Biomedical subjects

Nikola P Pavletich

Publications and source records attributed to Nikola P Pavletich.

At least 19 recordsLinked to original sources

PubMed2007

Ubiquitination regulates PTEN nuclear import and tumor suppression.

The PTEN tumor suppressor is frequently affected in cancer cells, and inherited PTEN mutation causes cancer-susceptibility conditions such as Cowden syndrome. PTEN acts as a plasma-membrane lipid-phosphatase antagonizing the phosphoinositide 3-kinase/AKT cell survival pathway. However, PTEN is also found in cell nuclei, but mechanism, function, and relevance of nuclear localization remain unclear. We show that nuclear PTEN is essential for tumor suppression and that PTEN nuclear import is mediated by its monoubiquitination. A lysine mutant of PTEN, K289E associated with Cowden syndrome, retains catalytic activity but fails to accumulate in nuclei of patient tissue due to an import defect. We identify this and another lysine residue as major monoubiquitination sites essential for PTEN import. While nuclear PTEN is stable, polyubiquitination leads to its degradation in the cytoplasm. Thus, we identify cancer-associated mutations of PTEN that target its posttranslational modification and demonstrate how a discrete molecular mechanism dictates tumor progression by differentiating between degradation and protection of PTEN.

Active Transport, Cell Nucleus↗

PubMed2005

Structure of the Rb C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F release.

The retinoblastoma (Rb) protein negatively regulates the G1-S transition by binding to the E2F transcription factors, until cyclin-dependent kinases phosphorylate Rb, causing E2F release. The Rb pocket domain is necessary for E2F binding, but the Rb C-terminal domain (RbC) is also required for growth suppression. Here we demonstrate a high-affinity interaction between RbC and E2F-DP heterodimers shared by all Rb and E2F family members. The crystal structure of an RbC-E2F1-DP1 complex reveals an intertwined heterodimer in which the marked box domains of both E2F1 and DP1 contact RbC. We also demonstrate that phosphorylation of RbC at serines 788 and 795 destabilizes one set of RbC-E2F-DP interactions directly, while phosphorylation at threonines 821 and 826 induces an intramolecular interaction between RbC and the Rb pocket that destabilizes the remaining interactions indirectly. Our findings explain the requirement of RbC for high-affinity E2F binding and growth suppression and establish a mechanism for the regulation of Rb-E2F association by phosphorylation.

Amino Acid Sequence↗

PubMed2005

Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase.

The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27(Kip1) phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27(Kip1) binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27(Kip1) is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27(Kip1) to the SCF(Skp2)-Cks1 complex.

Carrier Proteins↗

PubMed2005

Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54.

SWI2/SNF2 chromatin-remodeling proteins mediate the mobilization of nucleosomes and other DNA-associated proteins. SWI2/SNF2 proteins contain sequence motifs characteristic of SF2 helicases but do not have helicase activity. Instead, they couple ATP hydrolysis with the generation of superhelical torsion in DNA. The structure of the nucleosome-remodeling domain of zebrafish Rad54, a protein involved in Rad51-mediated homologous recombination, reveals that the core of the SWI2/SNF2 enzymes consist of two alpha/beta-lobes similar to SF2 helicases. The Rad54 helicase lobes contain insertions that form two helical domains, one within each lobe. These insertions contain SWI2/SNF2-specific sequence motifs likely to be central to SWI2/SNF2 function. A broad cleft formed by the two lobes and flanked by the helical insertions contains residues conserved in SWI2/SNF2 proteins and motifs implicated in DNA-binding by SF2 helicases. The Rad54 structure suggests that SWI2/SNF2 proteins use a mechanism analogous to helicases to translocate on dsDNA.

Amino Acid Motifs↗

PubMed2005

The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA junction.

The BRCA2 tumour suppressor is essential for the error-free repair of double-strand breaks (DSBs) in DNA by homologous recombination. This is mediated by RAD51, which forms a nucleoprotein filament with the 3' overhanging single-stranded DNA (ssDNA) of the resected DSB, searches for a homologous donor sequence, and catalyses strand exchange with the donor DNA. The 3,418-amino-acid BRCA2 contains eight approximately 30-amino-acid BRC repeats that bind RAD51 (refs 5, 6) and a approximately 700-amino-acid DBD domain that binds ssDNA. The isolated BRC and DBD domains have the opposing effects of inhibiting and stimulating recombination, respectively, and the role of BRCA2 in repair has been unclear. Here we show that a full-length BRCA2 homologue (Brh2) stimulates Rad51-mediated recombination at substoichiometric concentrations relative to Rad51. Brh2 recruits Rad51 to DNA and facilitates the nucleation of the filament, which is then elongated by the pool of free Rad51. Brh2 acts preferentially at a junction between double-stranded DNA (dsDNA) and ssDNA, with strict specificity for the 3' overhang polarity of a resected DSB. These results establish a BRCA2 function in RAD51-mediated DSB repair and explain the loss of this repair capacity in BRCA2-associated cancers.

Adenosine Triphosphatases↗

PubMed2005

High-level expression and purification of recombinant SCF ubiquitin ligases.

The SCF complexes are the prototype of a superfamily of cullin-dependent ubiquitin ligases, which regulate diverse cellular functions by promoting the ubiquitination of a large number of regulatory and signaling proteins. The SCF complexes are organized by the elongated scaffold protein subunit Cul1, which interacts with the Rbx1 RING finger protein at one end and the Skp1 adaptor protein at the other. By binding to Skp1, members of the F-box protein family are responsible for recruiting specific substrates to the ligase machine. This chapter describes methods that we have developed to achieve high-level expression and purification of two recombinant SCF complexes from both insect cells and bacteria. We emphasize the power of protein coexpression and a novel "Split-n-Coexpress" method in producing soluble and functional recombinant proteins and protein complexes. We propose that similar approaches can be used to obtain large quantities of other SCF and SCF-like complexes for biochemical and structural investigations.

Animals↗

PubMed2004

N-(cycloalkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumor agent.

N-Acyl-2-aminothiazoles with nonaromatic acyl side chains containing a basic amine were found to be potent, selective inhibitors of CDK2/cycE which exhibit antitumor activity in mice. In particular, compound 21 [N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide, BMS-387032], has been identified as an ATP-competitive and CDK2-selective inhibitor which has been selected to enter Phase 1 human clinical trials as an antitumor agent. In a cell-free enzyme assay, 21 showed a CDK2/cycE IC(50) = 48 nM and was 10- and 20-fold selective over CDK1/cycB and CDK4/cycD, respectively. It was also highly selective over a panel of 12 unrelated kinases. Antiproliferative activity was established in an A2780 cellular cytotoxicity assay in which 21 showed an IC(50) = 95 nM. Metabolism and pharmacokinetic studies showed that 21 exhibited a plasma half-life of 5-7 h in three species and moderately low protein binding in both mouse (69%) and human (63%) serum. Dosed orally to mouse, rat, and dog, 21 showed 100%, 31%, and 28% bioavailability, respectively. As an antitumor agent in mice, 21 administered at its maximum-tolerated dose exhibited a clearly superior efficacy profile when compared to flavopiridol in both an ip/ip P388 murine tumor model and in a s.c./i.p. A2780 human ovarian carcinoma xenograft model.

Animals↗

PubMed2004

Structural differences in the DNA binding domains of human p53 and its C. elegans ortholog Cep-1.

The DNA binding domains of human p53 and Cep-1, its C. elegans ortholog, recognize essentially identical DNA sequences despite poor sequence similarity. We solved the three-dimensional structure of the Cep-1 DNA binding domain in the absence of DNA and compared it to that of human p53. The two domains have similar overall folds. However, three loops, involved in DNA and Zn binding in human p53, contain small alpha helices in Cep-1. The alpha helix in loop L3 of Cep-1 orients the side chains of two conserved arginines toward DNA; in human p53, both arginines are mutation hotspots, but only one contacts DNA. The alpha helix in loop L1 of Cep-1 repositions the entire loop, making it unlikely for residues of this loop to contact bases in the major groove of DNA, as occurs in human p53. Thus, during evolution there have been considerable changes in the structure of the p53 DNA binding domain.

Amino Acid Sequence↗

PubMed2003

Werner syndrome protein contains three structure-specific DNA binding domains.

Werner syndrome (WS) is a premature aging syndrome caused by mutations in the WS gene (WRN) and a deficiency in the function of the Werner protein (WRN). WRN is a multifunctional nuclear protein that catalyzes three DNA-dependent reactions: a 3'-5'-exonuclease, an ATPase, and a 3'-5'-helicase. Deficiency in WRN results in a cellular phenotype of genomic instability. The biochemical characteristics of WRN and the cellular phenotype of WRN mutants suggest that WRN plays an important role in DNA metabolic pathways such as recombination, transcription, replication, and repair. The catalytic activities of WRN have been extensively studied and are fairly well understood. However, much less is known about the domain-specific interactions between WRN and its DNA substrates. This study identifies and characterizes three distinct WRN DNA binding domains using recombinant truncated fragments of WRN and five DNA substrates (long forked duplex, blunt-ended duplex, single-stranded DNA, 5'-overhang duplex, and Holliday junction). Substrate-specific DNA binding activity was detected in three domains, one N-terminal and two different C-terminal WRN fragments (RecQ conserved domain and helicase RNase D conserved domain-containing domains). The substrate specificity of each DNA binding domain may indicate that each protein domain has a distinct biological function. The importance of these results is discussed with respect to proposed roles for WRN in distinct DNA metabolic pathways.

Binding Sites↗

PubMed2003

Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding.

The Smad family of proteins mediates transforming growth factor-beta signaling from cell membrane to the nucleus. In the nucleus, Smads serve as transcription factors by directly binding to specific DNA sequences and regulating the expression of ligand-response genes. A previous structural analysis, at 2.8-A resolution, revealed a novel DNA-binding mode for the Smad MH1 domain but did not allow accurate assignment of the fines features of protein-DNA interactions. The crystal structure of a Smad3 MH1 domain bound to a palindromic DNA sequence, determined at 2.4-A resolution, reveals a surprisingly important role for water molecules. The asymmetric placement of the DNA-binding motif (a conserved 11-residue beta-hairpin) in the major groove of DNA is buttressed by seven well ordered water molecules. These water molecules make specific hydrogen bonds to the DNA bases, the DNA phosphate backbones, and several critical Smad3 residues. In addition, the MH1 domain is found to contain a bound zinc atom using four invariant residues among Smad proteins, three cysteines and one histidine. Removal of the zinc atom results in compromised DNA binding activity. These results define the Smad MH1 domain as a zinc-coordinating module that exhibits unique DNA binding properties.

Amino Acid Sequence↗

PubMed2003

Crystal structure of a human Mob1 protein: toward understanding Mob-regulated cell cycle pathways.

The Mob protein family comprises a group of highly conserved eukaryotic proteins whose founding member functions in the mitotic exit network. At the molecular level, Mob proteins act as kinase-activating subunits. We cloned a human Mob1 family member, Mob1A, and determined its three-dimensional structure by X-ray crystallography. The core of Mob1A consists of a four-helix bundle that is stabilized by a bound zinc atom. The N-terminal helix of the bundle is solvent exposed and together with adjacent secondary structure elements forms an evolutionarily conserved surface with a strong negative electrostatic potential. Several conditional mutant alleles of S. cerevisiae MOB1 target this surface and decrease its net negative charge. Interestingly, the kinases with which yeast Mob proteins interact have two conserved basic regions within their N-terminal lobe. Thus, Mob proteins may regulate their target kinases through electrostatic interactions mediated by conserved charged surfaces.

Amino Acid Sequence↗

PubMed2003

Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase.

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the >70 human F box proteins allowing the recognition of a wide range of substrates. The F box protein beta-TrCP1 recognizes the doubly phosphorylated DpSGphiXpS destruction motif, present in beta-catenin and IkappaB, and directs the SCF(beta-TrCP1) to ubiquitinate these proteins at specific lysines. The 3.0 A structure of a beta-TrCP1-Skp1-beta-catenin complex reveals the basis of substrate recognition by the beta-TrCP1 WD40 domain. The structure, together with the previous SCF(Skp2) structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant beta-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection.

Amino Acid Motifs↗

PubMed2003

The BRCA2-interacting protein DSS1 is vital for DNA repair, recombination, and genome stability in Ustilago maydis.

DSS1 encodes a small acidic protein shown in recent structural studies to interact with the DNA binding domain of BRCA2. Here we report that an ortholog of DSS1 is present in Ustilago maydis and associates with Brh2, the BRCA2-related protein, thus recapitulating the protein partnership in this genetically amenable fungus. Mutants of U. maydis deleted of DSS1 are extremely radiation sensitive, deficient in recombination, defective in meiosis, and disturbed in genome stability; these phenotypes mirror previous observations of U. maydis mutants deficient in Brh2 or Rad51. These findings conclusively show that Dss1 constitutes a protein with a significant role in the recombinational repair pathway in U. maydis, and imply that it plays a similar key role in the recombination systems of organisms in which recombinational repair is BRCA2 dependent.

BRCA2 Protein↗

PubMed2002

BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure.

Mutations in the BRCA2 (breast cancer susceptibility gene 2) tumor suppressor lead to chromosomal instability due to defects in the repair of double-strand DNA breaks (DSBs) by homologous recombination, but BRCA2's role in this process has been unclear. Here, we present the 3.1 angstrom crystal structure of a approximately 90-kilodalton BRCA2 domain bound to DSS1, which reveals three oligonucleotide-binding (OB) folds and a helix-turn-helix (HTH) motif. We also (i) demonstrate that this BRCA2 domain binds single-stranded DNA, (ii) present its 3.5 angstrom structure bound to oligo(dT)9, (iii) provide data that implicate the HTH motif in dsDNA binding, and (iv) show that BRCA2 stimulates RAD51-mediated recombination in vitro. These findings establish that BRCA2 functions directly in homologous recombination and provide a structural and biochemical basis for understanding the loss of recombination-mediated DSB repair in BRCA2-associated cancers.

Amino Acid Sequence↗

PubMed2002

Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling.

The ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) plays a central role in the cellular response to changes in oxygen availability. pVHL binds to HIF only when a conserved proline in HIF is hydroxylated, a modification that is oxygen-dependent. The 1.85 angstrom structure of a 20-residue HIF-1alpha peptide-pVHL-ElonginB-ElonginC complex shows that HIF-1alpha binds to pVHL in an extended beta strand-like conformation. The hydroxyproline inserts into a gap in the pVHL hydrophobic core, at a site that is a hotspot for tumorigenic mutations, with its 4-hydroxyl group recognized by buried serine and histidine residues. Although the beta sheet-like interactions contribute to the stability of the complex, the hydroxyproline contacts are central to the strict specificity characteristic of signaling.

Amino Acid Sequence↗

PubMed2002

Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold.

Amino Acid Motifs↗

PubMed2002

Structure of the 53BP1 BRCT region bound to p53 and its comparison to the Brca1 BRCT structure.

Brca1 C-terminal (BRCT) domains are a common protein-protein interaction motif in proteins involved in the DNA damage response and DNA repair. The DNA-damage response protein 53BP1 has two BRCT domains that bind to the DNA-binding domain of p53. The 53BP1 tandem-BRCT region is homologous to the tandem-BRCT region of Brca1, which is involved in double-strand break repair and homologous recombination and which binds BACH1, a member of the DEAH helicase family. Here we report the structures of a human 53BP1-p53 complex and of the rat Brca1 BRCT repeats. The 53BP1-p53 structure shows that the two BRCT repeats are arranged tandemly and pack extensively through an interface that also involves the inter-repeat linker. The first BRCT repeat and the linker together bind p53 on a region that overlaps with the DNA-binding surface of p53 and involves p53 residues that are mutated in cancer and are important for DNA binding. Comparison with the structure of the tandem-BRCT region of Brca1 shows a remarkable conservation of the repeat arrangement and of the inter-BRCT repeat interface. Analysis of human BRCA1 tumor-derived mutations and conservation identifies a potential protein-binding site that we show through mutagenesis is involved in BACH1 binding. The BACH1-binding region of Brca1 consists of a unique insertion in the first BRCT repeat and the inter-repeat linker and is analogous to the region of 53BP1 that binds p53.

Amino Acid Sequence↗

PubMed2002

Crystal structure of the FHA domain of the Chfr mitotic checkpoint protein and its complex with tungstate.

The Chfr mitotic checkpoint protein is frequently inactivated in human cancer. We determined the three-dimensional structure of its FHA domain in its native form and in complex with tungstate, an analog of phosphate. The structures revealed a beta sandwich fold similar to the previously determined folds of the Rad53 N- and C-terminal FHA domains, except that the Rad53 domains were monomeric, whereas the Chfr FHA domain crystallized as a segment-swapped dimer. The ability of the Chfr FHA domain to recognize tungstate suggests that it shares the ability with other FHA domains to bind phosphoproteins. Nevertheless, differences in the sequence and structure of the Chfr and Rad53 FHA domains suggest that FHA domains can be divided into families with distinct binding properties.

Amino Acid Sequence↗