beta-Lactamase binds to GroEL in a conformation highly protected against hydrogen/deuterium exchange.

Escherichia coli RTEM beta-lactamase reversibly forms a stable complex with GroEL, devoid of any enzymatic activity, at 48 degrees C. When beta-lactamase is diluted from this complex into denaturant solution, its unfolding rate is identical to that from the native state, while the unfolding rate from the molten globule state is too fast to be measured. Electrospray mass spectrometry shows that the rate of proton exchange in beta-lactamase in the complex at 48 degrees C is slower than in the absence of GroEL at the same temperature, and resembles the exchange of the native state at 25 degrees C. Similarly, the final number of protected deuterons is higher in the presence of GroEL than in its absence. We conclude that, for beta-lactamase, a state with significant native structure is bound to GroEL. Thus, different proteins are recognized by GroEL in very different states, ranging from totally unfolded to native-like, and this recognition may depend on which state can provide sufficient accessible hydrophobic amino acids in a suitably clustered arrangement. Reversible binding of native-like states with hydrophobic patches may be an important property of GroEL to protect the cell from aggregating protein after heat-shock.

Cloning of human acetyl-CoA carboxylase-beta and its unique features.

Acetyl-CoA carboxylase, which has a molecular mass of 265 kDa (ACC-alpha), catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids. In this study we report the complete amino acid sequence and unique features of an isoform of ACC with a molecular mass of 275 kDa (ACC-beta), which is primarily expressed in heart and skeletal muscles. In these tissues, ACC-beta may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis. ACC-beta contains an amino acid sequence at the N terminus which is about 200 amino acids long and may be uniquely related to the role of ACC-beta in controlling carnitine palmitoyltransferase I activity and fatty acid oxidation by mitochondria. If we exclude this unique sequence at the N terminus the two forms of ACC show about 75% amino acid identity. All of the known functional domains of ACC are found in the homologous regions. Human ACC-beta cDNA has an open reading frame of 7,343 bases, encoding a protein of 2,458 amino acids, with a calculated molecular mass of 276,638 Da. The mRNA size of human ACC-beta is approximately 10 kb and is primarily expressed in heart and skeletal muscle tissues, whereas ACC-alpha mRNA is detected in all tissues tested. A fragment of ACC-beta cDNA was expressed in Escherichia coli and antibodies against the peptide were generated to establish that the cDNA sequence that we cloned is that for ACC-beta.

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma). The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.

Betidamino acids: versatile and constrained scaffolds for drug discovery.

Betidamino acids (a contraction of "beta" position and "amide") are N'-monoacylated (optionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'acyl/alkyl group may mimic naturally occurring amino acid side chains or introduce novel functionalities. Betidamino acids are most conveniently generated on solid supports used for the synthesis of peptides by selective acylation of one of the two amino functions of orthogonally protected aminoglycine(s) to generate the side chain either prior to or after the elongation of the main chain. We have used unresolved Nalpha-tert-butyloxycarbonyl-N'alpha-fluorenylmethoxycarbonyl++ + aminoglycine, and Nalpha-(Nalpha-methyl)-tert-butyloxycarbonyl-N'alpha-fluo renylmethoxycarbonyl aminoglycine as the templates for the introduction of betidamino acids in Acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(A c)-Leu-Ilys-Pro-DAla-NH2, where 2Nal is 2-naphthylalanine, 4Cpa is 4-chlorophenylalanine, 3Pal is 3-pyridylalanine, Aph is 4-aminophenylalanine, and Ilys is Nepsilon-isopropyllysine], a potent gonadotropin-releasing hormone antagonist, in order to test biocompatibility of these derivatives. Diasteremneric peptides could be separated in most cases by reverse-phase HPLC. Biological results indicated small differences in relative potencies (<5-fold) between the D and L nonalkylated betidamino acid-containing Acyline derivatives. Importantly, most betide diastereomers were equipotent with Acyline. In an attempt to correlate structure and observed potency, Ramachandran-type plots were calculated for a series of betidamino acids and their methylated homologs. According to these calculations, betidamino acids have access to a more limited and distinct number of conformational states (including those associated with alpha-helices, beta-sheets, or turn structures), with deeper minima than those observed for natural amino acids.

A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the beta or gamma subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of beta and gamma subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the beta subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.

Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

Periodicity of polar and nonpolar amino acids is the major determinant of secondary structure in self-assembling oligomeric peptides.

The tendency of a polypeptide chain to form alpha-helical or beta-strand secondary structure depends upon local and nonlocal effects. Local effects reflect the intrinsic propensities of the amino acid residues for particular secondary structures, while nonlocal effects reflect the positioning of the individual residues in the context of the entire amino acid sequence. In particular, the periodicity of polar and nonpolar residues specifies whether a given sequence is consistent with amphiphilic alpha-helices or beta-strands. The importance of intrinsic propensities was compared to that of polar/nonpolar periodicity by a direct competition. Synthetic peptides were designed using residues with intrinsic propensities that favored one or the other type of secondary structure. The polar/nonpolar periodicities of the peptides were designed either to be consistent with the secondary structure favored by the intrinsic propensities of the component residues or in other cases to oppose these intrinsic propensities. Characterization of the synthetic peptides demonstrated that in all cases the observed secondary structure correlates with the periodicity of the peptide sequence--even when this secondary structure differs from that predicted from the intrinsic propensities of the component amino acids. The observed secondary structures are concentration dependent, indicating that oligomerization of the amphiphilic peptides is responsible for the observed secondary structures. Thus, for self-assembling oligomeric peptides, the polar/nonpolar periodicity can overwhelm the intrinsic propensities of the amino acid residues and serves as the major determinant of peptide secondary structure.

Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.

Two Drosophila nervous system antigens, Nervana 1 and 2, are homologous to the beta subunit of Na+,K(+)-ATPase.

A nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana (Nrv), has been purified on the basis of reactivity of its carbohydrate epitope(s) with anti-horseradish peroxidase (HRP) antibodies that are specific markers for Drosophila neurons. Anti-Nrv monoclonal antibodies (mAbs), specific for the protein moiety of Nrv, were used to screen a Drosophila embryo cDNA expression library. Three cDNA clones (designated Nrv1, Nrv2.1, and Nrv2.2) were isolated that code for proteins recognized by anti-Nrv mAbs on Western blots. DNA sequencing and Southern blot analyses established that the cDNA clones are derived from two different genes. In situ hybridization to Drosophila polytene chromosomes showed that the cDNA clones map to the third chromosome near 92C-D. Nrv1 and Nrv2.1/2.2 have open reading frames of 309 and 322/323 amino acids, respectively, and they are 43.4% identical at the amino acid level. The proteins deduced from these clones exhibit significant homology in both primary sequence and predicted topology to the beta subunit of Na+,K(+)-ATPase. Immunoaffinity-purified Nrv is associated with a protein (M(r) 100,000) recognized on Western blots by anti-ATPase alpha-subunit mAb. Our results suggest that the Drosophila nervous system-specific antigens Nrv1 and -2 are neuronal forms of the beta subunit of Na+,K(+)-ATPase.

Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.

The N-terminal region (A/B) of rat thyroid hormone receptors alpha 1, beta 1, but not beta 2 contains a strong thyroid hormone-dependent transactivation function.

In this study we have investigated the role of the N-terminal region of thyroid hormone receptors (TRs) in thyroid hormone (TH)-dependent transactivation of a thymidine kinase promoter containing TH response elements composed either of a direct repeat or an inverted palindrome. Comparison of rat TR beta 1 with TR beta 2 provides an excellent model since they share identical sequences except for their N termini. Our results show that TR beta 2 is an inefficient TH-dependent transcriptional activator. The degree of transactivation corresponds to that observed for the mutant TR delta N beta 1/2, which contains only those sequences common to TR beta 1 and TR beta 2. Thus, TH-dependent activation appears to be associated with two separate domains. The more important region, however, is embedded in the N-terminal domain. Furthermore, the transactivating property of TR alpha 1 was also localized to the N-terminal domain between amino acids 19 and 30. Using a coimmunoprecipitation assay, we show that the differential interaction of the N terminus of TR beta 1 and TR beta 2 with transcription factor IIB correlates with the TR beta 1 activation function. Hence, our results underscore the importance of the N-terminal region of TRs in TH-dependent transactivation and suggest that a transactivating signal is transmitted to the general transcriptional machinery via a direct interaction of the receptor N-terminal region with transcription factor IIB.

Functional interactions between the retinoblastoma (Rb) protein and Sp-family members: superactivation by Rb requires amino acids necessary for growth suppression.

The transient expression of the retinoblastoma protein (Rb) regulates the transcription of a variety of growth-control genes, including c-fos, c-myc, and the gene for transforming growth factor beta 1 via discrete promoter sequences termed retinoblastoma control elements (RCE). Previous analyses have shown that Sp1 is one of three RCE-binding proteins identified in nuclear extracts and that Rb functionally interacts with Sp1 in vivo, resulting in the "superactivation" of Sp1-mediated transcription. By immunochemical and biochemical criteria, we report that an Sp1-related transcription factor, Sp3, is a second RCE-binding protein. Furthermore, in transient cotransfection assays, we report that Rb "superactivates" Sp3-mediated RCE-dependent transcription in vivo and that levels of superactivation are dependent on the trans-activator (Sp1 or Sp3) studied. Using expression vectors carrying mutated Rb cDNAs, we have identified two portions of Rb required for superactivation: (i) a portion of the Rb "pocket" (amino acids 614-839) previously determined to be required for physical interactions between Rb and transcription factors such as E2F-1 and (ii) a novel amino-terminal region (amino acids 140-202). Since both of these regions of Rb are targets of mutation in human tumors, our data suggest that superactivation of Sp1/Sp3 may play a role in Rb-mediated growth suppression and/or the induction of differentiation.

The sulfur controller-2 negative regulatory gene of Neurospora crassa encodes a protein with beta-transducin repeats.

The sulfur regulatory system of Neurospora crassa is composed of a set of structural genes involved in sulfur catabolism controlled by a genetically defined set of trans-acting regulatory genes. These sulfur regulatory genes include cys-3+, which encodes a basic region-leucine zipper transcriptional activator, and the negative regulatory gene scon-2+. We report here that the scon-2+ gene encodes a polypeptide of 650 amino acids belonging to the expanding beta-transducin family of eukaryotic regulatory proteins. Specifically, SCON2 protein contains six repeated G beta-homologous domains spanning the C-terminal half of the protein. SCON2 represents the initial filamentous fungal protein identified in the beta-transducin group. Additionally, SCON2 exhibits a specific amino-terminal domain that potentially defines another subfamily of beta-transducin homologs. Expression of the scon-2+ gene has been examined using RNA hybridization and gel mobility-shift analysis. The dependence of scon-2+ expression on CYS3 function and the binding of CYS3 to the scon-2+ promoter indicate the presence of an important control loop within the N. crassa sulfur regulatory circuit involving CYS3 activation of scon-2+ expression. On the basis of the presence of beta-transducin repeats, the crucial role of SCON2 in the signal-response pathway triggered by sulfur limitation may be mediated by protein-protein interactions.