The folding pathway of a single domain in a multidomain protein is not affected by its neighbouring domain

Domains are the structural, functional, and evolutionary components of proteins. Most folding studies to date have concentrated on the folding of single domains, but more than 70% of human proteins contain more than one domain, and interdomain interactions can affect both the stability and the folding kinetics. Whether the folding pathway is altered by interdomain interactions is not yet known. Here we investigated the effect of a folded neighbouring domain on the folding pathway of spectrin R16 (the 16th α-helical repeat from chicken brain α-spectrin) by using the two-domain construct R1516. The R16 folds faster and unfolds more slowly in the presence of its folded neighbour R15 (the 15th α-helical repeat from chicken brain α-spectrin). An extensive Φ-value analysis of the R16 domain in R1516 was completed to compare the transition state of the R16 domain alone with that of the R16 domain in a multidomain construct. The results indicate that the folding pathways are the same. This result validates the current approach of breaking up larger proteins into domains for the study of protein folding pathways.     

Function of positive charges following signal-anchor sequences during translocation of the N-terminal domain

In topogenesis of membrane proteins on the endoplasmic reticulum, the orientation of the hydrophobic transmembrane (TM) segment is influenced by the charge of the flanking amino acid residues. We assessed the function of the positive charges downstream of the hydrophobic segment using synaptotagmin II. The positive charges were systematically replaced with non-charged residues. Although the original TM segment translocated the N terminus, the topology was inverted, depending on the mutations. Orientation was affected in mutants in which 6 Lys were shifted downstream, even when the 6 Lys were 25 residues from the hydrophobic segment. The Lys was functionally replaced by Arg, but not by Asp or Glu. The timing of action during polypeptide elongation indicated that the Lys functions at the ribosome exit sites. We suggest that the commitment of the TM segment to a particular orientation is influenced by far downstream parts of the polypeptide chain and that the positive charges are decoded after exiting the ribosome.     

Transition state stabilization by the N-terminal anticodon-binding domain of lysyl-tRNA synthetase

Lysyl-tRNA synthetase from Bacillus stearothermophilus (B.s. LysRS) (EC ) catalyzes aminoacylation of tRNA(Lys) with l-lysine, in which l-lysine was first activated with ATP to yield an enzyme (lysyladenylate complex), and then the lysine molecule was transferred from the complex to tRNA(Lys). B.s. LysRS is a homodimeric enzyme with a subunit that consists of two domains, an N-terminal tRNA anticodon-binding domain (TAB-ND: Ser(1)-Pro(144)) and a C-terminal Class II-specific catalytic domain (CAT-CD: Lys(151)-Lys(493)). CAT-CD alone retained catalytic activity, although at a low level; TAB-ND alone showed no activity. Size exclusion chromatography revealed that CAT-CD exists as a dimer, whereas TAB-ND was a monomer. The formation of a complex consisting of these domains was detected with the guidance of surface plasmon resonance. In accordance with this, the addition of TAB-ND to CAT-CD significantly enhanced both the l-lysine activation and the tRNA aminoacylation reactions. Kinetic analysis showed that deletion of TAB-ND resulted in a significant destabilization of the transition state of CAT-CD in the l-lysine activation reaction but had little effect on the ground state of substrate binding. A significant role of a cross-subunit interaction in the enzyme between TAB-ND and CAT-CD was proposed for the stabilization of the transition state in the l-lysine activation reaction.     

Biosynthesis of intestinal microvillar proteins. Surface expression of aminopeptidase N is not affected by chloroquine

The effect of chloroquine on the biosynthesis of pig intestinal aminopeptidase N (EC 3.4.11.2) was studied by labelling with [35S]methionine in organ cultured mucosal explants. The lysosomotropic agent did not alter the molecular size of either the transient or the mature form of the enzyme and did not markedly influence the relative intracellular distribution of the two forms. The microvillar expression of aminopeptidase N during labelling periods of 80-120 min was found to be unaffected by chloroquine. Together these data indicate that pH neutralization of the acidic compartments of the cell bears no consequence on the intracellular transport of the newly synthesized microvillar enzyme. This suggests that the acidic compartments are not involved in the post-Golgi transport and that this, in turn, probably occurs via a constitutive rather than a regulated pathway.     

The N-terminal domain of the reticulocyte-type 15-lipoxygenase is not essential for enzymatic activity but contains determinants for membrane binding

The rabbit reticulocyte-type 15-lipoxygenase is capable of oxygenating biomembranes and lipoproteins without the preceding action of ester lipid cleaving enzymes. This reaction requires an efficient membrane binding, and the N-terminal beta-barrel domain of the enzyme has been implicated in this process. To obtain detailed information on the structural requirements for membrane oxygenation, we expressed the rabbit wild-type 15-lipoxygenase, its beta-barrel deletion mutant (catalytic domain), and several lipoxygenase point mutations as His-tagged fusion proteins in Escherichia coli and tested their membrane binding characteristics. We found that: (i) the beta-barrel deletion mutant was catalytically active and its enzymatic properties (K(M), V(max), pH optimum, substrate specificity) were similar to those of the wild-type enzyme; (ii) when compared with the wild-type lipoxygenase, the membrane binding properties of the N-terminal truncation mutant were impaired but not abolished, suggesting a role of the catalytic domain in membrane binding; and (iii) Phe-70 and Leu-71 (constituents of the beta-barrel domain) but also Trp-181, which is located in the catalytic domain, were identified as sequence determinants for membrane binding. Mutation of these amino acids to more polar residues (F70H, L71K, W181E) impaired the membrane binding capacity of the recombinant enzyme. These data indicate that the C-terminal catalytic domain of the rabbit 15-lipoxygenase is enzymatically active and that the membrane binding properties of the enzyme are determined by a concerted action of the N-terminal beta-barrel and the C-terminal catalytic domain.     

Song-related brain regions in the red-winged blackbird are affected by sex and season but not repertoire size

Previous work in songbirds has delimited a neural system responsible for song production and control. Earlier studies have suggested that functional capacity in the song system may be related to the mass of the system in an animal's brain, and that adult plasticity in this neural system may be related to adult capacity for behavioral modification. We now test these hypotheses in adult red-winged blackbirds (Agelaius phoeniceus), a species in which song is produced primarily by males, new song types are added to the male's repertoire in adulthood, and there are substantial differences among males in song complexity. We find that the song system in males is much larger than in females. Song system nuclei become smaller in both sexes as the animals experience shorter days. We do not find any association between repertoire size and size of any of the song system structures examined. Thus, although sex differences in song may be related to differences between sexes in the mass of song system structures, individual differences in song do not appear to be directly related to mass within males. Seasonal change in song system structures in male redwings is consistent with there being a relation between adult plasticity in anatomy and in behavior; the large seasonal change in these structures in females suggests large seasonal changes in the function of these nuclei.     

pH-dependent interactions and the stability and folding kinetics of the N-terminal domain of L9. Electrostatic interactions are only weakly formed in the transition state for folding

The role of electrostatic interactions in the stability and the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) was investigated by determining the effects of varying the pH conditions. Urea denaturations and thermal unfolding experiments were used to measure the free energy of folding, DeltaG degrees, at 18 different pH values, ranging from pH 1.1 to pH 10.5. Folding rates were measured at 19 pH values between pH 2.1 and pH 9.5, and unfolding rates were determined at 15 pH values in this range using stopped-flow fluorescence experiments. The protein is maximally stable between pH 5.5 and 7.5 with a value of DeltaG degrees =4.45 kcal mol(-1). The folding rate reaches a maximum at pH 5.5, however the change in folding rates with pH is relatively modest. Over the pH range of 2.1 to 5.5 there is a small increase in folding rates, ln (k(f)) changes from 5.1 to 6.8. However, the change in stability is more dramatic, with a difference of 2.6 kcal mol(-1) between pH 2.0 and pH 5.4. The change in stability is largely due to the smaller barrier for unfolding at low pH values. The natural log of the unfolding rates varies by approximately four units between pH 2.1 and pH 5.5. The stability of the protein decreases above pH 7.5 and again the change is largely due to changes in the unfolding rate. ln (k(f)) varies by less than one unit between pH 5.5 and pH 9.5 while DeltaG degrees decreases by 2.4 kcal mol(-1) over the range of pH 5. 4 to pH 10.0, which corresponds to a change in ln K(eq) of 4.0. These studies show that pH-dependent interactions contribute significantly to the overall stability of the protein but have only a small effect upon the folding kinetics, indicating that electrostatic interactions are weakly formed in the transition state for folding.     

Gluconeogenesis in goat hepatocytes is affected by calcium, ammonia and other key metabolites but not primarily through cytosolic redox state

1. Gluconeogenesis from propionate and lactate was studied in caprine hepatocytes. 2. Reducing cytosol with additions of ETOH, ammonium, or lactate decreased [2-14C]propionate conversion to glucose. 3. Calcium oxidized the cytosol and increased gluconeogenesis from propionate by 198% and from lactate by 220%. 4. Cells isolated from lactating does and wethers differed quantitatively in propionate conversion to glucose and response to calcium. 5. Acetoacetate decreased and 3-OH-butyrate slightly increased glucose production from propionate. 6. Neither ketone body had any significant effect on gluconeogenesis from lactate. 7. Results reported herein suggest gluconeogenesis from propionate is not limited by lack of cytosolic reducing equivalents.     

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.     

Glucocorticoid receptor binding in twin pairs is affected by shared environment but not by shared genes

We set out to determine whether glucocorticoid receptor activity is affected mainly by genetic or environmental factors. The affinity and capacity of the glucocorticoid receptor was measured using dexamethasone binding in whole leukocytes from 53 monozygotic and 48 dizygotic twin pairs. Receptor binding characteristics assayed from twin pairs on the same day were highly correlated within twin pairs irrespective of zygosity. Apparent Kd was negatively correlated with environmental temperature (R²=0.13, P<0.0001) but this did not confound the intra-pair correlation, suggesting a strong familial component independent of zygosity. Receptor binding parameters were not more closely correlated in monozygotic twins than dizygotic twin pairs indicating that there is no major genetic contribution to receptor binding and that environmental influences predominate. The close similarity in binding between twin pairs in adulthood raises the possibility that familial, non-genetic, factors such as shared early life environment may programme the glucocorticoid receptor.     

Hydrophobic core packing in the SH3 domain folding transition state.

How tightly packed is the hydrophobic core of a folding transition state structure? We have addressed this question by characterizing the effects on folding kinetics of > 40 substitutions of both large and small amino acids in the hydrophobic core of the Fyn SH3 domain. Our results show that residues at three positions, which we designate as the 'core folding nucleus', are tightly packed in the transition state, and substitutions at these positions cause the largest changes in the folding rate. The other six positions examined appear to be loosely packed; thus, substitutions at these positions with larger hydrophobic residues generally accelerate folding, presumably by increasing the rate of nonspecific hydrophobic collapse. Surprisingly, the folding rate can be greatly accelerated by residues that also significantly destabilize the native state structure. Furthermore, mutants with identical thermodynamic stability can differ by up to 55-fold in their folding rates. These results highlight the importance of hydrophobic core composition, as opposed to only topology, in determining the folding rate of a protein. They also provide a new explanation for the 'abnormal' phi-values observed in many protein folding kinetics studies.     

pH jump studies of the folding of the multidomain ribosomal protein L9: the structural organization of the N-terminal domain does not affect the anomalously slow folding of the C-terminal domain

The folding kinetics of the multidomain ribosomal protein L9 were studied using pH jump stopped-flow fluorescence and circular dichroism (CD) in conjunction with guanidine hydrochloride (GdnHCl) jump stopped-flow CD experiments. Equilibrium CD and 1D (1)H NMR measurements demonstrated that the C-terminal domain unfolds below pH 4 while the N-terminal domain remains fully folded. Thus, the N-terminal domain remains folded during the pH jump experiments. The folding rate constant of the C-terminal domain was determined to be 3.5 s(-1) by pH jump experiments conducted in the absence of denaturant using stopped-flow CD and fluorescence. CD-detected GdnHCl jump measurements showed that the N- and C-terminal domains fold independently each by an apparent two-state mechanism. The folding rate constant for the N-terminal domain and the C-terminal domain in the absence of denaturant were calculated to be 760 and 4. 7 s(-1), respectively. The good agreement between the pH jump and the denaturant concentration jump experiments shows that the folding rate of the C-terminal domain is the same whether or not the N-terminal domain is folded. This result suggests that the slow folding of the C-terminal domain is not a consequence of unfavorable interactions with the rest of the protein chain during refolding. This is an interesting result since contact order analysis predicts that the folding rate of the C-terminal domain should be noticeably faster. The folding rate of the isolated N-terminal domain was also measured by stopped-flow CD and was found to be the same as the rate for the domain in the intact protein.     

Expansion of the octarepeat domain alters the misfolding pathway but not the folding pathway of the prion protein

A misfolded conformation of the prion protein (PrP), PrP (Sc), is the essential component of prions, the infectious agents that cause transmissible neurodegenerative diseases. Insertional mutations that lead to an increase in the number of octarepeats (ORs) in PrP are linked to familial human prion disease. In this study, we investigated how expansion of the OR domain causes PrP to favor a prion-like conformation. Therefore, we compared the conformational and aggregation modulating properties of wild-type versus expanded OR domains, either as a fusion construct with the protein G B1 domain (GB1-OR) or as an integral part of full-length mouse PrP (MoPrP). Using circular dichroism spectroscopy, we first demonstrated that ORs are not unfolded but exist as an ensemble of three distinct conformers: polyproline helix-like, beta-turn, and "Trp-related". Domain expansion had little effect on the conformation of GB1-OR fusion proteins. When part of MoPrP however, OR domain expansion changed PrP's folding landscape, not by hampering the production of native alpha-helical monomers but by greatly reducing the propensity to form amyloid and by altering the assembly of misfolded, beta-rich aggregates. These features may relate to subtle pH-dependent conformational differences between wild-type and mutant monomers. In conclusion, we propose that PrP insertional mutations are pathogenic because they enhance specific misfolding pathways of PrP rather than by undermining native folding. This idea was supported by a trial bioassay in transgenic mice overexpressing wild-type MoPrP, where intracerebral injection of recombinant MoPrP with an expanded OR domain but not wild-type MoPrP caused prion disease.     

Ruk is ubiquitinated but not degraded by the proteasome.

The regulator of ubiquitous kinase (Ruk) protein, also known as CIN85 or SETA, is an adaptor-type protein belonging to the CD2AP/CMS family. It was found in complexes with many signaling proteins, including phosphoinositol (PtdIns) 3-kinase (EC 2.7.1.137), Cbl, GRB2, p130Cas and Crk. Functional analysis of these interactions, implicated Ruk in the regulation of apoptosis, receptor endocytosis and cytoskeletal rearrangements. We have recently demonstrated that overexpression of Ruk induces apoptotic death in neurons, which could be reversed by activated forms of PtdIns 3-kinase and PKB/Akt. Furthermore, Ruk was shown to be a negative regulator of PtdIns 3-kinase activity through binding to its P85 regulatory subunit [Gout, I., Middleton, G., Adu, J., Ninkina, N. N., Drobot, L. B., Filonenko, V., Matsuka, G., Davies, A.M., Waterfield, M. & Buchman, V. L. (2000) Embo J.19, 4015-4025]. Here, we report for the first time, that all three isoforms of Ruk (L, M and S) are ubiquitinated. Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells. The interaction of Ruk M and S isoforms with Cbl was found to be mediated via heterodimerization with Ruk L. The use of proteosomal and lysosomal inhibitors clearly indicated that ubiquitination of Ruk L does not lead to its degradation. Based on this study, we propose a possible mechanism for the regulation of Ruk function by ubiquitination.     

CD36 N-terminal cytoplasmic domain is not required for the internalization of oxidized low-density lipoprotein

The uptake of OxLDLs (oxidized low density lipoproteins) by CD36-expressing macrophages in the arterial intima and the subsequent 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. The present study has addressed the function of the CD36 N-terminal cytoplasmic domain in the binding and internalization of OxLDL. A selection of CD36 N-terminal cytoplasmic domain mutants were generated and stably expressed in HEK-293 (human embryonic kidney) cells. The capacity of three mutants [CD36_C3/7-A (CD36-C3A/C7A), CD36_D4/R5-A (CD36-D4A/R5A) and CD36_nCPD(-) (CD36 lacking the N-terminal cytoplasmic domain)] to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluorimetry. Each of the CD36 constructs was expressed at differing levels at the cell surface, as measured by flow cytometry and Western blotting. Following incubation with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-OxLDL, cells bearing the CD36_wt (wild-type CD36), CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD(-) constructs all internalized DiI-OxLDL into endosomal structures, whereas empty-vector-transfected cells failed to do so, indicating that, unlike the C-terminal cytoplasmic domain, the N-terminal cytoplasmic domain is not essential for the endocytosis of OxLDL. In conclusion, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 N-terminal cytoplasmic domain. However, the N-terminal cytoplasmic domain may conceivably be implicated in the maturation of CD36.     

Transmembrane orientation of glycoproteins encoded by the v-fms oncogene

The retroviral oncogene v-fms encodes a glycoprotein whose transport to the plasma membrane is required for transformation. Tryptic digestion of microsomes from transformed cells yielded membrane-protected amino-terminal fragments 40 kd smaller than intact molecules. These fragments were glycosylated, and they included v-fms-coded epitopes expressed at the cell surface. Deletion of the predicted membrane-spanning peptide generated polypeptides that were completely sequestered within microsomes. The mutant glycoproteins acquired more asparagine-linked oligosaccharide chains than did wild-type molecules, lacked kinase activity in vitro, were not transported to the cell surface, and had no transforming activity. Thus, the membrane-spanning segment in the middle of the glycoprotein interrupts translocation of nascent chains into the endoplasmic reticulum, ultimately orienting the amino-terminal domain outside the cell and the carboxy-terminal kinase domain in the cytoplasm. These topological features are similar to those of several growth factor receptors, suggesting that v-fms transforms cells through modified receptor-mediated signals.     

The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain.

Lysyl-tRNA synthetase from higher eukaryotes possesses a lysine-rich N-terminal polypeptide extension appended to a classical prokaryotic-like LysRS domain. Band shift analysis showed that this extra domain provides LysRS with nonspecific tRNA binding properties. A N-terminally truncated derivative of LysRS, LysRS-DeltaN, displayed a 100-fold lower apparent affinity for tRNA(3)Lys and a 3-fold increase in K(m) for tRNA(3)Lys in the aminoacylation reaction, as compared with the native enzyme. The isolated N-domain of LysRS also displayed weak affinity for tRNA, suggesting that the catalytic and N-domains of LysRS act synergistically to provide a high affinity binding site for tRNA. A more detailed analysis revealed that LysRS binds and specifically aminoacylates an RNA minihelix mimicking the amino acid acceptor stem-loop structure of tRNA(3)Lys, whereas LysRS-DeltaN did not. As a consequence, merging an additional RNA-binding domain into a bacterial-like LysRS increases the catalytic efficiency of the enzyme, especially at the low concentration of deacylated tRNA prevailing in vivo. Our results provide new insights into tRNA(Lys) channeling in eukaryotic cells and shed new light on the possible requirement of native LysRS for triggering tRNA(3)Lys packaging into human immunodeficiency virus, type 1 viral particles.     

(Arg)3 within the N-terminal domain of glucosidase I contains ER targeting information but is not required absolutely for ER localization

Glucosidase I is an endoplasmic reticulum (ER) type II membrane enzyme that cleaves the distal alpha1,2-glucose of the asparagine-linked GlcNAc2-Man9-Glc3 precursor. To identify sequence motifs responsible for ER localization, we prepared a protein chimera by transferring the cytosolic and transmembrane domain of glucosidase I to the luminal domain of Golgi-Man9-mannosidase. The GIM9 hybrid was overexpressed in COS 1 cells as an ER-resident protein that displayed alpha1,2-mannosidase activity, excluding the possibility that the glucosidase I-specific domains interfere with folding of the Man9-mannosidase catalytic domain. After substitution of the Args in position 7, 8, or 9 relative to the N-terminus by leucine, the GIM9 mutants were transported to the cell surface indicating that the (Arg)3 sequence functions as an ER-targeting motif. Cell surface expression was also observed after substitution of Arg-7 or Arg-8 but not Arg-9 in GIM9 by either lysine or histidine. Thus the side chain structure, including its positive charge, appears to be essential for signal function. Analysis of the N-linked glycans suggests that the (Arg)3 sequence mediates ER localization through Golgi-to-ER retrograde transport. Glucosidase I remained localized in the ER after truncation or mutation of the N-terminal (Arg)3 signal, in contrast to comparable GIM9 mutants. ER localization was also observed with an M9GI chimera consisting of the cytosolic and transmembrane domain of Man9-mannosidase and the glucosidase I catalytic domain. ER-specific targeting information must therefore be provided by sequence motifs contained within the glucosidase I luminal domain. This structural information appears to direct ER localization by retention rather than by retrieval, as concluded from N-linked Man9-GlcNAc2 being the major glycan released from the wild-type enzyme.