Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B.subtilis RNase P protein

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5′-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain–P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of ∼0.1 µM. The C-domain–P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (∼0.9 min–1 at pH 7.8) and substrates with a hairpin–loop 3′ to the cleavage site (∼40 min–1). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10–500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem–loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.

Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli.

MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions of a lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.

Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.

We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2.

The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.

Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system.

Many biological processes rely upon protein-protein interactions. Hence, detailed analysis of these interactions is critical for their understanding. Due to the complexities involved, genetic approaches are often needed. In yeast and phage, genetic characterizations of protein complexes are possible. However, in multicellular organisms, such characterizations are limited by the lack of powerful selection systems. Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles. The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative selection for mutations that affect interaction, followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein (two-step selection). We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1. The mutations obtained identified a putative helix in the marked box, a region conserved among E2F family members, as an important determinant for interaction. This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system.

Linker histones affect patterns of digestion of supercoiled plasmids by single-strand-specific nucleases.

The effect of histone H1 binding on the cleavage of superhelical plasmids by single-strand-specific nucleases was investigated. Mapping of P1 cleavage sites in pBR322, achieved by EcoRI digestion after the original P1 attack, showed an intriguing phenomenon: preexisting susceptible sites became "protected," whereas some new sites appeared at high levels of H1. Similar results were obtained with another single-strand-specific nuclease, S1. Disappearance of cutting at preexisting sites and appearance of new sites was also observed in a derivative plasmid that contains a 36-bp stretch of alternating d(AT) sequence that is known to adopt an altered P1-sensitive conformation. On the other hand, H1 titration of a dimerized version of the d(AT)18-containing plasmid led to protection of all preexisting sites except the d(AT)18 inserts, which were still cut even at high H1 levels; in this plasmid no new sites appeared. The protection of preexisting sites is best explained by long-range effects of histone H1 binding on the superhelical torsion of the plasmid. The appearance of new sites, on the other hand, probably also involves a local effect of stabilization of specific sequences in Pl-sensitive conformation, due to direct H1 binding to such sequences. That such binding involves linker histone N- and/or C-terminal tails is indicated by the fact that titration with the globular domain of H5, while causing disappearance of preexisting sites, does not lead to the appearance of any new sites.

Three quaternary structures for a single protein.

The structure of a multisubunit protein (immunoglobulin light chain) was solved in three crystal forms, differing only in the solvent of crystallization. The three structures were obtained at high ionic strength and low pH, high ionic strength and high pH, and low ionic strength and neutral pH. The three resulting "snapshots" of possible structures show that their variable-domain interactions differ, reflecting their stabilities under specific solvent conditions. In the three crystal forms, the variable domains had different rotational and translational relationships, whereas no alteration of the constant domains was found. The critical residues involved in the observed effect of the solvent are tryptophans and histidines located between the two variable domains in the dimeric structure. Tryptophan residues are commonly found in interfaces between proteins and their subunits, and histidines have been implicated in pH-dependent conformation changes. The quaternary structure observed for a multisubunit protein or protein complex in a crystal may be influenced by the interactions of the constituents within the molecule or complex and/or by crystal packing interactions. The comparison of buried surface areas and hydrogen bonds between the domains forming the molecule and between the molecules forming the crystals suggest that, for this system, the interactions within the molecule are most likely the determining factors.

The embryonic transcription factor stage specific activator protein contains a potent bipartite activation domain that interacts with several RNA polymerase II basal transcription factors.

Stage specific activator protein (SSAP) is a member of a newly discovered class of transcription factors that contain motifs more commonly found in RNA-binding proteins. Previously, we have shown that SSAP specifically binds to its recognition sequence in both the double strand and the single strand form and that this DNA-binding activity is localized to the N-terminal RNA recognition motif domain. Three copies of this recognition sequence constitute an enhancer element that is directly responsible for directing the transcriptional activation of the sea urchin late histone H1 gene at the midblastula stage of embryogenesis. Here we show that the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain that can function in a variety of mammalian cell lines. This activity is as much as 3 to 5 times stronger than VP16 at activating transcription and requires a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. We present evidence that SSAP's activation domain shares targets that are also necessary for activation by E1a and VP16. Finally, SSAP's activation domain is found to participate in specific interactions in vitro with the basal transcription factors TATA-binding protein, TFIIB, TFIIF74, and dTAF(II) 110.

Activating transcription from single stranded DNA.

Sequence specific regulators of eukaryotic gene expression, axiomatically, act through double stranded DNA targets. Proteins that recognize DNA cis-elements as single strands but for which compelling evidence has been lacking to indicate in vivo involvement in transcription are orphaned in this scheme. We sought to determine whether sequence specific single strand binding proteins can find their cognate elements and modify transcription in vivo by studying heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the single stranded sequence (CCCTCCCCA; CT-element) of the human c-myc gene in vitro. To monitor its DNA binding in vivo, the ability of hnRNP K to activate a reporter gene was amplified by fusion with the VP16 transactivation domain. This chimeric protein was found to transactivate circular but not linear CT-element driven reporters, suggesting that hnRNP K recognizes a single strand region generated by negative supercoiling in circular plasmid. When CT-elements were engineered to overlap with lexA operators, addition of lexA protein, either in vivo or in vitro, abrogated hnRNP K binding most likely by preventing single strand formation. These results not only reveal hnRNP K to be a single strand DNA binding protein in vivo, but demonstrate how a segment of DNA may modify the transcriptional activity of an adjacent gene through the interconversion of duplex and single strands.

Molecular cloning of a phosphotyrosine-independent ligand of the p56lck SH2 domain.

A novel human cDNA encoding a cytosolic 62-kDa protein (p62) that binds to the Src homology 2 (SH2) domain of p56lck in a phosphotyrosine-independent manner has been cloned. The cDNA is composed of 2074 nucleotides with an open reading frame encoding 440 amino acids. Northern analysis suggests that p62 is expressed ubiquitously in all tissues examined. p62 is not homologous to any known protein in the data base. However, it contains a cysteine-rich region resembling a zinc finger motif, a potential G-protein-binding region, a PEST motif, and several potential phosphorylation sites. Using T7-epitope tagged p62 expression in HeLa cells, the expressed protein was shown to bind to the lck SH2 domain. Deletion of the N-terminal 50 amino acids abolished binding, but mutagenesis of the single tyrosine residue in this region had no effect on binding. Thus, the cloned cDNA indeed encodes the p62 protein, which is a phosphotyrosine-independent ligand for the lck SH2 domain. Its binding mechanism is unique with respect to binding modes of other known ligands for SH2 domains.

Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family.

The EVI1 gene, located at chromosome band 3q26, is overexpressed in some myeloid leukemia patients with breakpoints either 5' of the gene in the t(3;3)(q21;q26) or 3' of the gene in the inv(3)(q21q26). EVI1 is also expressed as part of a fusion transcript with the transcription factor AML1 in the t(3;21)(q26;q22), associated with myeloid leukemia. In cells with t(3;21), additional fusion transcripts are AML1-MDS1 and AML1-MDS1-EVI1. MDS1 is located at 3q26 170-400 kb upstream (telomeric) of EVI1 in the chromosomal region in which some of the breakpoints 5' of EVI1 have been mapped. MDS1 has been identified as a single gene as well as a previously unreported exon(s) of EVI1 We have analyzed the relationship between MDS1 and EVI1 to determine whether they are two separate genes. In this report, we present evidence indicating that MDS1 exists in normal tissues both as a unique transcript and as a normal fusion transcript with EVI1, with an additional 188 codons at the 5' end of the previously reported EVI1 open reading frame. This additional region has about 40% homology at the amino acid level with the PR domain of the retinoblastoma-interacting zinc-finger protein RIZ. These results are important in view of the fact that EVI1 and MDS1 are involved in leukemia associated with chromosomal translocation breakpoints in the region between these genes.

Proposed active site domain in estrogen sulfotransferase as determined by mutational analysis.

Point mutations were selectively introduced into a cDNA for guinea pig estrogen sulfotransferase (gpEST); each construct was then expressed in Chinese hamster ovary K1 cells. The molecular site chosen for study is a conserved GXXGXXK sequence that resembles the P-loop-type nucleotide-binding motif for ATP- and GTP-binding proteins and is located near the C terminus of all steroid and phenol(aryl) sulfotransferases for which the primary structures are known. Preliminary experiments demonstrated that the GXXGXXK motif is essential for binding the activated sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The present study was undertaken to ascertain the relative importance of each individual residue of the motif. While the mutation of a single motif residue had little effect on the interaction between gpEST and PAPS as determined by kinetic analysis and photoaffinity labeling, the mutation of any two residues in concert resulted in an approximate 10-fold increase in the Km for PAPS and reduced photoaffinity labeling. The mutation of all three motif residues resulted in an inactive enzyme and complete loss of photoaffinity labeling. Interestingly, several mutants also displayed a striking effect on the Km for the steroid substrate; double mutants, again, demonstrated greater perturbations (8- to 28-fold increase) than did single mutants. Unexpectedly, whereas the mutation of nonmotif residues had a negligible effect on the Km for PAPS, a marked increase in the Km for the estrogen substrate ( > 30-fold) was noted. On the basis of these findings, it is concluded that the sequence GISGDWKN within the C-terminal domain of gpEST represents a critical component of the active site.

A single amino acid in gamma-aminobutyric acid rho 1 receptors affects competitive and noncompetitive components of picrotoxin inhibition.

A class of bicuculline-insensitive gamma-aminobutyric acid (GABA) receptors, GABAC, has been identified in retina. Several lines of evidence indicate that GABAC receptors are formed partially or wholly of GABA rho subunits. These receptors generate a Cl- current in response to GABA but differ from GABAA receptors in a number of ways. Picrotoxin, widely accepted as a noncompetitive antagonist of GABAA receptors, displays competitive and noncompetitive antagonism of GABAC receptors in perch and bovine retina and GABA rho 1 receptors expressed in Xenopus oocytes. The aim of this study was to identify the molecular basis of the two components of picrotoxin inhibition of GABA rho 1 receptors. By using a domain-swapping and mutagenesis strategy, a difference in picrotoxin sensitivity between rho 1 and rho 2 receptors was localized to a single amino acid in the putative second transmembrane domain. Substitution of this amino acid with residues found in the analogous position in highly picrotoxin-sensitive glycine alpha and GABAA subunits increased the sensitivity of rho 1 mutants 10- to 500-fold. Importantly, the competitive component of picrotoxin inhibition of the rho 1 mutant receptors was almost eliminated. These findings demonstrate that an amino acid in the putative channel domain of GABA rho 1 receptors influences picrotoxin sensitivity and mediates agonist binding by an allosteric mechanism.

Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA.

Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD) complexed to a single consensus pentamer sequence and of the isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered. To better understand these relationships, we have examined the binding of the p53DBD to two biologically important full-response elements (the WAF1 and ribosomal gene cluster sites) by using DNA circularization and analytical ultracentrifugation. We show that the p53DBD binds DNA strongly and cooperatively with p53DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the mean apparent Kd is (8.3 +/- 1.4) x 10(-8) M, and no intermediate species of lower stoichiometries can be detected. We show further that complex formation induces an axial bend of at least 60 degrees in both response elements. These results, taken collectively, demonstrate that p53DBD possesses the ability to direct the formation of a tight nucleoprotein complex having the same 4:1 DNA-binding stoichiometry as wild-type p53 which is accompanied by a substantial conformational change in the response-element DNA. This suggests that the p53DBD may play a role in the tetramerization function of p53. A possible role in this regard is proposed.

Mutational analysis of phytochrome B identifies a small COOH-terminal-domain region critical for regulatory activity.

Overexpression of phytochrome B (phyB) in transgenic Arabidopsis results in enhanced deetiolation in red light. To define domains of phyB functionally important for its regulatory activity, we performed chemical mutagenesis of a phyB-overexpressing line and screened for phenotypic revertants in red light. Four phyB-transgene-linked revertants that retain parental levels of full-length, dimeric, and spectrally normal overexpressed phyB were identified among 101 red-light-specific revertants. All carry single amino acid substitutions in the transgene-encoded phyB that reduce activity by 40- to 1000-fold compared to the nonmutagenized parent. The data indicate that the mutant molecules are fully active in photosignal perception but defective in the regulatory activity responsible for signal transfer to downstream components. All four mutations fall within a 62-residue region in the COOH-terminal domain of phyB, with two independent mutations occurring in a single amino acid, Gly-767. Accumulating evidence indicates that the identified region is a critical determinant in the regulatory function of both phyB and phyA.