Recombinant alpha and beta subunits of M.AquI constitute an active DNA methyltransferase

AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed alpha and beta) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the alpha and beta subunits of M.AquI into expression vectors. The overexpressed His-fusion alpha and beta subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The alpha and beta subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-Lmethionine to DNA is reconstituted. We also showed that the beta subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the alpha subunit     

Sequence-specific DNA binding by the MspI DNA methyltransferase.

The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the fist C-residue. We have investigated the sequence-specific DNA-binding properties of M.MspI under equilibrium conditions, using gel-mobility shift assays and DNasel footprinting. M.MspI binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methionine as well as the analogues, sinefungin and S-adenosyl-L-homocysteine. In the presence of S-adenosyl-L-homocysteine, M.MspI shows the highest binding affinity to DNA containing a hemimethylated recognition sequence (Kd = 3.6 x 10(-7) M), but binds less well to unmethylated DNA (Kd = 8.3 x 10(-7) M). Surprisingly it shows specific, although poor, binding to fully methylated DNA (Kd = 4.2 x 10(-6) M). M.MspI binds approximately 5-fold more tightly to DNA containing its recognition sequence, CCGG, than to nonspecific sequences in the absence of cofactors. In the presence of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine or sinefungin the discrimination between specific and non-specific sequences increases up to 100-fold. DNasel footprinting studies indicate that 16 base pairs of DNA are covered by M.MspI, with the recognition sequence CCGG located asymmetrically within the footprint.     

Functional studies of the small subunit of EcoHK31I DNA methyltransferase

EcoHK31I DNA methyltransferase recognizes the sequence 5'-YGGCCR-3' and adds a methyl group to the fifth position of the internal cytosine to protect the DNA from cleavage by its cognate endonuclease. M.EcoHK31I is composed of polypeptides alpha and beta. Polypeptide beta only contains the conserved IX motif of the C5-MTase family, and provides a unique example to show that this motif alone may be dislocated to another polypeptide. By electromobility shift assay, we found that the alpha/beta complex recognizes specific oligonucleotide substrates. Polypeptide alpha formed aggregates with DNA, while polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists in the proper binding of polypeptide alpha to DNA substrate. The complex of polypeptide alpha and a polypeptide beta variant with an N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive methyltransferase. The dissociation equilibrium constant (Kd) of the alpha/beta complex was 56.4 nM, while the Kd value for the alpha/deltaN46-polypeptide beta complex was increased approximately 95-fold, caused by a drastic decrease in dissociate rate constant (kd) and an increase in the association rate constant (ka). This indicates that the N-terminal region of polypeptide beta takes part in subunit interaction, while the C-terminal region is involved in DNA binding.     

Cloning, expression, and purification of a thermostable nonhomodimeric restriction enzyme, BslI

BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The BslI restriction-modification system from Bacillus species was cloned and expressed in Escherichia coli. The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), the bslIRalpha gene, and the bslIRbeta gene. The alpha and beta subunits of BslI can be expressed independently in E. coli in the absence of BslI methylase (M.BslI) protection. BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (alphabeta), heterotetramers (alpha(2)beta(2)), and possibly oligomers in solution. Two beta subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer BslI complex (alpha(2)beta(2)). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is alpha(2)beta(2). M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the beta group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 in BslI sites in the first or second cytosine are resistant to BslI digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion. Two putative zinc fingers are found in the alpha subunit of BslI endonuclease.     

Organization of the multiple coenzymes and subunits and role of the covalent flavin link in the complex heterotetrameric sarcosine oxidase

Heterotetrameric (alphabetagammadelta) sarcosine oxidase from Corynebacterium sp. P-1 (cTSOX) contains noncovalently bound FAD and NAD(+) and covalently bound FMN, attached to beta(His173). The beta(His173Asn) mutant is expressed as a catalytically inactive, labile heterotetramer. The beta and delta subunits are lost during mutant enzyme purification, which yields a stable alphagamma complex. Addition of stabilizing agents prevents loss of the delta but not the beta subunit. The covalent flavin link is clearly a critical structural element and essential for TSOX activity or preventing FMN loss. The alpha subunit was expressed by itself and purified by affinity chromatography. The alpha and beta subunits each contain an NH(2)-terminal ADP-binding motif that could serve as part of the binding site for NAD(+) or FAD. The alpha subunit and the alphagamma complex were each found to contain 1 mol of NAD(+) but no FAD. Since NAD(+) binds to alpha, FAD probably binds to beta. The latter could not be directly demonstrated since it was not possible to express beta by itself. However, FAD in TSOX from Pseudomonas maltophilia (pTSOX) exhibits properties similar to those observed for the covalently bound FAD in monomeric sarcosine oxidase and N-methyltryptophan oxidase, enzymes that exhibit sequence homology with beta. A highly conserved glycine in the ADP-binding motif of the alpha(Gly139) or beta(Gly30) subunit was mutated in an attempt to generate NAD(+)- or FAD-free cTSOX, respectively. The alpha(Gly139Ala) mutant is expressed only at low temperature (t(optimum) = 15 degrees C), but the purified enzyme exhibited properties indistinguishable from the wild-type enzyme. The much larger barrier to NAD(+) binding in the case of the alpha(Gly139Val) mutant could not be overcome even by growth at 3 degrees C, suggesting that NAD(+) binding is required for TSOX expression. The beta(Gly30Ala) mutant exhibited subunit expression levels similar to those of the wild-type enzyme, but the mutation blocked subunit assembly and covalent attachment of FMN, suggesting that both processes require a conformational change in beta that is induced upon FAD binding. About half of the covalent FMN in recombinant preparations of cTSOX or pTSOX is present as a reversible covalent 4a-adduct with a cysteine residue. Adduct formation is not prevented by mutating any of the three cysteine residues in the beta subunit of cTSOX to Ser or Ala. Since FMN is attached via its 8-methyl group to the beta subunit, the FMN ring must be located at the interface between beta and another subunit that contains the reactive cysteine residue.     

DNA duplexes containing photoactive derivatives of 2'-deoxyuridine as photocrosslinking probes for EcoRII DNA methyltransferase-substrate interaction

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.     

Covalent binding of 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) to the isolated alpha and beta subunits and the alpha 3 beta 3 core complex of TF1. Covalent binding of BzATP prevents association of alpha and beta subunits and induces dissociation of the alpha 3 beta 3 core complex.

Binding of the photoreactive ATP analog, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP), to the isolated alpha and beta subunits of TF1 and to the alpha 3 beta 3 "core" complex of the holoenzyme is described. About 1 mol of BzATP/mol of subunit was incorporated to isolated alpha and beta subunits. The incorporation of BzATP was prevented by ATP. Covalent binding of BzATP to the alpha subunit was in general somewhat lower than that observed with the beta subunit. No complex was formed upon mixing of either of the modified subunits with the complementary nontreated subunits. Covalent binding of 3 mol of BzATP/alpha 3 beta 3 complex completely inhibited ATPase activity and resulted in the dissociation of the complex. The labeled nucleotide analog was specifically incorporated into the beta subunit of the complex. The holoenzyme TF1, in contrast to the core complex, did not dissociate to the individual subunits upon covalent binding of BzATP. These results are discussed in relation to the location of the catalytic nucleotide binding site(s) and the conformation stability of the alpha 3 beta 3 core complex of TF1.     

Domain structure and subunit interactions in the type I DNA methyltransferase M.EcoR124I.

The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed independently of HsdM. Two soluble fragments of the HsdS subunit have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S3) comprising the N-terminal target recognition domain together with the central conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 subunit exists principally as a dimer of 50 kDa. Gel retardation and competition assays show that both S3 and S11 are able to bind to HsdM, each with a subunit stoichiometry of 1:1. The tetrameric complex (S3/HsdM)(2) is required for effective DNA binding. Cooperative binding is observed and at low enzyme concentration, the multisubunit complex dissociates, leading to a loss of DNA binding activity. The (S3/HsdM)(2) complex is able to bind to both the EcoR124I DNA recognition sequence GAAN(6)RTCG and a symmetrical DNA sequence GAAN(7)TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease III footprinting of the (S3/HsdM)(2) -DNA complex indicates that 29 nucleotides are protected on each strand, corresponding to a region 8 bp on both the 3' and 5' sides of the recognition sequence bound by the (S3/HsdM)(2) complex.     

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA–M.HhaI–AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA.     

Isolation of a deoxycytidylate methyl transferase capable of protecting DNA uniquely against cleavage by endonuclease R.Aqu I (isoschizomer of Ava I).

A sequence-specific modification methylase (M.AquI) was isolated and purified from Agmenellum quadruplicatum (Synechococcus PCC 7002). This enzyme uniquely methylates the deoxycytidylate residue in the sequence *CYCGRG indicated by the asterisk. It was shown to protect DNA against cleavage by restriction endonucleases AvaI, SmaI and XhoI, which recognize the sequences CYCGRG, CCCGGG, and CTCGAG, respectively.     

Molecular basis of ligand recognition by integrin alpha5beta 1. II. Specificity of arg-gly-Asp binding is determined by Trp157 OF THE alpha subunit

Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the alpha subunit in determining ligand specificity. Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-alpha(5) monoclonal antibody (mAb) 16 to alpha(5)beta(1). In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the alpha(5) subunit that contains Ser(156)-Trp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation also abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose that Trp(157) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of alpha(5)beta(1) for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the beta(1) subunit, our results suggest that Trp(157) of alpha(5) must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1).     

Molecular basis of ligand recognition by integrin alpha 5beta 1. I. Specificity of ligand binding is determined by amino acid sequences in the second and third NH2-terminal repeats of the alpha subunit

The NH(2)-terminal portion (putative ligand-binding domain) of alpha subunits contains 7 homologous repeats, the last 3 or 4 of which possess divalent cation binding sequences. These repeats are predicted to form a seven-bladed beta-propeller structure. To map ligand recognition sites on the alpha(5) subunit we have taken the approach of constructing and expressing alpha(V)/alpha(5) chimeras. Although the NH(2)-terminal repeats of alpha(5) and alpha(V) are >50% identical at the amino acid level, alpha(5)beta(1) and alpha(V)beta(1) show marked differences in their ligand binding specificities. Thus: (i) although both integrins recognize the Arg-Gly-Asp (RGD) sequence in fibronectin, the interaction of alpha(5)beta(1) but not of alpha(V)beta(1) with fibronectin is strongly dependent on the "synergy" sequence Pro-His-Ser-Arg-Asn; (ii) alpha(5)beta(1) binds preferentially to RGD peptides in which RGD is followed by Gly-Trp (GW) whereas alpha(V)beta(1) has a broader specificity; (iii) only alpha(5)beta(1) recognizes peptides containing the sequence Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Therefore, amino acid residues involved in ligand recognition by alpha(5)beta(1) can potentially be identified in gain-of-function experiments by their ability to switch the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1). By introducing appropriate restriction enzyme sites, or using site-directed mutagenesis, parts of the NH(2)-terminal repeats of alpha(V) were replaced with the corresponding regions of the alpha(5) subunit. Chimeric subunits were expressed on the surface of Chinese hamster ovary-B2 cells (which lack endogenous alpha(5)) as heterodimers with hamster beta(1). Stable cell lines were generated and tested for their ability to attach to alpha(5)beta(1)-selective ligands. Our results demonstrate that: (a) the first three NH(2)-terminal repeats contain the amino acid sequences that determine ligand binding specificity and the same repeats include the epitopes of function blocking anti-alpha subunit mAbs; (b) the divalent cation-binding sites (in repeats 4-7) do not confer alpha(5)beta(1)- or alpha(V)beta(1)-specific ligand recognition; (c) amino acid residues Ala(107)-Tyr(226) of alpha(5) (corresponding approximately to repeats 2 and 3) are sufficient to change all the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1); (d) swapping a small part of a predicted loop region of alpha(V) with the corresponding region of alpha(5) (Asp(154)-Ala(159)) is sufficient to confer selectivity for RGDGW and the ability to recognize RRETAWA.     

The HsdS polypeptide of the Type IC restriction enzyme Eco R124 is a sequence-specific DNA-binding protein

The HsdS and HsdM polypeptides of the type IC restriction enzyme EcoR124 have been purified independently and used in a set of gel retardation experiments to determine the minimum requirements for sequence-specific recognition of DNA by this enzyme. The HsdS polypeptide alone is able to bind to DNA in a sequence-specific manner. In addition, whilst the presence of the HsdM polypeptide gives rise to a stimulation of DNA binding by the HsdS subunit it is not clear whether, under the conditions of the experiments reported here, the HsdS subunit maintains the same interactions with the HsdM subunits observed in the absence of DNA.     

The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities.

The integration host factor (IHF) of Escherichia coli is a small, basic protein that is required for lambda site-specific recombination and a variety of cellular processes. It is composed of two subunits, alpha and beta, that are encoded by the himA and hip (himD) genes, respectively. IHF is a sequence-specific DNA-binding protein and bends the DNA when it binds. We have used the bacteriophage P22-based challenge phage selection to isolate suppressor mutants with altered, expanded DNA binding specificities. The suppressors were isolated by selecting mutants that recognize variants of the phage lambda H'IHF recognition site. Two of the mutants recognize both the wild-type and a single variant site and contain amino acid substitutions at positions 64 (Pro to Leu) or 65 (Lys to Ser) of the alpha subunit. These substitutions are in a region of the protein that is predicted to contain a flexible arm that interacts with DNA. Three other mutants, which recognize the wild-type and a different variant site, contain amino acid substitutions at position 44 (Glu to Lys, Val or Gly) of the beta subunit. These substitutions are in the middle of a predicted beta-strand of the subunit. We discuss the possible mechanisms of suppression by the mutants in terms of a model of the IHF-DNA complex proposed by Yang and Nash [Cell, 57, 869-880 (1989)].     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Insulin-dependent intermolecular subunit communication between isolated alpha beta heterodimeric insulin receptor complexes

The dissociation of the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex into an alpha beta heterodimeric state was found to occur in a pH- and dithiothreitol (DTT)-dependent manner. Formation of the alpha beta heterodimeric complex, under conditions which preserved tracer insulin binding and protein kinase activities (pH 8.75 for 25 min followed by 2.0 mM DTT for 5 min) occurred with an approximate 50% efficiency. The resulting nondissociated alpha 2 beta 2 heterotetrameric complexes could then be separated effectively by Bio-Gel A-1.5m gel filtration chromatography at neutral pH. The isolated DTT-treated but nondissociated alpha 2 beta 2 heterotetrameric complex was resistant to any further dissociation by a second round of DTT and alkaline pH treatment, whereas the isolated alpha beta heterodimeric complex was stable to spontaneous reassociation for at least 72 h at pH 7.60. Kinetic analyses of the insulin receptor protein kinase activity demonstrated that the insulin stimulation of glutamic acid:tyrosine (4:1) synthetic polymer phosphorylation for both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes occurred via an increase in Vmax without any significant change in Km. Examination of beta subunit autophosphorylation of the alpha beta heterodimeric complex, in the presence but not in the absence of insulin, demonstrated the appearance of the covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Further, the initial rate of insulin-stimulated beta subunit autophosphorylation in the isolated alpha beta heterodimeric complex occurred in a dilution-dependent (intermolecular) manner. These data demonstrate that the isolated alpha beta heterodimeric insulin receptor complex is fully capable of expressing insulin-dependent activation of the beta subunit protein kinase domain with the covalent reassociation of the alpha beta heterodimeric complex into an alpha 2 beta 2 heterotetrameric disulfide-linked state.     

Subunit interactions inhibit the binding of beta nerve growth factor to receptors on embryonic chick sensory neurons

The binding of the beta nerve growth factor subunit in the 7 S nerve growth factor complex to the two nerve growth factor receptors on chick embryo dorsal root ganglion cells was investigated. Under conditions where the 7 S nerve growth factor complex is maximally stable (in the presence of excess alpha and gamma subunits and of 20 to 30 microM zinc ion), no binding to either receptor was detectable. The time course of the decrease in the binding of beta nerve growth factor to the receptors upon addition of alpha and gamma subunits and zinc ion paralleled the formation of the 7 S complex. Addition of alpha and gamma subunits and zinc ion to the bisdes-arginine118-nerve growth factor, which does not re-form the 7 S complex, failed to inhibit the binding of the derivative to either receptor. While the alpha subunit alone had no effect on beta nerve growth factor binding, the gamma subunit decreased its binding in proportion to the amount of complex formed between these two subunits, suggesting that the beta . gamma complex, like the 7 S complex, does not bind to nerve growth factor receptors.     

Mechanisms of specific and nonspecific binding of architectural proteins in prokaryotic gene regulation

IHF and HU are small basic proteins of eubacteria that bind as homodimers to double-stranded DNA and bend the duplex to promote architectures required for gene regulation. These architectural proteins share a common alpha/beta fold but exhibit different nucleic acid binding surfaces and distinct functional roles. With respect to DNA-binding specificity, for example, IHF is sequence specific, while HU is not. We have employed Raman difference spectroscopy and gel mobility assays to characterize the molecular mechanisms underlying such differences in DNA recognition. Parallel studies of solution complexes of IHF and HU with the same DNA nonadecamer (5' --> 3' sequence: TC TAAGTAGTTGATTCATA, where the phage lambda H1 consensus sequence of IHF is underlined) show the following. (i) The structure of the targeted DNA site is altered much more dramatically by IHF than by HU binding. (ii) In the IHF complex, the structural perturbations encompass both the sugar-phosphate backbone and the bases of the consensus sequence, whereas only the DNA backbone is altered by HU binding. (iii) In the presence of excess protein, complexes of order higher than 1 dimer per duplex are detected for HU:DNA, though not for IHF:DNA. The results differentiate structural motifs of IHF:DNA and HU:DNA solution complexes, provide Raman signatures of prokaryotic sequence-specific and nonspecific recognition, and suggest that the architectural role of HU may involve the capability to recruit additional binding partners to even relatively short DNA sequences.