Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly.

Two invariant tryptophan residues on the N-terminal extracellular region of the rat alpha1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA(A) (gamma-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA(A) receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant alpha1 subunits, coexpressed with beta2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [(3)H]muscimol, a GABA site ligand, but no binding to [(35)S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [(3)H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with beta2 and gamma2 polypeptides, the mutant alpha1 polypeptides did not form [(3)H]flunitrazepam binding sites though wild-type alpha1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant alpha1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between alpha and beta subunits, but not of the GABA binding site.     

Carboxyl-terminal sequences critical for inositol 1,4,5-trisphosphate receptor subunit assembly

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of conserved subunits that each contains six transmembrane regions (TMRs) localized near the carboxyl terminus. Receptor subunit assembly into a tetramer appears to be a multideterminant process involving an additive contribution of membrane spanning helices and the short cytosolic carboxyl terminus (residues 2590-2749). Previous studies have shown that of the six membrane-spanning regions in each subunit, the 5th and 6th transmembrane regions, and the carboxyl terminus are strong determinants for assembly. The fifth and sixth TMRs contain numerous beta-branched amino acids that may participate in coiled/coil formation via putative leucine zipper motifs. InsP(3)R truncation mutants were expressed in COS-1 cells and analyzed by sucrose density gradient sedimentation and gel filtration for their ability to assemble. Chemical cross-linking with the homobifunctional reagents sDST or DMS of mammalian and bacterially expressed carboxyl-terminal containing receptor fragments reveals that sequences within the carboxyl terminus confer the formation of subunit dimers. A series of InsP(3) receptor carboxyl-terminal fragments and glutathione S-transferase (GST)/InsP(3)R chimeras were expressed in Escherichia coli and used in an in vitro assay to elucidate the minimal sequence responsible for association of the carboxyl termini into dimers. The results presented here indicate that this minimal sequence is approximately 30 residues in length and is localized between residues 2629 and 2654. These residues are highly conserved between the three InsP(3)R isoforms ( approximately 80% identity) as well as the ryanodine receptor ( approximately 40% identity) and suggest that a conserved assembly motif may exist between the two intracellular receptor families. We propose that assembly of the InsP(3) receptor to a tetramer involves intersubunit interactions mediated through both the membrane-spanning regions and residues 2629-2654 of the carboxyl terminus possibly through the formation of a dimer of dimers.     

Structural and functional characterization of inositol 1,4,5-trisphosphate receptor channel from mouse cerebellum.

The cerebellar inositol 1,4,5-trisphosphate (InsP3) receptor is a high molecular weight glycoprotein abundantly expressed in Purkinje cells. The subunit structure of the InsP3 receptor protein was examined by cross-linking experiments. Agarose-polyacrylamide gel electrophoresis of the cross-linked materials demonstrated that the cerebellar InsP3 receptor protein is composed of four noncovalently bound identical subunits each with a Mr of 320,000 in both purified and microsome-bound states. Chromatography of the purified receptor on a calmodulin-Sepharose column demonstrated a Ca2(+)-dependent interaction of the InsP3 receptor with calmodulin. Photoaffinity labeling of the cerebellar microsomal fraction with [alpha-32P]8-azidoadenosine 5'-triphosphate revealed the presence of ATP-binding site in the InsP3 receptor. Scatchard analysis of the purified InsP3 receptor revealed the Bmax and Kd values for ATP binding of 2.3 pmol/micrograms and 17 microM, respectively. Reconstitution of the purified InsP3 receptor into the planar lipid bilayer indicated channel activity in the purified receptor. It exhibited a calcium conductance (26 pS in 53 mM Ca2+) and sodium conductance (21 pS in 100-500 mM asymmetric Na+ solutions) with permeability ratios of PCa/PTris = 6.3 and PNa/PCl = 5.4. The purified channel was activated with submillimolar ATP in the presence of InsP3 and modified to reach a large conductance state.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

Hydroperoxidase II of Escherichia coli exhibits enhanced resistance to proteolytic cleavage compared to other catalases

Catalase (hydroperoxidase) HPII of Escherichia coli is the largest catalase so far characterized, existing as a homotetramer of 84 kDa subunits. Each subunit has a core structure that closely resembles small subunit catalases, supplemented with an extended N-terminal sequence and compact flavodoxin-like C-terminal domain. Treatment of HPII with trypsin, chymotrypsin, or proteinase K, under conditions of limited digestion, resulted in cleavage of 72-74 residues from the N-terminus of each subunit that created a homotetramer of 76 kDa subunits with 80% of wild-type activity. Longer treatment with proteinase K removed the C-terminal domain, producing a transient 59 kDa subunit which was subsequently cleaved into two fragments, 26 and 32 kDa. The tetrameric structure was retained despite this fragmentation, with four intermediates being observed between the 336 kDa native form and the 236 kDa fully truncated form corresponding to tetramers with a decreasing complement of C-termini (4, 3, 2, and 1). The truncated tetramers retained 80% of wild-type activity. The T(m) for loss of activity during heating was decreased from 85 to 77 degrees C by removal of the N-terminal sequence and to 59 degrees C by removal of the C-terminal domain, revealing the importance of the C-terminal domain in enzyme stability. The sites of cleavage were determined by N- and C-terminal sequencing, and two were located on the surface of the tetramer with a third being exposed by removal of the C-terminal domain.     

Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy

The structure of monomeric human chemokine IL-8 (residues 1–66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1–72), with the main differences being the location of the N-loop (residues 10–22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67–72). NMR was used to analyze the interactions of monomeric IL-8 (1–66) with ND-CXCR1 (residues 1–38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1–72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.     

Single channel function of recombinant type-1 inositol 1,4,5-trisphosphate receptor ligand binding domain splice variants.

In this study we describe the expression and function of the two rat type-1 inositol 1,4,5-trisphosphate receptor (InsP3R) ligand binding domain splice variants (SI+/-/SII+). Receptor protein from COS-1 cells transfected with the type-1 InsP3R expression plasmids (pInsP3R-T1, pInsP3R-T1ALT) or control DNA were incorporated into planar lipid bilayers and the single channel properties of the recombinant receptors were defined. The unitary conductance of the two splice variants were approximately 290 pS with Cs+ as charge carrier and approximately 65 pS with Ca2+ as charge carrier. Both InsP3R expression products consistently behaved like those of the native type-1 receptor isoform isolated from cerebellum in terms of their InsP3, Ca2+, and heparin sensitivity. An InsP3 receptor ligand binding domain truncation lacking the 310 amino-terminal amino acids (pInsP3R-DeltaT1ALT) formed tetrameric complexes but failed to bind InsP3 with high affinity, and did not form functional Ca2+ channels when reconstituted in lipid bilayers. These data suggest that 1) the ligand binding alternative splice site is functionally inert in terms of InsP3 binding and single channel function, and 2) the single channel properties of the expressed recombinant type-1 channel are essentially identical to those of the native channel. This work establishes a foundation from which molecular/biophysical approaches can be used to define the structure-function properties of the InsP3 receptor channel family.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Identification of the bovine gamma-aminobutyric acid type A receptor alpha subunit residues photolabeled by the imidazobenzodiazepine [3H]Ro15-4513

Ligands binding to the benzodiazepine-binding site in gamma-aminobutyric acid type A (GABA(A)) receptors may allosterically modulate function. Depending upon the ligand, the coupling can either be positive (flunitrazepam), negative (Ro15-4513), or neutral (flumazenil). Specific amino acid determinants of benzodiazepine binding affinity and/or allosteric coupling have been identified within GABA(A) receptor alpha and gamma subunits that localize the binding site at the subunit interface. Previous photolabeling studies with [(3)H]flunitrazepam identified a primary site of incorporation at alpha(1)His-102, whereas studies with [(3)H]Ro15-4513 suggested incorporation into the alpha(1) subunit at unidentified amino acids C-terminal to alpha(1)His-102. To determine the site(s) of photoincorporation by Ro15-4513, we affinity-purified ( approximately 200-fold) GABA(A) receptor from detergent extracts of bovine cortex, photolabeled it with [(3)H]Ro15-4513, and identified (3)H-labeled amino acids by N-terminal sequence analysis of subunit fragments generated by sequential digestions with a panel of proteases. The patterns of (3)H release seen after each digestion of the labeled fragments determined the number of amino acids between the cleavage site and labeled residue, and the use of sequential proteolytic fragmentation identified patterns of cleavage sites unique to the different alpha subunits. Based upon this radiochemical sequence analysis, [(3)H]Ro15-4513 was found to selectively label the homologous tyrosines alpha(1)Tyr-210, alpha(2)Tyr-209, and alpha(3)Tyr-234, in GABA(A) receptors containing those subunits. These results are discussed in terms of a homology model of the benzodiazepine-binding site based on the molluscan acetylcholine-binding protein structure.     

Subunit oligomerization, and topology of the inositol 1,4, 5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP(3)R, a series of membrane-spanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membrane-spanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP(3)R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membrane-spanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor's 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization.     

Membrane tethering enables an extracellular domain of the acetylcholine receptor alpha subunit to form a heterodimeric ligand-binding site

The first step of assembly of the nicotinic acetylcholine receptor (AChR) of adult skeletal muscle is the specific association of the alpha subunit with either delta or epsilon subunits to form a heterodimer with a ligand-binding site. Previous experiments have suggested that het erodimer formation in the ER arises from interaction between the luminal, NH2-terminal domains of the subunits. When expressed in COS cells with the delta subunit, however, the truncated NH2-terminal domain of the subunit folded correctly but did not form a heterodimer. Association with the delta subunit occurred only when the NH2-terminal domain was retained in the ER and was tethered to the membrane by its own M1 transmembrane domain, by the transmembrane domain of another protein, or by a glycolipid link. In each case, the ligand-binding sites of the resulting heterodimers were indistinguishable from that formed when the full-length alpha subunit was used. Attachment to the membrane may promote interaction by concentrating or orienting the subunit; alternatively, a membrane-bound factor may facilitate subunit association.     

Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.     

Recombinant interleukin-2 analogs. Dynamic probes for receptor structure.

Interleukin-2 (IL-2) and its receptor complex have become one of the most studied members of a growing family of protein hormones characterized by structural similarities in both ligands and their receptors. Structure-function studies of IL-2 have been complicated by the multimeric nature of its receptor. Two receptor subunits (55- and 75-kDa type I cell surface proteins) can participate to form the high affinity binding site. Although the IL-2 is apparently unique in some respects, similar subunit cooperativity has now been shown to be a common feature for other members of this receptor family. The availability of cell lines expressing the individual IL-2 receptor subunits has allowed detailed analysis of subunit binding characteristics. Results regarding the relationship of molecular recognition at each subunit to the mechanism of ligand binding at the high affinity site, however, have led to different interpretations. In this study we have employed previously prepared C-terminal IL-2 mutant proteins to examine receptor binding at all three classes using a variety of equilibrium and kinetic techniques. These results indicate that the high affinity IL-2 receptor complex includes the p55/p75 heterodimer prior to IL-2 binding and that both receptor subunits participate simultaneously in ligand capture.     

Functional role of the extracellular N-terminal domain of neuropeptide Y subfamily receptors in membrane integration and agonist-stimulated internalization

The N terminus is the most variable element in G protein-coupled receptors (GPCRs), ranging from seven residues up to approximately 5900 residues. For family B and C GPCRs it is described that at least part of the ligand binding site is located within the N terminus. Here we investigated the role of the N terminus in the neuropeptide Y receptor family, which belongs to the class A of GPCRs. We cloned differentially truncated Y receptor mutants, in which the N terminus was partially or completely deleted. We found, that eight amino acids are sufficient for full ligand binding and signal transduction activity. Interestingly, we could show that no specific amino acids but rather the extension of the first transmembrane helix by any residues is sufficient for receptor activity but also for membrane integration in case of the hY(1) and the hY(4) receptors. In contrast, the complete deletion of the N terminus in the hY(2) receptors resulted in a mutant that is fully integrated in the membrane but does not bind the ligand very well and internalizes much slower compared to the wild type receptor. Interestingly, also these effects could be reverted by any N-terminal extension. Accordingly, the most important function of the N termini seems to be the stabilization of the first transmembrane helix to ensure the correct receptor structure, which obviously is essential for ligand binding, integration into the cell membrane and receptor internalization.     

Structure/function analysis of the integrin beta 1 subunit by epitope mapping

Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti- chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.     

Chimeric receptors of the human C3a receptor and C5a receptor (CD88)

Chimeras were generated between the human anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to define the structural requirements for ligand binding and discrimination. Chimeric receptors were generated by systematically exchanging between the two receptors four receptor modules (the N terminus, transmembrane regions 1 to 4, the second extracellular loop, and transmembrane region 5 to the C terminus). The mutants were transiently expressed in HEK-293 cells (with or without Galpha-16) and analyzed for cell surface expression, binding of C3a and C5a, and functional responsiveness (calcium mobilization) toward C3a, C5a, and a C3a as well as a C5a analogue peptide. The data indicate that in both anaphylatoxin receptors the transmembrane regions and the second extracellular loop act as a functional unit that is disrupted by any reciprocal exchange. N-terminal substitution confirmed the two-binding site model for the human C5aR, in which the receptor N terminus is required for high affinity binding of the native ligand but not a C5a analogue peptide. In contrast, the human C3a receptor did not require the original N terminus for high affinity binding of and activation by C3a, a result that was confirmed by N-terminal deletion mutants. This indicates a completely different binding mode of the anaphylatoxins to their corresponding receptors. The C5a analogue peptide, but not C5a, was an agonist of the C3aR. Replacement of the C3aR N terminus by the C5aR sequence, however, lead to the generation of a true hybrid C3a/C5a receptor, which bound and functionally responded to both ligands, C3a and C5a.     

The C-terminal C1 cassette of the N -methyl-D-aspartate receptor 1 subunit contains a bi-partite nuclear localization sequence

The N -methyl-D-aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C-terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C-terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C-terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi-partite NLS does in fact exist within the NR1-1 C-terminus. Computer algorithms identified a putative helix-loop-helix motif that spanned the C0C1 cassettes of the C-terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation.     

Structural requirements for ligand binding by a probable plant vacuolar sorting receptor

How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.     

Rat liver glycine methyltransferase. Cooperative binding of S-adenosylmethionine and loss of cooperativity by removal of a short NH2-terminal segment

Rat liver glycine methyltransferase, a homotetramer, exhibits sigmoidal rate behavior with respect to S-adenosylmethionine (Ogawa, H., and Fujioka, M. (1982) J. Biol. Chem. 257, 3447-3452). The binding experiment shows that the sigmoidicity observed in initial velocity kinetics is explained by the cooperative binding of S-adenosylmethionine to the catalytic sites residing on each subunit. Limited proteolysis of glycine methyltransferase with trypsin in the presence of S-adenosylmethionine yields an enzyme lacking the NH2-terminal 8 residues. The proteolytically modified enzyme retains a tetrameric structure. The truncated enzyme shows no cooperativity with respect to S-adenosylmethionine binding and kinetics. It has values of Vmax and Km for glycine identical to those of the native enzyme, but a 3-fold lower [S]0.5 value for S-adenosylmethionine. The proteolytic modification is without effect on the circular dichroism and fluorescence spectra. Furthermore, the protein fluorescence of the modified enzyme is quenched upon addition of S-adenosylmethionine to the same extent as observed with the native enzyme. These results suggest that a short NH2-terminal segment, which lies outside the active site, is important for communication between subunits.     

The C-terminal domain of dnaQ contains the polymerase binding site

The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.