, B cells, and antibody are found in the white matter of the central nervous system in multiple sclerosis. The epitope center for the antibody response to human myelin basic protein (MBP) fits precisely the minimal epitope Pro85-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro96 for that reported for HLA DR2b (DRB1*1501)-restricted T cells that recognize MBP [Wucherpfenning, K.W., Sette, A., Southwood, S., Oseroff, C., Matsui, M., Strominger, J. & Hafler, D. (1994) J. Exp. Med. 179, 279-290], and overlaps with the reported DR2a-restricted epitope for T cells reactive to MBP [Martin, R., Howell, M. D., Jaraquemada, D., Furlage, M., Richert, J., Brostoff, S., Long, E. O., McFarlin, D. E. & McFarland, H. F. (1991) J. Exp. Med. 173, 19-24]. We describe a molecular model of this epitope.

Expression and signaling specificity of the IFNAR chain of the type I interferon receptor complex.

The IFNAR chain of the type I interferon (IFN) receptor (IFNIR) undergoes rapid ligand-dependent tyrosine phosphorylation and acts as a species-specific transducer for type I IFN action. Using the vaccinia/T7 expression system to amplify IFNAR expression, we found that human HeLa-S3 cells transiently express high levels of cell surface IFNAR chains (approximately 250,000 chains per cell). Metabolic labeling and immunoblot analysis of transfected HeLa cells show that the IFNAR chain is initially detected as 65-kDa and 98-kDa precursors, and then as the 130-kDa mature protein. Due to variation in N-glycosylation, the apparent molecular mass of the mature IFNAR chain varies from 105 to 135 kDa in different cells. IFNIR structure was characterized in various human cell lines by analyzing 125I-labeled IFN cross-linked complexes recognized by various antibodies against IFNIR subunits and JAK protein-tyrosine kinases. Precipitation of cross-linked material from Daudi cells with anti-IFNAR antibodies showed that IFNAR was present in a 240-kDa complex. Precipitation of cross-linked material from U937 cells with anti-TYK2 sera revealed a 240-kDa complex, which apparently did not contain IFNAR and was not present in IFN-resistant HEC1B cells. The tyrosine phosphorylation and down-regulation of the IFNAR chain were induced by type I IFN in several human cell lines of diverse origins but not in HEC1B cells. However, of type I IFNs, IFN-beta uniquely induced the tyrosine phosphorylation of a 105-kDa protein associated with the IFNAR chain in two lymphoblastoid cell lines (Daudi and U266), demonstrating the specificity of transmembrane signaling for IFN-beta and IFN-alpha through the IFNAR chain.

Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering.

The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired. The ability to modify TE specificity should allow the production of additional "designer oils" in genetically engineered plants.

Specificity of dimer formation in tropomyosins: influence of alternatively spliced exons on homodimer and heterodimer assembly.

Tropomyosins consist of nearly 100% alpha-helix and assemble into parallel and in-register coiled-coil dimers. In vitro it has been established that nonmuscle as well as native muscle tropomyosins can form homodimers. However, a mixture of muscle alpha and beta tropomyosin subunits results in the formation of the thermodynamically more stable alpha/beta heterodimer. Although the assembly preference of the muscle tropomyosin heterodimer can be understood thermodynamically, the presence of multiple tropomyosin isoforms expressed in nonmuscle cells points toward a more complex principle for determining dimer formation. We have investigated the dimerization of rat tropomyosins in living cells by the use of epitope tagging with a 16-aa sequence of the influenza hemagglutinin. Employing transfection and immunoprecipitation techniques, we have analyzed the dimers formed by muscle and nonmuscle tropomyosins in rat fibroblasts. We demonstrate that the information for homo- versus heterodimerization is contained within the tropomyosin molecule itself and that the information for the selectivity is conferred by the alternatively spliced exons. These results have important implications for models of the regulation of cytoskeletal dynamics.

Redirecting the specificity of ubiquitination by modifying ubiquitin-conjugating enzymes.

Depletion of specific cellular proteins is a powerful tool in biological research and has many medical and agricultural benefits. In contrast to genetic methods currently available to attenuate protein levels, we describe an alternative approach that redirects the ubiquitin-dependent proteolytic pathway to facilitate specific proteolytic removal. Degradation via the ubiquitin pathway requires the prior attachment of multiple ubiquitins to the target protein. This attachment is accomplished, in part, by a family of enzymes designated E2s (or ubiquitin-conjugating enzymes), some of which use domains near their C termini for target recognition. Here, we demonstrate that E2 target recognition can be redefined by engineering E2s to contain appropriate protein-binding peptides fused to their C termini. In five dissimilar examples, chimeric E2s were created that recognized and ubiquitinated their respective binding partners with high specificity. We also show that ubiquitination of one protein targeted by this method led to its ATP-dependent degradation in vitro. Thus, by exploiting interacting domains derived from natural and synthetic ligands, it may be possible to design E2s capable of directing the selective removal of many intracellular proteins.

RNA polymerase II/III transcription specificity determined by TATA box orientation.

The TATA box sequence in eukaryotes is located about 25 bp upstream of many genes transcribed by RNA polymerase II (Pol II) and some genes transcribed by RNA polymerase III (Pol III). The TATA box is recognized in a sequence-specific manner by the TATA box-binding protein (TBP), an essential factor involved in the initiation of transcription by all three eukaryotic RNA polymerases. We have investigated the recognition of the TATA box by the Pol II and Pol III basal transcription machinery and its role in establishing the RNA polymerase specificity of the promoter. Artificial templates were constructed that contained a canonical TATA box as the sole promoter element but differed in the orientation of the 8-bp TATA box sequence. As expected, Pol II initiated transcription in unfractionated nuclear extracts downstream of the "forward" TATA box. In distinct contrast, transcription that initiated downstream of the "reverse" TATA box was carried out specifically by Pol III. Importantly, this effect was observed regardless of the source of the DNA either upstream or downstream of the TATA sequence. These findings suggest that TBP may bind in opposite orientations on Pol II and Pol III promoters and that opposite, yet homologous, surfaces of TBP may be utilized by the Pol II and Pol III basal machinery for the initiation of transcription.