Intrinsically unstructured proteins by design—electrostatic interactions can control binding,folding, and function of a helix‐loop‐helix heterodimer

Intrinsically disordered proteins that exist as unordered monomeric structures in aqueous solution at pH 7 but fold into four‐helix bundles upon binding to recognized polypeptide targets have been designed. NMR and CD spectra of the monomeric polypeptides show the hallmarks of unordered structures, whereas in the bound state they are highly helical. Analytical ultracentrifugation data shows that the polypeptides bind to their targets to form exclusively heterodimers at neutral pH. To demonstrate the relationship between binding, folding, and function, a catalytic site for ester hydrolysis was introduced into an unordered and largely inactive monomer, but that was structured and catalytically active in the presence of a specific polypeptide target. Electrostatic interactions between surface‐exposed residues inhibited the binding and folding of the monomers at pH 7. Charge–charge repulsion between ionizable amino acids was thus found to be sufficient to disrupt binding between polypeptide chains despite their inherent propensities for structure formation and may be involved in the folding and function of inherently disordered proteins in biology. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Electron transfer in monomeric forms of beef and shark heart cytochrome c oxidase

Beef heart cytochrome c oxidase is dimeric in reconstituted membranes and in nonionic detergents at physiological pH [Henderson, R., Capaldi, R. A., & Leigh, J. (1977) J. Mol. Biol. 112, 631; Robinson, N.C., & Capaldi, R. A. (1977) Biochemistry 16, 375], raising the possibility that this aggregation state is a prerequisite for enzymatic activity. A procedure for dissociating the enzyme into monomers is presented. This involves treating the protein with high concentrations of Triton X-100 at pH 8.5. The electron transfer activity of the monomer is comparable to that of the dimer under identical assay conditions. The beef heart cytochrome c oxidase monomer was found to be heterogeneous in hydrodynamic studies, probably due to dissociation of associated polypeptides, including subunit III. Monomer molecular weights in the range 129 000-160 000 were obtained. Previous studies have indicated that shark heart cytochrome c oxidase is monomeric under physiological conditions. Sedimentation equilibrium studies reported here confirm this. The elasmobranch enzyme, with a similar polypeptide composition to that of beef enzyme, was determined to have a molecular weight of 158 000.     

Studies on cytochrome c oxidase, X. Isolation and amino-acid sequence of polypeptide VIIIb

The isolation and sequence determination of the cytoplasmically synthesized polypeptide VIIIb from beef heart cytochrome c oxidase is described. Several methods for isolating polypeptide VIIIb with gelchromatographic technics are presented. The complete amino-acid sequence is deduced from a N-terminal sequencer run, overlapping tryptic peptides and peptides obtained after tryptophan specific cleavage with cyanogen bromide in heptafluorobutyric acid/formic acid. The small protein consists of 46 amino acids and has a molecular mass of 4 962 Da. The existence of a hydrophobic segment with a length of 20 residues characterizes it as a membrane penetrating protein. The stoichiometry of this polypeptide in the functional monomer of cytochrome c oxidase (complex IV) is 2 and is thus different from all the other polypeptides constituting the respiratory complex IV. The function of this component of the terminal oxidase is as yet unknown.     

Aspartate transcarbamoylase genes of Pseudomonas putida: requirement for an inactive dihydroorotase for assembly into the dodecameric holoenzyme.

The nucleotide sequences of the genes encoding the enzyme aspartate transcarbamoylase (ATCase) from Pseudomonas putida have been determined. Our results confirm that the P. putida ATCase is a dodecameric protein composed of two types of polypeptide chains translated coordinately from overlapping genes. The P. putida ATCase does not possess dissociable regulatory and catalytic functions but instead apparently contains the regulatory nucleotide binding site within a unique N-terminal extension of the pyrB-encoded subunit. The first gene, pyrB, is 1,005 bp long and encodes the 334-amino-acid, 36.4-kDa catalytic subunit of the enzyme. The second gene is 1,275 bp long and encodes a 424-residue polypeptide which bears significant homology to dihydroorotase (DHOase) from other organisms. Despite the homology of the overlapping gene to known DHOases, this 44.2-kDa polypeptide is not considered to be the functional product of the pyrC gene in P. putida, as DHOase activity is distinct from the ATCase complex. Moreover, the 44.2-kDa polypeptide lacks specific histidyl residues thought to be critical for DHOase enzymatic function. The pyrC-like gene (henceforth designated pyrC') does not complement Escherichia coli pyrC auxotrophs, while the cloned pyrB gene does complement pyrB auxotrophs. The proposed function for the vestigial DHOase is to maintain ATCase activity by conserving the dodecameric assembly of the native enzyme. This unique assembly of six active pyrB polypeptides coupled with six inactive pyrC' polypeptides has not been seen previously for ATCase but is reminiscent of the fused trifunctional CAD enzyme of eukaryotes.     

The relationship between the glucose oxidase subunit structure and its thermostability

The thermostability of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) at 60 degrees C has been studied as a function of its concentration in various media (pure water and pure deuterium oxide). In deuterium oxide, glucose oxidase is more stable than in water, and two kinds of stabilizing effect have been observed: the medium-organization effect and the enzyme-concentration effect. This effect has been related to the glucose oxidase subunit structure. This enzyme contains four forms of subunit: monomer, dimer, trimer, and tetramer, which are all composed of the identical monomer. The monomers of glucose oxidase subunits are linked by the non-covalent bond. Only dimer and trimer possess the enzymatic activity. During glucose oxidase denaturing, monomers assemble into dimer, trimer, or tetramer. This redistribution behavior depends on the enzyme concentration and the nature of the medium.     

Evidence for an essential role of intradimer interaction in catalytic function of carnosine dipeptidase II using electrospray‐ionization mass spectrometry

Carnosine dipeptidase II (CN2/CNDP2) is an M20 family metallopeptidase that hydrolyses various dipeptides including β‐alanyl‐l ‐histidine (carnosine). Crystallographic analysis showed that CN2 monomer is composed of one catalytic and one dimerization domains, and likely to form homodimer. In this crystal, H228 residue of the dimerization domain interacts with the substrate analogue bestatin on the active site of the dimer counterpart, indicating that H228 is involved in enzymatic reaction. In the present study, the role of intradimer interaction of CN2 in its catalytic activity was investigated using electrospray‐ionization time‐of‐flight mass spectrometry (ESI‐TOF MS). First, a dimer interface mutant I319K was prepared and shown to be present as a folded monomer in solution as examined by using ESI‐TOF MS. Since the mutant was inactive, it was suggested that dimer formation is essential to its enzymatic activity. Next, we prepared H228A and D132A mutant proteins with different N‐terminal extended sequences, which enabled us to monitor dimer exchange reaction by ESI‐TOF MS. The D132A mutant is a metal ligand mutant and also inactive. But the activity was partially recovered time‐dependently when H228A and D132A mutant proteins were incubated together. In parallel, H228A/D132A heterodimer was formed as detected by ESI‐TOF MS, indicating that interaction of a catalytic center with H228 residue of the other subunit is essential to the enzymatic reaction. These results provide evidence showing that intradimer interaction of H228 with the reaction center of the dimer counterpart is essential to the enzymatic activity of CN2.     

Calmodulin-activated cyclic nucleotide phosphodiesterase from brain. Relationship of subunit structure to activity assessed by radiation inactivation

The apparent target sizes of the basal and calmodulin-dependent activities of calmodulin-activated phosphodiesterase from bovine brain were estimated using target theory analysis of data from radiation inactivation experiments. Whether crude or highly purified samples were irradiated, the following results were obtained. Low doses of radiation caused a 10 to 15% increase in basal activity, which, with further irradiation, decayed with an apparent target size of approximately 60,000 daltons. Calmodulin-dependent activity decayed with an apparent target size of approximately 105,000 daltons. The percentage stimulation of enzyme activity by calmodulin decreased markedly as a function of radiation dosage. These observations are consistent with results predicted by computer-assisted modeling based on the assumptions that: 1) the calmodulin-activated phosphodiesterase exists as a mixture of monomers which are fully active in the absence of calmodulin and dimers which are inactive in the absence of calmodulin; 2) in the presence of calmodulin, a dimer exhibits activity equal to that of two monomers; 3) on radiations destruction of a dimer, an active monomer is generated. This monomer-dimer hypothesis provides a plausible explanation for and definition of basal and calmodulin-dependent phosphodiesterase activity.     

Despite its high similarity with monomeric arginine kinase, muscle creatine kinase is only enzymatically active as a dimer

Although having highly similar primary to tertiary structures, the different guanidino kinases exhibit distinct quaternary structures: monomer, dimer or octamer. However, no evidence for communication between subunits has yet been provided, and reasons for these different levels of quaternary complexity that can be observed from invertebrate to mammalian guanidino kinases remain elusive. Muscle creatine kinase is a dimer and disruption of the interface between subunits has been shown to give rise to destabilized monomers with slight residual activity; this low activity could, however, be due to a fraction of protein molecules present as dimer. CK monomer/monomer interface involves electrostatic interactions and increasing salt concentrations unfold and inactivate this enzyme. NaCl and guanidine hydrochloride show a synergistic unfolding effect and, whatever the respective concentrations of these compounds, inactivation is associated with a dissociation of the dimer. Using an interface mutant (W210Y), protein concentration dependence of the NaCl-induced unfolding profile indicates that the active dimer is in equilibrium with an inactive monomeric state. Although highly similar to muscle CK, horse shoe crab (Limulus polyphemus) arginine kinase (AK) is enzymatically active as a monomer. Indeed, high ionic strengths that can monomerize and inactivate CK, have no effect on AK enzymatic activity or on its structure as judged from intrinsic fluorescence data. Our results indicate that expression of muscle creatine kinase catalytic activity is dependent on its dimeric state which is required for a proper stabilization of the monomers.     

The processing of a cathepsin L precursor in vitro

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.     

Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.     

Functional overexpression of gamma-secretase reveals protease-independent trafficking functions and a critical role of lipids for protease activity

Presenilins appear to form the active center of gamma-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue gamma-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, gamma-secretase appears to play a role in the trafficking of the beta-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized gamma-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. Gamma-secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the gamma-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive gamma-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of gamma-secretase.     

The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer

Hyaluronan (HA), a linear polysaccharide composed of beta1,3-GlcNAc-beta1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membrane-associated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V--VII were rather resistant to degradation. From the relative inaccessibility of subunits V--VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

Dihydrofolate reductase as a new "affinity handle".

Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives. All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity. The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step. The use of methotrexate-bound affinity chromatography was very effective. In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence. The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system. Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.     

DNA topoisomerase I from calf thymus mitochondria is associated with a DNA binding, inner membrane protein.

During purification of the type I DNA topoisomerase from calf thymus mitochondria, two polypeptides, p78 and p63, cofractionate with the enzymatic activity (Lazarus et al., (1987) Biochemistry 26, 6195-6203). The two polypeptides are released from a mitochondrial inner membrane preparation by nonionic detergent lysis and both adsorb strongly to a single-stranded DNA agarose column. We have attempted to characterize the relationship between these two polypeptides and have found the following: (i) the mitochondrial topoisomerase is active in free (monomer) and associated (heterodimer) form; (ii) the catalytic activity resides solely in p78, as adjudged by both the covalent linkage of the enzyme to substrate DNA and the ability of the enzyme to relax supercoils; (iii) at low ionic strength the enzyme is active in monomer form with p78 alone being sufficient for activity; (iv) in high salt, the high molecular weight species is a 140-kDa heterodimer composed of one p78 and one p63; and (v) the two polypeptides are not structurally related as digestion with V8 protease results in distinct proteolytic fragment patterns. These results suggest that p63 may have an important role in the metabolism of the mitochondrial topoisomerase.     

Redesigning the procaspase‐8 dimer interface for improved dimerization

Caspase‐8 is a cysteine directed aspartate‐specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase‐8 depends on adaptor‐induced dimerization of procaspase‐8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase‐8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase‐3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase‐8 is a monomer while procaspase‐3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase‐8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase‐8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase‐8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase‐8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.     

Effect of subunit dissociation, denaturation, aggregation, coagulation, and decomposition on enzyme inactivation kinetics: II. Biphasic and grace period behavior

A model previously developed to characterize enzymatic in activation behavior was used to explain the non-first-order biphasic and grace period phenomena that are often observed with oligomeric enzymes. Luciferase and urease were used as model enzyme such as luciferase, the oligomer initially dissociates reversibly into two native monomer species. These native monomers can then reversibly denature and irreversibly aggregate and coagulate. With the hexamer, urease, two trimers are formed that can subsequently aggregate to form an inactive hexamer. The dissociated monomer species of luciferase do not possess catalytic activity, so the inactivation mechanism, is biphasic; the first slope of a first-order kinetic plot is influenced by the reversible oligomer/monomer/denatured-monomer transition. Whereas the second slope is associated with either irreversible aggregation or coagulation. In contrast, the trimer of urease has the same activity as the hexamer; therefore, during the intitial hexamer-trimer transition, little activity loss occurs. However, as the trimer concentration increases, activity decreases as a result of trimer aggregation. As a result, grace period inactivation behavior is observed. (c) 1992 John Wiley & Sons, Inc.     

Determination of molecular mass of the aroid alternative oxidase by radiation-inactivation analysis.

The functional molecular mass of the cyanide-resistant salicylhydroxamate-sensitive duroquinol oxidase activity from Sympocarpus foetidus (skunk cabbage) and Sauromatum guttatum spadix mitochondria was determined by radiation-inactivation analysis. The functional molecular mass for the oxidase activity was found to be 26,700 Da for skunk cabbage and 29,700 Da for Sauromatum guttatum mitochondria frozen at -70 degrees C. Irradiation of dried mitochondrial samples resulted in a larger target size of 38,000 Da, and in some cases, a stimulation of activity at low dose of radiation. The functional molecular mass of cytochrome c oxidase activity from skunk-cabbage and bovine heart mitochondria was also investigated. Dried and frozen mitochondrial samples from both species yielded similar target sizes, in the range 70,900-73,400 Da. Purified bovine heart cytochrome c oxidase was also irradiated, and yielded a functional molecular mass of 66,400 Da. The target size of cytochrome c oxidase agrees with literature values insofar as the target size is considerably smaller than the molecular mass of the entire complex.     

The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer

Hyaluronan (HA), a linear polysaccharide composed of β1,3-GlcNAc-β1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membrane-associated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.