首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.  相似文献   

2.
The effect of hydrophobic core packing on sidechain dynamics was analyzed by comparing the dynamics of wild-type (WT) ubiquitin to those of a variant which has seven core mutations. This variant, 1D7, was designed to resemble WT by having a well-packed core of similar volume, and we find that its overall level of dynamics is only subtly different from WT. However, the mutations caused a redistribution in the positions of core residues that are dynamic. This correlates with the tendency of these residues to populate unfavorable rotamers, suggesting that strain from poor sidechain conformations may promote increased flexibility as a mechanism to relieve unfavorable steric interactions. The results demonstrate that even when core volume is conserved, different packing arrangements in mutants can alter dynamic behavior.  相似文献   

3.
The mucosotrophic human papillomaviruses (HPVs) are classified as high-risk (HR) or low-risk (LR) genotypes based on their neoplastic properties. We have demonstrated previously that the E7 protein destabilizes p130, a pRb-related pocket protein, thereby promoting S-phase reentry in postmitotic, differentiated keratinocytes of squamous epithelia, and that HR HPV E7 does so more efficiently than LR HPV E7. The E7 proteins of LR HPV-11 and -6b uniquely possess lysine residues following a casein kinase II phosphorylation motif which is critical for the biological function of E7. We now show that mutations of these lysine residues elevated the efficiency of S-phase reentry, independent of their charge. An 11E7 K39,42R mutation moderately increased the association with and the destabilization of p130. Unexpectedly, polyubiquitination on these lysine residues did not attenuate E7 activity, as their mutation caused elevated proteasomal degradation and decreased protein stability. In this regard, the biologically more potent HR HPV E7 proteins were also less stable than the LR HPV E7 proteins. We infer that these lysine residues impede functional protein-protein interactions. A G22D mutation of 11E7 at the pocket protein binding motif possessed augmented efficiency in promoting S-phase reentry and strongly enhanced association with p130 and pRb. The combined effects of these two classes of 11E7 mutations exhibited an efficiency of S-phase reentry comparable to that of HR HPV E7. Thus, these nonconserved residues are primarily responsible for the differential abilities of LR and HR HPV E7 proteins to promote unscheduled DNA replication in organotypic raft cultures.  相似文献   

4.
Boswell S  Mathew J  Beach M  Osuna R  Colón W 《Biochemistry》2004,43(10):2964-2977
The diverse roles of tyrosine residues in proteins may be attributed to their dual hydrophobic and polar nature, which can result in hydrophobic and ring stacking interactions, as well as hydrogen bonding. The small homodimeric DNA binding protein, factor for inversion stimulation (FIS), contains four tyrosine residues located at positions 38, 51, 69, and 95, each involved in specific intra- or intermolecular interactions. To investigate their contributions to the stability, flexibility, and spectroscopic properties of FIS, each one was independently mutated to phenylalanine. Equilibrium denaturation experiments show that Tyr95 and Tyr51 stabilize FIS by about 2 and 1 kcal/mol, respectively, as a result of their involvement in a hydrogen bond-salt bridge network. In contrast, Tyr38 destabilizes FIS by about 1 kcal/mol due to the placement of a hydroxyl group in a hydrophobic environment. The stability of FIS was not altered when the solvent-exposed Tyr69 was mutated. Limited proteolysis with trypsin and V8 proteases was used to monitor the flexibility of the C-terminus (residues 71-98) and the dimer core (residues 26-70), respectively. The results for Y95F and Y51F FIS revealed a different proteolytic susceptibility of the dimer core compared to the C-terminus, suggesting an increased flexibility of the latter. DNA binding affinity of the various FIS mutants was only modestly affected and correlated inversely with the C-terminal flexibility probed by trypsin proteolysis. Deconvolution of the fluorescence contribution of each mutant revealed that it varies in intensity and direction for each tyrosine in WT FIS, highlighting the role of specific interactions and the local environment in determining the fluorescence of tyrosine residues. The significant changes in stability, flexibility, and signals observed for the Y51F and Y95F mutations are attributed to their coupled participation in the hydrogen bond-salt bridge network. These results highlight the importance of tyrosine hydrogen-bonding and packing interactions for the stability of FIS and demonstrate the varying roles that tyrosine residues can play on the structural and spectroscopic properties of even small proteins.  相似文献   

5.
6.
Alphavirus budding from the plasma membrane occurs through the specific interaction of the nucleocapsid core with the cytoplasmic domain of the E2 glycoprotein (cdE2). Structural studies of the Sindbis virus capsid protein (CP) have suggested that these critical interactions are mediated by the binding of cdE2 into a hydrophobic pocket in the CP. Several molecular genetic studies have implicated amino acids Y400 and L402 in cdE2 as important for the budding of alphaviruses. In this study, we characterized the role of cdE2 residues in structural polyprotein processing, glycoprotein transport, and capsid interactions. Along with hydrophobic residues, charged residues in the N terminus of cdE2 were critical for the effective interaction of cores with cdE2, a process required for virus budding. Mutations in the C-terminal signal sequence region of cdE2 affected E2 protein transport to the plasma membrane, while nonbudding mutants that were defective in cdE2-CP interaction accumulated E2 on the plasma membrane. The interaction of cdE2 with cytoplasmic cores purified from infected cells and in vitro-assembled core-like particles suggests that cdE2 interacts with assembled cores to mediate budding. We hypothesize that these cdE2 interactions induce a change in the organization of the nucleocapsid core upon binding leading to particle budding and priming of the nucleocapsid cores for disassembly that is required for virus infection.  相似文献   

7.
SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8–43) and a short cytoplasmic helix (residues 53–60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6–18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5’ position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.  相似文献   

8.
The C2 domain of cytosolic phospholipase A2 (cPLA2) is involved in the Ca2+-dependent membrane binding of this protein. To identify protein residues in the C2 domain of cPLA2 essential for its Ca2+ and membrane binding, we selectively mutated Ca2+ ligands and putative membrane-binding residues of cPLA2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration. The mutations of five Ca2+ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane binding and activation of cPLA2, indicating that two calcium ions bound to the C2 domain have differential roles. The mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A) in the calcium binding loops show that the membrane binding of cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane. Leu39 and Val97 are fully inserted into the membrane, whereas Phe35 and Tyr96 are partially inserted. Finally, the mutations of four cationic residues in a beta-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA2 to zwitterionic and anionic membranes, respectively, indicating that they are not directly involved in membrane binding. In conjunction with our previous study on the C2 domain of protein kinase C-alpha (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), these results demonstrate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetration of protein and that individual Ca2+ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins.  相似文献   

9.
The E6 protein of human papillomavirus (HPV) exhibits complex interaction patterns with several host proteins, and their roles in HPV-mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein synapse-associated protein 97 (SAP97). All of the potential binding sites in SAP97 bind E6 with micromolar affinity. The dissociation rate constants govern the different affinities of HPV16 and HPV18 E6 for SAP97. Unexpectedly, binding is not mutually exclusive, and all three PDZ domains can simultaneously bind E6. Intriguingly, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues distal to the canonical binding pocket in the PDZ(2) domain exhibited noncanonical interactions with the E6 protein. This is consistent with a larger proportion of the protein surface defining binding specificity, as compared with that reported previously.  相似文献   

10.
Hydrophobic residues outside the active site of HIV-1 protease frequently mutate in patients undergoing protease inhibitor therapy; however, the mechanism by which these mutations confer drug resistance is not understood. From analysis of molecular dynamics simulations, 19 core hydrophobic residues appear to facilitate the conformational changes that occur in HIV-1 protease. The hydrophobic core residues slide by each other, exchanging one hydrophobic van der Waal contact for another, with little energy penalty, while maintaining many structurally important hydrogen bonds. Such hydrophobic sliding may represent a general mechanism by which proteins undergo conformational changes. Mutation of these residues in HIV-1 protease would alter the packing of the hydrophobic core, affecting the conformational flexibility of the protease. Therefore these residues impact the dynamic balance between processing substrates and binding inhibitors, and thus contribute to drug resistance.  相似文献   

11.
Huang Y  Liu Z 《PloS one》2010,5(11):e15375
Proteins function by interacting with other molecules, where both native and nonnative interactions play important roles. Native interactions contribute to the stability and specificity of a complex, whereas nonnative interactions mainly perturb the binding kinetics. For intrinsically disordered proteins (IDPs), which do not adopt rigid structures when being free in solution, the role of nonnative interactions may be more prominent in binding processes due to their high flexibilities. In this work, we investigated the effect of nonnative hydrophobic interactions on the coupled folding and binding processes of IDPs and its interplay with chain flexibility by conducting molecular dynamics simulations. Our results showed that the free-energy profiles became rugged, and intermediate states occurred when nonnative hydrophobic interactions were introduced. The binding rate was initially accelerated and subsequently dramatically decreased as the strength of the nonnative hydrophobic interactions increased. Both thermodynamic and kinetic analysis showed that disordered systems were more readily affected by nonnative interactions than ordered systems. Furthermore, it was demonstrated that the kinetic advantage of IDPs ("fly-casting" mechanism) was enhanced by nonnative hydrophobic interactions. The relationship between chain flexibility and protein aggregation is also discussed.  相似文献   

12.
The HPV E6 oncoprotein maintains the malignant phenotype of HPV-positive cancer cells and represents an attractive therapeutic target. E6 forms a complex with the cellular E6AP ubiquitin ligase, ultimately leading to p53 degradation. The recently elucidated x-ray structure of a HPV16 E6/E6AP complex showed that HPV16 E6 forms a distinct binding pocket for E6AP. This discovery raises the question whether the E6AP binding pocket is druggable, i. e. whether it provides a docking site for functional E6 inhibitors. To address these issues, we performed a detailed analysis of the HPV16 E6 interactions with two small peptides: (i) E6APpep, corresponding to the E6 binding domain of E6AP, and (ii) pep11**, a peptide that binds to HPV16 E6 and, in contrast to E6APpep, induces apoptosis, specifically in HPV16-positive cancer cells. Surface plasmon resonance, NMR chemical shift perturbation, and mammalian two-hybrid analyses coupled to mutagenesis indicate that E6APpep contacts HPV16 E6 amino acid residues within the E6AP pocket, both in vitro and intracellularly. Many of these amino acids were also important for binding to pep11**, suggesting that the binding sites for the two peptides on HPV16 E6 overlap. Yet, few E6 amino acids were differentially involved which may contribute to the higher binding affinity of pep11**. Data from the HPV16 E6/pep11** interaction allowed the rational design of single amino acid exchanges in HPV18 and HPV31 E6 that enabled their binding to pep11**. Taken together, these results suggest that E6 molecular surfaces mediating E6APpep binding can also accommodate pro-apoptotic peptides that belong to different sequence families. As proof of concept, this study provides the first experimental evidence that the E6AP binding pocket is druggable, opening new possibilities for rational, structure-based drug design.  相似文献   

13.
Abstract

TATA-box binding protein (TBP) in a monomelic form and the complexes it forms with DNA have been elucidated with molecular dynamics simulations. Large TBP domain motions (bend and twist) are detected in the monomer as well as in the DNA complexes; these motions can be important for TBP binding of DNA. TBP interacts with guanine bases flanking the TATA element in the simulations of the complex; these interactions may explain the preference for guanine observed at these DNA positions. Side chains of some TBP residues at the binding interface display significant dynamic flexibility that results in ‘flipflop’ contacts involving multiple base pairs of the DNA. We discuss the possible functional significance of these observations.  相似文献   

14.
Horner SM  DiMaio D 《Journal of virology》2007,81(12):6254-6264
Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells.  相似文献   

15.
The first crystal structure of a complex between a hepatitis C virus (HCV) core protein-derived peptide (residues 13-40) and the Ab fragment of a murine mAb (19D9D6) has been solved, allowing determination of the recognized epitope and elucidation of its conformation. This Ab, raised against the first 120 residues of the core protein, recognizes core particles and strongly competes with anticore human Abs, suggesting that it is highly representative of the human anti-HCV core response. Its epitope lies within the first 45 aa of the protein, the major antigenic segment of core recognized both by murine and human Abs. Surprisingly, the recognized epitope (29-37: QIVGGVYLL) has an unusual preponderance of hydrophobic residues, some of which are buried in a small hydrophobic core in the nuclear magnetic resonance structure of the peptide (2-45) in solution, suggesting that the Ab may induce a structural rearrangement upon recognition. The flexibility may reside entirely within the Ag, since the Fab'-peptide complex structure at 2.34 A shows that the Ab binding site is hardly perturbed by complexation. Given that the recognized residues are unlikely to be solvent exposed, we are left with the interesting possibility that Ab-core interactions may take place in a nonaqueous environment.  相似文献   

16.
Yang Q  Berton N  Manning MC  Catalano CE 《Biochemistry》1999,38(43):14238-14247
The terminase enzyme from bacteriophage lambda is responsible for the insertion of a dsDNA genome into the confines of the viral capsid. The holoenzyme is composed of gpA and gpNu1 subunits in a gpA(1) x gpNu1(2) stoichiometry. While genetic studies have described regions within the two proteins responsible for DNA binding, capsid binding, and subunit interactions in the holoenzyme complex, biochemical characterization of these domains is limited. We have previously described the cloning, expression, and biochemical characterization of a soluble DNA binding domain of the terminase gpNu1 subunit (Met1 to Lys100) and suggested that the hydrophobic region spanning Lys100 to Pro141 defines a domain responsible for self-association interactions, and that is important for cooperative DNA binding [Yang et al. (1999) Biochemistry 38, 465-477]. We further suggested that the genetically defined gpA-interactive domain in the C-terminal half of the protein is limited to the C-terminal approximately 40 amino acids of gpNu1. Here we describe the cloning, expression, and biochemical characterization of gpNu1DeltaP141, a deletion mutant of gpNu1 that comprises the DNA binding domain and the putative hydrophobic self-assembly domain of the full-length protein. Purified gpNu1DeltaP141 shows a strong tendency to aggregate in solution; However, the protein remains soluble in 0.4 M guanidine hydrochloride, and circular dichroism (CD) and fluorescence spectroscopic studies demonstrate that the protein is folded under these conditions. Moreover, CD spectroscopy and thermally induced unfolding studies suggest that the DNA binding domain and the self-association domain represent independent folding domains of gpNu1DeltaP141. The mutant protein interacts weakly with the gpA subunit, but does not form a catalytically competent holoenzyme complex, suggesting that the C-terminal 40 residues are important for appropriate subunit interactions. Importantly, gpNu1DeltaP141 binds DNA tightly, but with less specificity than does full-length protein, and the data suggest that the C-terminal residues are further required for specific DNA binding activity. The implications of these results in the assembly of a functional holoenzyme complex are discussed.  相似文献   

17.
Using Brownian dynamics simulations, all of the charged residues in Chlamydomonas reinhardtii cytochrome c(6) (cyt c(6)) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f (cyt f) were modeled. Systematic mutation of charged residues on both PC and cyt c(6) confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c(6) displayed similar inhibition classes. Our results indicate a difference between the two acidic clusters on PC. Mutations D44A and E43A of the lower cluster showed greater inhibition than do any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c(6) that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. In PC residues D42, E43, D44, D53, D59, D61, and E85, and in cyt c(6) residues D2, E54, K57, D65, R66, E70, E71, and the heme had significant electrostatic contacts with cyt f charged residues. PC and cyt c(6) showed different binding sites and orientations on cyt f. As there are no experimental cyt c(6) mutation data available for algae, our results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement, which supports the predictive power of Brownian dynamics simulations in mutagenesis studies.  相似文献   

18.
Interaction between the E2 protein and E1 helicase of human papillomaviruses (HPVs) is essential for the initiation of viral DNA replication. We recently described a series of small molecules that bind to the N-terminal transactivation domain (TAD) of HPV type 11 E2 and inhibits its interaction with E1 in vitro and in cellular assays. Here we report the crystal structures of both the HPV11 TAD and of a complex between this domain and an inhibitor, at 2.5- and 2.4-A resolution, respectively. The HPV11 TAD structure is very similar to that of the analogous domain of HPV16. Inhibitor binding caused no significant alteration of the protein backbone, but movements of several amino acid side chains at the binding site, in particular those of Tyr-19, His-32, Leu-94, and Glu-100, resulted in the formation of a deep hydrophobic pocket that accommodates the indandione moiety of the inhibitor. Mutational analysis provides functional evidence for specific interactions between Tyr-19 and E1 and between His-32 and the inhibitor. A second inhibitor molecule is also present at the binding pocket. Although evidence is presented that this second molecule makes only weak interactions with the protein and is likely an artifact of crystallization, its presence defines additional regions of the binding pocket that could be exploited to design more potent inhibitors.  相似文献   

19.
The class 1 PDZ domain Tip-1 protein was first identified as a binding partner for the Human T-Cell Leukaemia Virus type 1 (HTLV-1) Tax oncoprotein. It was later shown to interact with: the RhoA signaling effector Rhotekin, the Wnt signaling effector β-catenin and the E6 oncoprotein from high-risk HPV16 but not low-risk HPV6. These observations suggested that Tip-1 may be an important “hub” protein that is involved in pathways with a proven link to carcinogenesis. Based on these findings, it was decided to further characterize the cellular role of Tip-1 by carrying out a yeast two-hybrid screen to identify new binding partners in order to uncover potentially novel functions of this protein. This identified an intracellular fragment of the trans-membrane receptor plexin D1 and a C-terminal fragment of the AT hook DNA binding containing 1 (AHDC1) protein which had a carboxyl terminal PDZ binding domain consensus sequence. Both of these interactions were confirmed by yeast mating assay which was also used to show that mutant constructs of AHDC1 lacking the carboxyl PDZ binding site did not bind Tip-1. Immunofluorescent imaging of these proteins in HPV16 E6 expressing human C33A cervical carcinoma cells suggested they may co-localize.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号