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1.
The binding of high-mannose oligosaccharides to the domain-swapped dimeric form of the potent HIV-inactivating protein cyanovirin-N (CV-N) was investigated in solution by NMR, complementing recent structural studies by X-ray crystallography on similar complexes [J. Biol. Chem. 277 (2002) 34336]. The crystal structures of CV-N dimer complexed with Man-9 and hexamannoside revealed two carbohydrate binding sites on opposite ends of the molecule. No binding was observed at site 1, previously identified on the solution monomer of CV-N [Structure 9 (2001) 931; Shenoy et al., Chem. Biol. 9 (2002) 1109]. Here, we report the presence of four sugar binding sites on the CV-N dimer in solution, identified by chemical shift mapping with hexamannoside and nonamannoside, synthetic substructures of Man-9. Our results demonstrate that in solution the domain-swapped CV-N dimer, like the CV-N monomer, contains two types of sites that are available for carbohydrate binding, suggesting that the occlusion of the primary sites in the crystal is due to specific features of the solid state.  相似文献   

2.
Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 A resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 A resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.  相似文献   

3.
Han Z  Simpson JT  Fivash MJ  Fisher R  Mori T 《Peptides》2004,25(4):551-561
Cyanovirin-N (CV-N) exerts a potent human immunodeficiency virus (HIV)-inactivating activity against diverse strains of HIV by binding to the viral surface envelope glycoprotein gp120 and blocking its essential interactions with cellular receptors. Based on previous thermodynamic analyses, it has been speculated that discrete protein-protein interactions might play an important ancillary role in the CV-N/gp120 binding event, in addition to the interactions of CV-N with specific oligosaccharides present on gp120. Here, we report the identification and characterization of CV-N-binding peptides, which were isolated by screening of M13 phage-displayed peptide libraries. After performing three rounds of biopanning of the libraries against biotinylated CV-N, a CV-N-binding motif, X3CX6(W/F)(Y/F)CX2(Y/F), was evident. A vector was designed to express CV-N-binding peptides as a fusion with thioredoxin (Trx) containing a penta-His affinity tag. The CV-N-binding peptides fused with His-tagged Trx inhibited binding of the corresponding peptide-bearing phages to CV-N, confirming that the peptides possessed CV-N-binding activity. Optical biosensor binding studies showed that the one of the CV-N-binding peptide, TN10-1, bound to CV-N with a KD value of 1.9 microM. The results of alanine scanning mutagenesis of the peptide showed that aromatic residues at positions 11, 12, and 16, as well as the conformational structure of the peptide secured by a disulfide bond, were important for the binding interactions. A series of competitive binding assays confirmed that gp120 inhibited CV-N binding of the corresponding peptide-bearing phages, and suggested that TN10-1 peptides were mimicking the protein component of gp120 rather than mimicking specific oligosaccharides present on gp120.  相似文献   

4.
The cyanobacterial lectin Cyanovirin-N (CV-N) exhibits antiviral activity against HIV at a low nanomolar concentration by interacting with high-mannose oligosaccharides on the virus surface envelope glycoprotein gp120. Atomic structures of wild-type CV-N revealed a monomer in solution and a domain-swapped dimer in the crystal, with the monomer comprising two independent carbohydrate binding sites that individually bind with micromolar affinity to di- and trimannoses. In the mutant CVN(mutDB), the binding site on domain B was abolished and the protein was found to be completely inactive against HIV. We determined the solution NMR and crystal structures of this variant and characterized its sugar binding properties. In solution and the crystal, CVN(mutDB) is a monomer and no domain-swapping was observed. The protein binds to Man-3 and Man-9 with similar dissociation constants ( approximately 4 muM). This confirms that the nanomolar activity of wild-type CV-N is related to the multisite nature of the protein carbohydrate interaction.  相似文献   

5.
The 36 kDa vesicular-integral membrane protein, VIP36, has been originally isolated from MDCK cells as a component of glycolipid-enriched detergent-insoluble complexes containing apical marker proteins, and its luminal domain shows homology to leguminous plant lectins and ERGIC-53. As the first step to identify the functional role of VIP36, the carbohydrate binding specificity of VIP36 was investigated using a fusion protein of glutathione- S -transferase and luminal domain of VIP36 (Vip36). It was found that VIP36 recognizes high-mannose type glycans containing alpha1-->2 Man residues and alpha-amino substituted asparagine. The binding of Vip36 to high-mannose type glycans was independent of Ca(2+)and theoptimal condition was pH 6.0 at 37 degrees C. The concentration at which half inhibition of the binding by Man(7-9).GlcNAc(2). N Ac. Asn occurred was 1.0 x 10(-9)M. The association constant between Man(7-9).GlcNAc(2)in porcine thyroglobulin and immobilized Vip36 was 2.1 x 10(8)M(-1)as determined by means of a biosensor based on surface plasmon resonance. These results indicate that VIP36 functions as an intracellular lectin recognizing glycoproteins which possess high-mannose type glycans, (Manalpha1-->2)(2-4).Man(5). GlcNAc(2).  相似文献   

6.
Bonay P  Molina R  Fresno M 《Glycobiology》2001,11(9):719-729
The sugar binding specificity of the recently described mannose-specific carbohydrate-binding proteins (CBP) isolated to homogeneity from both the epimastigote and trypomastigote stages of the pathogenic protozoa Trypanosoma cruzi has been studied by quantitative hapten inhibition of the biotinylated CBPs to immobilized thyroglobulin using model oligosaccharides. The results clearly show a differential specificity toward high-mannose glycans between the CBPs from the two developmental stages. Thus, the isolated CBP from epimastigotes exhibited stronger affinity for higher mannose oligomers containing the Manalpha1-2Manalpha1-6Manalpha1-6 structure. Its affinity decreased, as did the number of mannose residues on the oligomer or removal of the terminal Manalpha1-2-linked mannose. By contrast the CBP isolated from the trypomastigote stage showed about 400-fold lower avidity than the epimastigote form, and contrary to it, it was slightly more specific toward Man5GlcNAc than Man9GlcNAc. Analysis of the interaction of epimastigote-Man-CBP with its ligands by UV difference spectroscopy indicates the existence of an extended binding site in that protein with a large enthalpic contribution to the binding. The thermodynamic parameters of binding were obtained by isothermal titration calorimetry and been found that the DeltaH values to be in good agreement with the van't Hoff values. The binding reactions are mainly enthalpically driven and exhibit enthalpy-enthropy compensation. In addition, analysis of the high-mannose glycans from different parts of the digestive tract of the reduviid insect vector of T. cruzi suggest a role of the CBP in the retention of the epimastigote stage in the anterior portion of the gut.  相似文献   

7.
The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77-G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.  相似文献   

8.
The crystal structure of cyanovirin-N (CV-N), a protein with potent antiviral activity, was solved at 1.5 A resolution by molecular replacement using as the search model the solution structure previously determined by NMR. The crystals belong to the space group P3221 with one monomer of CV-N in each asymmetric unit. The primary structure of CV-N contains 101 residues organized in two domains, A (residues 1 to 50) and B (residues 51 to 101), with a high degree of internal sequence and structural similarity. We found that under the conditions of the crystallographic experiments (low pH and 26 % isopropanol), two symmetrically related monomers form a dimer by domain swapping, such that domain A of one monomer interacts with domain B' of its crystallographic symmetry mate and vice versa. Because the two swapped domains are distant from each other, domain swapping does not result in additional intramolecular interactions. Even though one of the protein sample solutions that was used for crystallization clearly contained 100 % monomeric CV-N molecules, as judged by various methods, we were only able to obtain crystals containing domain-swapped dimers. With the exception of the unexpected phenomenon of domain swapping, the crystal structure of CV-N is very similar to the NMR structure, with a root-mean-square deviation of 0.55 A for the main-chain atoms, the best agreement reported to date for structures solved using both techniques.  相似文献   

9.
The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s).  相似文献   

10.
The development of anti-human immunodeficiency virus (HIV) microbicides for either topical or ex vivo use is of considerable interest, mainly due to the difficulties in creating a vaccine that would be active against multiple clades of HIV. Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium (blue-green algae) Nostoc ellipsosporum with potent virucidal activity, was identified in the search for such antiviral agents. The binding of CV-N to the heavily glycosylated HIV envelope protein gp120 is carbohydrate-dependent. Since previous CV-N-dimannose structures could not fully explain CV-N-oligomannose binding, we determined the crystal structures of recombinant CV-N complexed to Man-9 and a synthetic hexamannoside, at 2.5- and 2.4-A resolution, respectively. CV-N is a three-dimensional domain-swapped dimer in the crystal structures with two primary sites near the hinge region and two secondary sites on the opposite ends of the dimer. The binding interface is constituted of three stacked alpha1-->2-linked mannose rings for Man-9 and two stacked mannose rings for hexamannoside with the rest of the saccharide molecules pointing to the solution. These structures show unequivocally the binding geometry of high mannose sugars to CV-N, permitting a better understanding of carbohydrate binding to this potential new lead for the design of drugs against AIDS.  相似文献   

11.
Wang BJ  Tang W  Zhang P  Wei Q 《IUBMB life》2011,63(7):574-581
Previous deletion studies have suggested that Tyr306 has an important influence on the properties of protein phosphatase-1 (PP1). To test this inference, we constructed three site-directed mutants, PP1Y306A, PP1Y306K, and PP1Y306E. The specific activity of PP1Y306A was 3.5-fold higher than that of PP1wt, especially with K-R-Tp-I-R-R as substrate, and it also had a 13-fold higher Kcat and a 43-fold higher Kcat /Km . PP1Y306K and PP1Y306E, in that order, and were next in terms of increased activity. Use of the denaturant guanidine hydrochloride (GdnHCl) demonstrated that mutation of this site decreased enzyme stability. PP1Y306A and PP1Y306E lost all activity when incubated for 24 h in 0.6 M GdnHCl, and their fluorescence spectra confirmed the loss of stability. Because all three substitutions had a similar effect, we infer that the aromatic group of Tyr plays a crucial role in maintaining enzyme stability. Our results show that Tyr306 does affect the spatial conformation of the catalytic subunit of PP1 molecule. The IC50 of PP1Y306A for the inhibitor microcystin-LR was threefold higher than that of PP1wt, whereas those of PP1Y306E for tautomycin and norcantharidin were 15-fold and 10-fold higher, respectively. We conclude that Tyr306 plays an important role in enzyme stability and inhibitor binding.  相似文献   

12.
The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man(8)GlcNAc(2), and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole Delta och1 Delta mnn1 Delta mnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Man alpha1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Man alpha1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.  相似文献   

13.
Matei E  Louis JM  Jee J  Gronenborn AM 《Proteins》2011,79(5):1538-1549
Members of the cyanovirin-N homolog (CVNH) lectin family are found in bacteria, fungi and plants. As part of our ongoing work on CVNH structure-function studies, we determined the high-resolution NMR solution structure of the homolog from the wheat head blight disease causing ascomycetous fungus Gibberella zeae (or Fusarium graminearum), hereafter called GzCVNH. Like cyanovirin-N (CV-N), GzCVNH comprises two tandem sequence repeats and the protein sequence exhibits 30% identity with CV-N. The overall structure is similar to those of other members of the CVNH family, with the conserved pseudo-symmetric halves of the structure, domains A and B, closely resembling recently determined structures of Tuber borchii, Neurospora crassa, and Ceratopteris richardii CVNH proteins. Although GzCVNH exhibits a similar glycan recognition profile to CV-N and specifically binds to Manα(1-2)Manα, its weak carbohydrate binding affinity to only one binding site is insufficient for conferring anti-HIV activity.  相似文献   

14.
Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 A of the free and of the dimannose-bound forms of P51G-m4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.  相似文献   

15.
The prokaryotic lectin cyanovirin-N (CV-N) is a potent inhibitor of HIV envelope-mediated cell entry, and thus is a leading candidate among a new class of potential anti-HIV microbicides. The activity of CV-N is a result of interactions with the D1 arm of high-mannose oligosaccharides on the viral glycoprotein gp120. Here, we present computationally refined models of CV-N recognition of the di- and trisaccharides that represent the terminal three sugars of the D1 arm by each CV-N binding site. These models complement existing structural data, both from NMR spectroscopy and X-ray crystallography. When used with a molecular dynamics/continuum electrostatic (MD/PBSA) approach to compute binding free energies, these models explain the relative affinity of each site for the two saccharides. This work presents the first validation of the application of continuum electrostatic models to carbohydrate-protein association. Taken as a whole, the results both provide models of CV-N sugar recognition and demonstrate the utility of these computational methods for the study of carbohydrate-binding proteins.  相似文献   

16.
The psbO gene encoding the extrinsic 33 kDa protein of oxygen-evolving photosystem II (PSII) complex was cloned and sequenced from a red alga, Cyanidium caldarium. The gene encodes a polypeptide of 333 residues, of which the first 76 residues served as transit peptides for transfer across the chloroplast envelope and thylakoid membrane. The mature protein consists of 257 amino acids with a calculated molecular mass of 28,290 Da. The sequence homology of the mature 33 kDa protein was 42.9-50.8% between the red alga and cyanobacteria, and 44.7-48.6% between the red alga and higher plants. The cloned gene was expressed in Escherichia coli, and the recombinant protein was purified, subjected to protease-treatments. The cleavage sites of the 33 kDa protein by chymotrypsin or V8 protease were determined and compared among a cyanobacterium (Synechococcus elongatus), a euglena (Euglena gracilis), a green alga (Chlamydomonas reinhardtii) and two higher plants (Spinacia oleracea and Oryza sativa). The cleavage sites by chymotrypsin were at 156F and 190F for the cyanobacterium, 159M, 160F and 192L for red alga, 11Y and 151F for euglena, 10Yand 150F for green alga, and 16Y for spinach, respectively. The cleavage sites by V8 protease were at 181E (cyanobacterium), 182E and 195E (red alga), 13E, 67E, 69E, 153D and 181E (euglena), 176E and 180E (green alga), and 18E or 19E (higher plants). Since most of the residues at these cleavage sites were conserved among the six organisms, the results indicate that the structure of the 33 kDa protein, at least the structure based on the accessibility by proteases, is different among these organisms. In terms of the cleavage sites, the structure of the 33 kDa protein can be divided into three major groups: cyanobacterial and red algal-type has cleavage sites at residues around 156-195, higher plant-type at residues 16-19, and euglena and green algal-type at residues of both cyanobacterial and higher plant-types.  相似文献   

17.
We have previously described a hybrid protein, GHHA, that contains a fragment of the influenza hemagglutinin joined to the C terminus of a nearly complete rat growth hormone (Rizzolo, L.J., Finidori, J., Gonzalez, A., Arpin, M., Ivanov, I.E., Adesnik, M., and Sabatini, D.D. (1985) J. Cell Biol. 101, 1351-1362). GHHA was transported from the rough endoplasmic reticulum (ER) to a smooth cisterna, continuous with the rough ER, but proximal to the Golgi apparatus. We have now labeled GHHA with [3H]palmitate, demonstrating that fatty acylation can occur in the ER. As expected for a thioester linkage, the label was released from GHHA by hydroxylamine and identified as palmitic acid by thin-layer chromatography. In a second study, we analyzed the structure of the N-linked carbohydrate chain of GHHA. The N-linked oligosaccharides, all high-mannose type, were released by endoglycosidase H and size-fractionated by high pressure liquid chromatography. The predominant structures were Glc1Man8GlcNAc and Man8GlcNAc, indicating that only 2 or 3 glucose and 1 mannose residues were removed from the original Glc3Man9GlcNAc2. Determination of the structure by acetolysis fragmentation indicated that a single Man8GlcNAc isomer was formed by a deoxymannojirimycin-sensitive alpha-mannosidase. This contrasts with a previously characterized ER alpha-mannosidase (Bischoff, J., Liscum, L., and Kornfeld, R. (1986) J. Biol. Chem. 261, 4766-4774) that generates the same isomer, but is deoxymannojirimycin-resistant. These data suggest the possibility that different enzymes are partitioned within the ER.  相似文献   

18.
Previous studies have indicated the existence of separate binding sites of ubiquitin-protein ligase, E3, specific for basic (Type I) or bulky hydrophobic (Type II) NH2-terminal amino acid residues of proteins. Another class (Type III) of protein substrates appeared to interact with E3 at regions other than the NH2 terminus (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698). In the present study we have used affinity chromatography on immobilized protein substrates to examine the question of whether the different binding sites belong to one E3 enzyme, or to different E3 species. Another objective was to develop a procedure for the extensive purification of E3. When a crude extract of reticulocytes is applied to Type I or Type II protein substrates linked to Sepharose, E3 becomes strongly bound to the affinity columns and is not eluted with salt at high concentration. However, the enzyme can be specifically eluted by a dipeptide that has an NH2-terminal residue similar to that of matrix-bound protein substrate. A 350-fold purification is obtained in this single step. Preparations of E3 purified on either Type I or Type II protein substrate affinity columns act on both types of protein substrates, indicating that the separate binding sites for basic and hydrophobic NH2-terminal residues belong to one enzyme. Another species of E3 that acts strongly on some Type III protein substrates does not bind to Type I or Type II protein substrate affinity columns.  相似文献   

19.
Previous studies on the catalytic subunit of cAMP-dependent protein kinase (PKA) identified a conserved interaction pair comprised of Tyr204 from the P+1 loop and Glu230 at the end of the alphaF-helix. Single-point mutations of Tyr204 to Ala (Y204A) and Glu230 to Gln (E230Q) both resulted in alterations in enzymatic kinetics. To understand further the molecular basis for the altered kinetics and the structural role of each residue, we analyzed the Y204A and the E230Q mutants using hydrogen/deuterium (H/D) exchange coupled with mass spectrometry and other biophysical techniques. The fact that the mutants exhibit distinct molecular properties, supports previous hypotheses that these two residues, although in the same interaction node, contribute to the same enzymatic functions through different molecular pathways. The Tyr204 mutation appears to affect the dynamic properties, while the Glu230 mutation affects the surface electrostatic profile of the enzyme. Furthermore, H/D exchange analysis defines the dynamic allosteric range of Tyr204 to include the catalytic loop and three additional distant surface regions, which exhibit increased deuterium exchange in the Y204A but not the E230Q mutant. Interestingly, these are the exact regions that previously showed decreased deuterium exchange upon binding of the RIalpha regulatory subunit of PKA. We propose that these sites, coupled with the P+1 loop through Tyr204, represent one of the major allosteric networks in the kinase. This coupling provides a coordinated response for substrate binding and enzyme catalysis. H/D exchange analysis also further defines the stable core of the catalytic subunit to include the alphaE, alphaF and alphaH-helix. All these observations lead to an interesting new way to view the structural architecture and allosteric conformational regulation of the protein kinase molecule.  相似文献   

20.
The major phosphorylation sites of the bovine papillomavirus E2 transactivator protein are two serine residues, 298 and 301, that are located in a flexible hinge region between the DNA binding and transactivation domains. Phosphorylation of serine residue 301 promotes ubiquitination and rapid degradation of the E2 protein by the proteasome pathway. To understand the mechanism through which phosphorylation regulates the intracellular levels of this unique papillomavirus regulatory protein, we have carried out an extensive mutational analysis of the region surrounding the phosphorylation sites of the E2 protein. Our results indicate that casein kinase II phosphorylates serine 301. However, phosphorylation of serine 301 is not a sufficient recognition motif for proteasomal degradation; other residues that directly surround the phosphorylation sites are crucial for E2 degradation. The phenotypes of E2 proteins mutated in this region indicate that phosphorylation of serine 301 induces a conformational change that leads to degradation of the E2 protein. In support of this model, circular dichroism studies of the conformational tendencies of peptides from this region indicate that phosphorylation at position 301 decreases the local thermodynamic stability of this region. Thus, this region appears to have evolved to display a marginal local thermodynamic stability that can be regulated by phosphorylation, leading to targeted degradation of the E2 protein.  相似文献   

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