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Results for “Protein Structure, Quaternary”

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PubMed2006

High-level expression and purification of human thymidine kinase 1: quaternary structure, stability, and kinetics.

Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His(6)-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (approximately 35 mg) and truncated hTK1 (approximately 23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP-MgCl(2) incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures (T(m)) of 71.7 and 73.4 degrees C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (DeltaT(m) ranging from 5.6 to 12.5 degrees C) and hTK1M (DeltaT(m) ranging from 0.8 to 5.3 degrees C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K(M) of 0.51 microM and a k(cat) of 0.28 s(-1) for wild-type hTK1. The truncated hTK1M has a K(M) of 0.87 microM and k(cat) of 1.65 s(-1), thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.

Amino Acid Sequence↗

PubMed2003

Quaternary structure of alpha-crustacyanin from lobster as seen by small-angle X-ray scattering.

The structure of alpha-crustacyanin, the blue carotenoprotein of lobster (Homarus gammarus) carapace, has been investigated for the first time using small-angle X-ray scattering. In this paper, we have determined the dimensions of this protein composed of eight heterodimeric subunits of beta-crustacyanin. Analysis of the scattering spectra and estimation of the shape of alpha-crustacyanin show that the protein fits into a cylinder with an axial length of 238 A and a radius of 47.5 A, in which the eight beta-crustacyanin molecules are probably arranged in a helical manner.

Animals↗

PubMed2006

Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center.

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt(III)-containing enzyme catalyzing the degradation of thiocyanate to carbonyl sulfide and ammonia. We determined the crystal structures of the apo- and native SCNases at a resolution of 2.0 A. SCNases in both forms had a conserved hetero-dodecameric structure, (alphabetagamma)(4). Four alphabetagamma hetero-trimers were structurally equivalent. One alphabetagamma hetero-trimer was composed of the core domain and the betaN domain, which was located at the center of the molecule and linked the hetero-trimers with novel quaternary interfaces. In both the apo- and native SCNases, the core domain was structurally conserved between those of iron and cobalt-types of nitrile hydratase (NHase). Native SCNase possessed the post-translationally modified cysteine ligands, gammaCys131-SO(2)H and gammaCys133-SOH like NHases. However, the low-spin cobalt(III) was found to be in the distorted square-pyramidal geometry, which had not been reported before in any protein. The size as well as the electrostatic properties of the substrate-binding pocket was totally different from NHases with respect to the charge distribution and the substrate accessibility, which rationally explains the differences in the substrate preference between SCNase and NHase.

Amino Acid Motifs↗

PubMed2001

A recombinant glycine receptor fragment forms homo-oligomers distinct from its GABA(A) counterpart.

The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABA(A) receptors are inhibitory. In this study, a fragment of the human glycine receptor alpha1 subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyperexpressed for the first time in Escherichia coli. This fragment is highly homologous in sequence to the corresponding fragment of the GABA(A) receptor. The recombinant fragment was found to have stable beta-rich secondary structure, similar to that found for the homologous GABA(A) receptor fragment, and ordered tertiary packing, suggesting a stable structural domain. Results from laser scattering studies suggest that the fragment forms trimers in solution. In addition, SDS-induced changes in secondary structure were found to occur prior to changes in oligomerization status, suggesting that oligomerization was secondary structure dependent. A study of quaternary structure using single particle analysis electron microscopy (EM) also suggested that the fragment formed homo-trimers. One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across. This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric assembly of the equivalent GABA(A) receptor fragment reported by us earlier. The fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomerization of the glycine receptor in vivo. Furthermore, the finding that two cousin receptor fragments form distinct quaternary structures indicates that sequence similarity does not necessarily imply quaternary structure similarity and, hence, care must be taken when applying a structure model derived from studies of individual receptors to the whole ligand-gated ion channel superfamily.

Amino Acid Sequence↗

PubMed2004

The oxidation process of Antarctic fish hemoglobins.

Analysis of the molecular properties of proteins extracted from organisms living under extreme conditions often highlights peculiar features. We investigated by UV-visible spectroscopy and X-ray crystallography the oxidation process, promoted by air or ferricyanide, of five hemoglobins extracted from Antarctic fishes (Notothenioidei). Spectroscopic analysis revealed that these hemoglobins share a common oxidation pathway, which shows striking differences from the oxidation processes of hemoglobins from other vertebrates. Indeed, simple exposure of these hemoglobins to air leads to the formation of a significant amount of the low-spin hexacoordinated form, denoted hemichrome. This hemichrome form, which is detected under a variety of experimental conditions, can be reversibly transformed to either carbomonoxy or deoxygenated forms with reducing agents. Interestingly, the spectra of the fully oxidized species, obtained by treating the protein with ferricyanide, show the simultaneous presence of peaks corresponding to different hexacoordinated states, the aquomet and the hemichrome. In order to assign the heme region state of the alpha and beta chains, the air-oxidized and ferricyanide-oxidized forms of Trematomus bernacchii hemoglobin were crystallized. Crystallographic analysis revealed that these forms correspond to an alpha(aquomet)-beta(bishistidyl-hemichrome) state. This demonstrates that the alpha and beta chains of Antarctic fish hemoglobins follow very different oxidation pathways. As found for Trematomus newnesi hemoglobin in a partial hemichrome state [Riccio, A., Vitagliano, L., di Prisco, G., Zagari, A. & Mazzarella, L. (2002) Proc. Natl Acad. Sci. USA99, 9801-9806], the quaternary structures of these alpha(aquomet)-beta(bishistidyl-hemichrome) forms are intermediate between the physiological R and T hemoglobin states. Together, these structures provide information on the general features of this intermediate state.

Animals↗

PubMed2006

Physico-chemical properties of molten dimer ascorbate oxidase.

The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.

Ascorbate Oxidase↗

PubMed2001

Identification of an RNA-binding Site in the ATP binding domain of Escherichia coli Rho by H2O2/Fe-EDTA cleavage protection studies.

Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA binding domain of known structure as well as an ATP binding domain for which a structure has been proposed based on homology modeling. Treatment of Rho with H2O2 in the presence of Fe-EDTA caused single-cut cleavage at a number of points that coincide with solvent-exposed loops in both the known and predicted structures, thereby providing support for the validity of the tertiary and quaternary structural models of Rho. The binding of ATP caused one distinct change in the cleavage pattern, a strong protection at a cleavage point in the P-loop of the ATP binding domain. Binding of RNA and single-stranded DNA (poly(dC)) caused strong protection at several accessible parts of the oligosaccharide/oligonucleotide binding (OB) fold in the RNA binding domain. RNA molecules but not DNA molecules also caused a strong, ATP-dependent protection at a cleavage site in the predicted Q-loop of the ATP binding domain. These results suggest that Rho has two distinct binding sites for RNA. Besides the site composed of multiples of the RNA binding domain, to which single-stranded DNA as well as RNA can bind, it has a separate, RNA-specific site on the Q-loop in the ATP binding domain. In the proposed quaternary structure of Rho, the Q-loops from the six subunits form the upper entrance to the hole in the ring-shaped hexamer through which the nascent transcript is translocated by actions coupled to ATP hydrolyses.

Adenine↗

PubMed2001

Dimer stabilization upon activation of the transcriptional antiterminator LicT.

LicT belongs to the BglG/SacY family of transcriptional antiterminators that induce the expression of sugar metabolizing operons in Gram positive and Gram negative bacteria. These proteins contain a N-terminal RNA-binding domain and a regulatory domain called PRD which is phosphorylated on conserved histidine residues by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Although it is now well established that phosphorylation of PRD-containing transcriptional regulators tunes their functional response, the molecular and structural basis of the regulation mechanism remain largely unknown.A constitutively active LicT variant has been obtained by introducing aspartic acid in replacement of His207 and His269, the two phosphorylatable residues of the PRD2 regulatory sub-domain. Here, the functional and structural consequences of these activating mutations have been evaluated in vitro using various techniques including surface plasmon resonance, limited proteolysis, analytical centrifugation and X-ray scattering. Comparison with the native, unphosphorylated form shows that the activating mutations enhance the RNA-binding activity and induce tertiary and quaternary structural changes. Both mutant and native LicT form dimers in solution but the native dimer exhibits a less stable and more open conformation than the activated mutant form. Examination of the recently determined crystal structure of mutant LicT regulatory domain suggests that dimer stabilization is accomplished through salt-bridge formation at the PRD2:PRD2 interface, resulting in domain motion and dimer closure propagating the stabilizing effect from the protein C-terminal end to the N-terminal effector domain. These results suggest that LicT activation arises from a conformational switch inducing long range rearrangement of the dimer interaction surface, rather than from an oligomerization switch converting an inactive monomer into an active dimer.

Bacillus subtilis↗

PubMed2004

Oxidized GroEL can function as a chaperonin.

Here, we report on the facilitated reactivation (85%) of oxidatively inactivated rhodanese by an oxidized form of the molecular chaperone GroEL (ox-GroEL). Reactivation by ox-GroEL required a reductant, and the enzyme substrate, sodium thiosulfate. Also, we found that ox-GroEL formed a complex with oxidatively inactivated rhodanese as shown by differential centrifugation and fluorescence spectroscopy. Ox-GroEL was obtained upon incubation of native GroEL for 16 h with 5 mM hydrogen peroxide. Under these conditions, GroEL was shown to retain its quaternary and secondary structures, but it displayed an increased exposure of hydrophobic surfaces as detected with 1,1'-bis(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS) fluorescence. Additionally, ox-GroEL was significantly more sensitive towards proteolysis with trypsin compared to the native form of the protein. The oxidatively inactivated form of rhodanese, also had an increased exposure of hydrophobic surfaces, as previously reported. Thus, the proteins binding appeared to be mediated by hydrophobic interactions. Unlike in prior reactivation studies that involved native GroEL or alpha-crystallin, we have clearly shown that an oxidized form of GroEL can function as a molecular chaperone in the reactivation of oxidatively inactivated rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.

Animals↗

PubMed2001

A novel tryptophan synthase beta-subunit from the hyperthermophile Thermotoga maritima. Quaternary structure, steady-state kinetics, and putative physiological role.

Tryptophan synthase catalyzes the last two steps in the biosynthesis of the amino acid tryptophan. The enzyme is an alpha beta beta alpha complex in mesophilic microorganisms. The alpha-subunit (TrpA) catalyzes the cleavage of indoleglycerol phosphate to glyceraldehyde 3-phosphate and indole, which is channeled to the active site of the associated beta-subunit (TrpB1), where it reacts with serine to yield tryptophan. The TrpA and TrpB1 proteins are encoded by the adjacent trpA and trpB1 genes in the trp operon. The genomes of many hyperthermophilic microorganisms, however, contain an additional trpB2 gene located outside of the trp operon. To reveal the properties and potential physiological role of TrpB2, the trpA, trpB1, and trpB2 genes of Thermotoga maritima were expressed heterologously in Escherichia coli, and the resulting proteins were purified and characterized. TrpA and TrpB1 form the familiar alpha beta beta alpha complex, in which the two different subunits strongly activate each other. In contrast, TrpB2 forms a beta(2)-homodimer that has a high catalytic efficiency k(cat)/K(m)(indole) because of a very low K(m)(indole) but does not bind to TrpA. These results suggest that TrpB2 acts as an indole rescue protein, which prevents the escape of this costly hydrophobic metabolite from the cell at the high growth temperatures of hyperthermophiles.

Amino Acid Sequence↗

PubMed2002

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function.

The mechanism of cooperativity in the human hemoglobin tetramer (a dimer of alpha beta dimers) has historically been modeled as a simple two-state system in which a low-affinity structural form (T) switches, on ligation, to a high-affinity form (R), yielding a net loss of hydrogen bonds and salt bridges in the dimer-dimer interface. Modifications that weaken these cross-dimer contacts destabilize the quaternary T tetramer, leading to decreased cooperativity and enhanced ligand affinity, as demonstrated in many studies on symmetric double modifications, i.e., a residue site modified in both alpha- or both beta-subunits. In this work, hybrid tetramers have been prepared with only one modified residue, yielding molecules composed of a wild-type dimer and a modified dimer. It is observed that the cooperative free energy of ligation to the modified dimer is perturbed to the same extent whether in the hybrid tetramer or in the doubly modified tetramer. The cooperative free energy of ligation to the wild-type dimer is unperturbed, even in the hybrid tetramer, and despite the overall destabilization of the T tetramer by the modification. This asymmetric response by the two dimers within the same tetramer shows that loss of dimer-dimer contacts is not communicated across the dimer-dimer interface, but is transmitted through the dimer that bears the modified residue. These observations are interpreted in terms of a previously proposed dimer-based model of cooperativity with an additional quaternary (T/R) component.

Binding Sites↗

PubMed2001

Structural similarity and functional diversity in proteins containing the legume lectin fold.

Knowledge of structural relationships in proteins is increasingly proving very useful for in silico characterizations and is also being exploited as a prelude to almost every investigation in functional and structural genomics. A thorough understanding of the crucial features of a fold becomes necessary to realize the full potential of such relationships. To illustrate this, structures containing the legume lectin-like fold were chosen for a detailed analysis since they exhibit a total lack of sequence similarity among themselves and also belong to diverse functional families. A comparative analysis of 15 different families containing this fold was therefore carried out, which led to the determination of the minimal structural principles or the determining region of the fold. A critical evaluation of the structural features, such as the curvature of the front sheet, the presence of the hydrophobic cores and the binding site loops, suggests that none of them are crucial for either the formation or the stability of the fold, but are required to generate diversity and specificity to particular carbohydrates. In contrast, the presence of the three sheets in a particular geometry and also their topological connectivities seem to be important. The fold has been shown to tolerate different types of protein-protein associations, most of them exhibiting different types of quaternary associations and some even existing as complexes with other folds. The function of every family in this study is discussed with respect to its fold, leading to the suggestion that this fold can be linked to carbohydrate recognition in general.

Carbohydrates↗

PubMed2002

Structure of the DNA-bound T-box domain of human TBX3, a transcription factor responsible for ulnar-mammary syndrome.

T-box genes encode transcription factors involved in morphogenesis and organogenesis of vertebrates and invertebrates. Mutations in human T-box genes TBX3, TBX5, and TBX1 cause severe genetic disorders known as Ulnar-Mammary syndrome (UMS), Holt-Oram syndrome (HOS), and DiGeorge syndrome, respectively. The crystal structure of the T-box domain of the first human T-box transcription factor, TBX3, in complex with DNA at 1.7 A resolution explains structural consequences of T-box domain point mutations observed in UMS and HOS patients. Comparison with the structure of the T-box domain from Xenopus laevis (Xbra) bound to DNA shows differences in several secondary structure elements and in the quaternary structure of the two complexes. TBX3 independently recognizes the two binding sites present in the palindromic DNA duplex, whereas in Xbra, binding to the palindrome is stabilized through interactions between the two monomers. The different quaternary structures suggest different DNA binding modes for T-box transcription factors.

Abnormalities, Multiple↗

PubMed2004

Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes.

The crystal structure of the sulfolactate dehydrogenase from the hyperthermophilic and methanogenic archaeon Methanocaldococcus jannaschii was solved at 2.5 A resolution (PDB id. 1RFM). The asymmetric unit contains a tetramer of tight dimers. This structure, complexed with NADH, does not contain a cofactor-binding domain with 'Rossmann-fold' topology. Instead, the tertiary and quaternary structures indicate a novel fold. The NADH is bound in an extended conformation in each active site, in a manner that explains the pro-S specificity. Cofactor binding involves residues belonging to both subunits within the tight dimers, which are therefore the smallest enzymatically active units. The protein was found to be a homodimer in solution by size-exclusion chromatography, analytical ultracentrifugation and small-angle neutron scattering. Various compounds were tested as putative substrates. The results indicate the existence of a substrate discrimination mechanism, which involves electrostatic interactions. Based on sequence homology and phylogenetic analyses, several other enzymes were classified as belonging to this novel family of homologous (S)-2-hydroxyacid dehydrogenases.

Binding Sites↗

PubMed2006

The HC fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disulphide bond.

Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.

Binding Sites↗

PubMed2002

Increasing the thermal stability of an oligomeric protein, beta-glucuronidase.

The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80 degrees C), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads to enzymes that have kinetic parameters that are optimized for high temperatures.

Directed Molecular Evolution↗

PubMed2003

Crystal structure of creatininase from Pseudomonas putida: a novel fold and a case of convergent evolution.

Creatinine amidohydrolase (creatininase; EC 3.5.2.10) from Pseudomonas putida, a homohexameric enzyme with a molecular mass of 28.4 kDa per subunit, is a cyclic amidohydrolase catalysing the reversible conversion of creatinine to creatine. The enzyme plays a key role in the bacterial degradation of creatinine. The three-dimensional structure of creatininase from P.putida was determined and refined to 2.1A. The structure shows the six subunits arranged as a trimer of dimers and definitely disproves previous reports that the enzyme has an octameric quaternary structure. Each monomer consists of a central, four-stranded, parallel beta-sheet flanked by two alpha-helices on both sides of the beta-sheet. This topology is unique within the superfamily of amidohydrolases. Moreover, creatininase possesses a novel fold with no close structural relatives within the Protein Data Bank. Each creatininase monomer contains a binuclear zinc centre near the C termini of the beta-strands and the N termini of the main alpha-helices. These zinc ions indicate the location of the active site unambiguously. The active site is entirely buried and is not accessible from the solution without movement of parts of the protein. The two zinc ions are bridged by a water molecule and by an aspartate residue, which acts as a bidentate ligand. They differ from each other in the number and the spatial arrangement of their ligands. One of them is tetrahedrally and the other trigonal-bipyramidally ligated. Using two water molecules of the first coordination sphere as anchor points, a creatinine-water adduct resembling the transition state of the hydrolysation reaction was modelled into the active site. The resulting complex in combination with structural comparisons with other amidohydrolases enabled us to identify the most probable candidate for the catalytic base and to suggest a putative reaction mechanism. Surprisingly these structural comparisons revealed a similarity in the active-site arrangement between creatininase and the hydantoinase-like cyclic amidohydrolases that was unexpected, given the completely unrelated primary and tertiary structures. In particular, the zinc-bridging aspartate residue of creatininase is a spatially and functionally analogue to a carboxylated lysine residue found in dihydroorotase and the hydantoinases. Hence, creatininase and the hydantoinase-like cyclic amidohydrolases represent a further example of convergent evolution within the enzyme class of hydrolases.

Amidohydrolases↗

PubMed2003

Dimerization of cGMP-dependent protein kinase Ibeta is mediated by an extensive amino-terminal leucine zipper motif, and dimerization modulates enzyme function.

All mammalian cGMP-dependent protein kinases (PKGs) are dimeric. Dimerization of PKGs involves sequences located near the amino termini, which contain a conserved, extended leucine zipper motif. In PKG Ibeta this includes eight Leu/Ile heptad repeats, and in the present study, deletion and site-directed mutagenesis have been used to systematically delete these repeats or substitute individual Leu/Ile. The enzymatic properties and quaternary structures of these purified PKG mutants have been determined. All had specific enzyme activities comparable to wild type PKG. Simultaneous substitution of alanine at four or more of the Leu/Ile heptad repeats ((L3A/L10A/L17A/I24A), (L31A/I38A/L45A/I52A), (L17A/I24A/L31A/I38A/L45A/I52A), and (L3A/L10A/L45A/I52A)) of the motif produces a monomeric PKG Ibeta. Mutation of two Leu/Ile heptad repeats can produce either a dimeric (L3A/L10A) or monomeric (L17A/I24A and L31A/I38A) PKG. Point mutation of Leu-17 or Ile-24 (L17A or I24A) does not disrupt dimerization. These results suggest that all eight Leu/Ile heptad repeats are involved in dimerization of PKG Ibeta. Six of the eight repeats are sufficient to mediate dimerization, but substitutions at some positions (Leu-17, Ile-24, Leu-31, and Ile-38) appear to have greater impact than others on dimerization. The Ka of cGMP for activation of monomeric mutants (PKG Ibeta (delta1-52) and PKG Ibeta L17A/I24A/L31A/I38A/L45A/I52A) is 2- to 3-fold greater than that for wild type dimeric PKG Ibeta, and there is a corresponding 2- to 3-fold increase in cGMP-dissociation rate of the high affinity cGMP-binding site (site A) of these monomers. These results indicate that dimerization increases sensitivity for cGMP activation of the enzyme.

Amino Acid Motifs↗