A Zinc Site in the C-Terminal Domain of RAG1 Is Essential for DNA Cleavage Activity

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn²⁺ ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn²⁺ ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn²⁺ ions. Stripping the full complement of bound Zn²⁺ ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn²⁺ core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn²⁺ ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn²⁺ ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn²⁺ coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn²⁺ serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.     

Characterising the Binding Specificities of the Subunits Associated with the KMT2/Set1 Histone Lysine Methyltransferase

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5′-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.     

The Ribosomal Grip of the Peptidyl-tRNA is Critical for Reading Frame Maintenance

If a ribosome shifts to an alternative reading frame during translation, the information in the message is usually lost. We have selected mutants of Salmonella typhimurium with alterations in tRNA_cmo5UGG^Pro that cause increased frameshifting when present in the ribosomal P-site. In 108 such mutants, two parts of the tRNA molecule are altered: the anticodon stem and the D-arm, including its tertiary interactions with the variable arm. Some of these alterations in tRNA_cmo5UGG^Pro are in close proximity to ribosomal components in the P-site. The crystal structure of the 30S subunit suggests that the C-terminal end of ribosomal protein S9 contacts nucleotides 32-34 of peptidyl-tRNA. We have isolated mutants with defects in the C-terminus of S9 that induce + 1 frameshifting. Combinations of changes in tRNA_cmo5UGG^Pro and S9 suggest that an interaction occurs between position 32 of the peptidyl-tRNA and the C-terminal end of S9. Together, our results suggest that the cause of frameshifting is an aberrant interaction between the peptidyl-tRNA and the P-site environment. We suggest that the “ribosomal grip” of the peptidyl-tRNA is pivotal for maintaining the reading frame.     

A nucleosome interaction module is required for normal function of Arabidopsis thaliana BRAHMA

The BRAHMA (BRM) gene encodes the SNF2-type ATPase of the putative Arabidopsis thaliana SWI/SNF chromatin remodelling complex. This family of ATPases is characterized by the presence of a conserved catalytic domain and an arrangement of auxiliary domains, whose functions in the remodelling activity remains unclear. Here, we characterize, at the molecular and functional level, the carboxy-terminal part of Arabidopsis BRM. We have found three DNA-binding regions that bind various free DNA and nucleosomal probes with different specificity. One of these regions contains an AT-hook motif. The carboxy terminus also contains a bromodomain able to bind histones H3 and H4. We propose that this array of domains constitute a nucleosome interaction module that helps BRM to interact with its substrate. We also characterize an Arabidopsis mutant that expresses a BRM protein lacking the last 454 amino acid residues (BRM-DeltaC), encompassing the bromodomain and two of the three DNA-binding activities identified. This mutant displays an intermediate phenotype between those of the wild-type and a null allele mutant, suggesting that the nucleosome interaction module is required for the normal function of BRM but it is not essential for the remodelling activity of BRM-containing SWI/SNF complexes.     

Alteration of the C-Terminal Ligand Specificity of the Erbin PDZ Domain by Allosteric Mutational Effects

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.     

A LIM-9 (FHL) / SCPL-1 (SCP) Complex Interacts with the C-terminal Protein Kinase Regions of UNC-89 (Obscurin) in Caenorhabditis elegans Muscle

The C. elegans gene unc-89 encodes a set of mostly giant polypeptides (up to 900 kDa) that contain multiple immunoglobulin (Ig) and fibronectin type 3 (Fn3), a triplet of SH3-DH-PH, and two protein kinase domains. The loss of function mutant phenotype and localization of antibodies to UNC-89 proteins indicate that the function of UNC-89 is to help organize sarcomeric A-bands, especially M-lines. Recently, we reported that each of the protein kinase domains interacts with SCPL-1, which contains a CTD-type protein phosphatase domain. Here, we report that SCPL-1 interacts with LIM-9 (FHL), a protein that we first discovered as an interactor of UNC-97 (PINCH) and UNC-96, components of an M-line costamere in nematode muscle. We show that LIM-9 can interact with UNC-89 through its first kinase domain and a portion of unique sequence lying between the two kinase domains. All the interactions were confirmed by biochemical methods. A yeast three-hybrid assay demonstrates a ternary complex between the two protein kinase regions and SCPL-1. Evidence that the UNC-89/SCPL-1 interaction occurs in vivo was provided by showing that over-expression of SCPL-1 results in disorganization of UNC-89 at M-lines. We suggest two structural models for the interactions of SCPL-1 and LIM-9 with UNC-89 at the M-line.     

Electrostatic interactions mediate binding of obscurin to small ankyrin 1: biochemical and molecular modeling studies

Small ankyrin 1 (sAnk1; also known as Ank1.5) is an integral protein of the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle cells, where it is thought to bind to the C-terminal region of obscurin, a large modular protein that surrounds the contractile apparatus. Using fusion proteins in vitro, in combination with site-directed mutagenesis and surface plasmon resonance measurements, we previously showed that the binding site on sAnk1 for obscurin consists, in part, of six lysine and arginine residues. Here we show that four charged residues in the high-affinity binding site on obscurin for sAnk1 (between residues 6316 and 6345), consisting of three glutamates and a lysine, are necessary, but not sufficient, for this site on obscurin to bind to sAnk1 with high affinity. We also identify specific complementary mutations in sAnk1 that can partially or completely compensate for the changes in binding caused by charge-switching mutations in obscurin. We used molecular modeling to develop structural models of residues 6322-6339 of obscurin bound to sAnk1. The models, based on a combination of Brownian and molecular dynamics simulations, predict that the binding site on sAnk1 for obscurin is organized as two ankyrin-like repeats, with the last α-helical segment oriented at an angle to nearby helices, allowing lysine 6338 of obscurin to form an ionic interaction with aspartate 111 of sAnk1. This prediction was validated by double-mutant cycle experiments. Our results are consistent with a model in which electrostatic interactions between specific pairs of side chains on obscurin and sAnk1 promote binding and complex formation.     

Mechanism for Attenuation of DNA Binding by MarR Family Transcriptional Regulators by Small Molecule Ligands

Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands.     

Folding of Tetrameric p53: Oligomerization and Tumorigenic Mutations Induce Misfolding and Loss of Function

The physiologically active form of p53 consists of a tetramer of four identical 393-amino-acid subunits associated via their tetramerization domains (TDs; residues 325-355). One in two human tumors contains a point mutation in the DNA binding domain (DBD) of p53 (residues 94-312). Most existing studies on the effects of these mutations on p53 structure and function have been carried out on the isolated DBD fragment, which is monomeric. Recent structural evidence, however, suggests that DBDs may interact with each other in full-length tetrameric forms of p53. Here, we investigate the effects of tumorigenic DBD mutations on the folding of p53 in its tetrameric form. We employ the construct consisting of DBD and TD (amino acids 94-360). We characterize the stability and conformational state of the tumorigenic DBD mutants R248Q, R249S, and R282Q using equilibrium denaturation and functional assays. Destabilizing mutations cause DBD to misfold when it is part of the p53 tetramer, but not when it is monomeric. This conformation is populated under moderately destabilizing conditions (10 °C in 2 M urea, and at physiological temperature in the absence of denaturant). Under those same conditions, it is not present in the isolated DBD fragment or in the presence of the TD mutation L344P, which abolishes tetramerization. Misfolding appears to involve intramolecular DBD-DBD association within a single tetrameric molecule. This association is promoted by destabilization of DBD (caused by mutation or elevated temperature) and by the high local DBD concentration enforced by tetramerization of TD. Disrupting the nonnative DBD-DBD interaction or transiently inhibiting tetramerization and allowing p53 to fold as a monomer may be potential strategies for pharmacological intervention in cancer.     

棉花脱水应答元件(DRE)结合蛋白GhDBP1的原核表达、纯化及其DNA结合活性

棉花是一种重要的经济作物,其生产和产量要受到干旱、低温和高盐等环境胁迫的影响,因此提高棉花对这些胁迫的抗性非常重要.脱水应答元件(DRE-dehydrationresponsiveelement)结合蛋白(DBP)在调节植物对环境胁迫的抗性中起到非常重要的作用.而且过量表达DBP类基因的转基因植株能够很好抵抗这些环境胁迫,所以研究棉花中此类DRE元件结合蛋白对棉花生产有非常重要的意义.在以前的工作中,从棉花中分离一个DBP基因,命名为GhDBP1并在转录水平上分析它在棉花植株中的表达特征.在研究中,报道了GhDBP1的原核表达、纯化和它的DNA结合特性.GhDBP1基因的编码区用PCR技术扩增出来插入到原核表达载体pET28a中,并转化到大肠杆菌菌株BL21(DE3)中.经过IPTG诱导,GhDBP1融合蛋白在BL21(DE3)菌株中成功进行表达.利用Ni-NTA亲和层析技术得到了纯化的融合蛋白.在非同位素的凝胶滞留实验中,纯化的GhDBP1融合蛋白能够结合到含有DRE元件的DNA片段上.另外,用SWISS-MODEL软件对GhDBP1蛋白的DNA结合区的三维结构进行了计算机模拟.模拟的结果显示,GhDBP1蛋白的DNA结合区的主链结构和折叠模式与已知的拟南芥GCC盒结合蛋白AtERF1的DNA结合区结构很相似.这些结果显示了GhDBP1是一个脱水应答元件(DRE)结合的转录因子,并可能运用与AtERF1的DNA结合区相似的结构和它的目标序列脱水应答元件(DRE)相结合.     

A proteasome assembly defect in rpn3 mutants is associated with Rpn11 instability and increased sensitivity to stress

Rpn11 is a proteasome-associated deubiquitinating enzyme that is essential for viability. Recent genetic studies showed that Rpn11 is functionally linked to Rpn10, a major multiubiquitin chain binding receptor in the proteasome. Mutations in Rpn11 and Rpn10 can reduce the level and/or stability of proteasomes, indicating that both proteins influence its structural integrity. To characterize the properties of Rpn11, we examined its interactions with other subunits in the 19S regulatory particle and detected strong binding to Rpn3. Two previously described rpn3 mutants are sensitive to protein translation inhibitors and an amino acid analog. These mutants also display a mitochondrial defect. The abundance of intact proteasomes was significantly reduced in rpn3 mutants, as revealed by strongly reduced binding between 20S catalytic with 19S regulatory particles. Proteasome interaction with the shuttle factor Rad23 was similarly reduced. Consequently, higher levels of multiUb proteins were associated with Rad23, and proteolytic substrates were stabilized. The availability of Rpn11 is important for maintaining adequate levels of intact proteasomes, as its depletion caused growth and proteolytic defects in rpn3. These studies suggest that Rpn11 is stabilized following its incorporation into proteasomes. The instability of Rpn11 and the defects of rpn3 mutants are apparently caused by a failure to recruit Rpn11 into mature proteasomes.     

A mitotic kinase TOPK enhances Cdk1/cyclin B1-dependent phosphorylation of PRC1 and promotes cytokinesis

A MAPKK-like mitotic kinase, TOPK, implies the formation of mitotic spindles and spindle midzone and accomplishing cytokinesis, however, its underlying mechanism remains unclear. A microtubule bundling protein, PRC1, plays a pivotal role in the formation of mitotic spindles and spindle midzone. Because of their functional resemblance, we attempted to clarify the links between these two molecules. TOPK supported mitotic advance via the cdk1/cyclin B1-dependent phosphorylation of PRC1. TOPK induced the phosphorylation of PRC1 at T481 in vivo, however, TOPK did not phosphorylate PRC1 in vitro. TOPK induced the phosphorylation of PRC1 at T481 only when the cdk1/cyclin B1 existed simultaneously in vitro. Both the enzymatic activity of TOPK and association competence of TOPK with PRC1 were mandatory for this phosphorylation. TOPK binds to cdk1/cyclin B1, microtubules and PRC1 via its unique region near the C terminus. TOPK co-localized closely with cdk1 throughout the cell cycle in vivo. Collectively, these data indicate that TOPK, which makes a kinase-substrate complex with cdk1/cyclin B1 and PRC1 on microtubules during mitosis, enhances the cdk1/cyclin B1-dependent phosphorylation of PRC1 and thereby strongly promotes cytokinesis.     

Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning.