Solution structure of Rap1 BRCT domain from Saccharomyces cerevisiae reveals a novel fold

Rap1 (repressor-activator protein 1) from Saccharomyces cerevisiae, containing a BRCT domain at its N-terminus, is a multifunctional protein that controls telomere function, silencing, and the activation of glycolytic and ribosomal protein genes. In this work, we determined the solution structure of Rap1 BRCT domain, which contains three β-strands and three α-helices. Structural comparison indicated that Rap1 BRCT domain adopts a global fold similar to other BRCT domains, implying some common structural aspects of BRCT domain family. On the other hand, Rap1 BRCT domain displays structural characteristics significantly different from other BRCT domains in that Rap1 BRCT domain adopts a rather flexible conformation with less secondary structure elements, revealing a novel fold of the BRCT domain family.     

Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa

SUMO E3 ligase of the Siz/PIAS family that promotes sumoylation of target proteins contains SAP motif in its N-terminal region. The SAP motif with a consensus sequence of 35 residues was first proposed to be as a new DNA binding motif found in diverse nuclear proteins involved in chromosomal organization. We have determined solution structures of the SAP domains of SUMO ligases Siz1 from yeast and rice by NMR spectroscopy, showing that the structure of the SAP domain (residues 2-105) of rice Siz1 is a four-helix bundle with an up-down-extended loop-down-up topology, whereas the SAP domain (residues 1-111) of yeast Siz1 is comprised of five helices where the fifth helix alpha5 causes a significant change in the alignment of the four-helix bundle characteristic to the SAP domains of the Siz/PIAS family. We have also demonstrated that both SAP domains have binding ability to an A/T-rich DNA, but that binding affinity of yeast Siz1 SAP is at least by an order of magnitude higher than that of rice Siz1 SAP. Our NMR titration experiments clearly showed that yeast Siz1 SAP uses alpha2-helix for DNA binding more effectively than rice Siz1 SAP, which would result from the dislocation of this helix due to the existence of the extra helix alpha5. In addition, based on the structures of the SAP domains determined here and registered in Protein Data Bank, general features of structures of the SAP domains are discussed in conjunction with equivocal nature of their DNA binding.     

Insights into nonspecific binding of homeodomains from a structure of MATalpha2 bound to DNA

The 2.1-A resolution crystal structure of the MATalpha2 homeodomain bound to DNA reveals the unexpected presence of two nonspecifically bound alpha2 homeodomains, in addition to the two alpha2 homeodomains bound to canonical alpha2 binding sites. One of the extra homeodomains makes few base-specific contacts, while the other extra homeodomain binds to DNA in a previously unobserved manner. This unusually bound homeodomain is rotated on the DNA, making possible major groove contacts by side-chains that normally do not contact the DNA. This alternate docking may represent one way in which homeodomains sample nonspecific DNA sequences.     

Solution structure of the DNA-binding domain of interleukin enhancer binding factor 1 (FOXK1a)

Interleukin enhancer binding factor (ILF) binds to the interleukin-2 (IL-2) promoter and regulates IL-2 gene expression. In this study, the 3D structure of the DNA-binding domain of ILF was determined by multidimensional NMR spectroscopy. NMR structure analysis revealed that the DNA-binding domain of ILF is a new member of the winged helix/forkhead family, and that its wing 2 contains an extra alpha-helix. This is the first study to report the presence of a C-terminal alpha-helix in place of a typical wing 2 in a member of this family. This structural difference may be responsible for the different DNA-binding specificity of ILF compared to other winged helix/forkhead proteins. Our deletion studies of the fragments of ILF also suggest that the C-terminal region plays a regulatory role in DNA binding.     

Solution structure of the ubiquitin-binding domain in Swa2p from Saccharomyces cerevisiae

The SWA2/AUX1 gene has been proposed to encode the Saccharomyces cerevisiae ortholog of mammalian auxilin. Swa2p is required for clathrin assembly/dissassembly in vivo, thereby implicating it in intracellular protein and lipid trafficking. While investigating the 287-residue N-terminal region of Swa2p, we found a single stably folded domain between residues 140 and 180. Using binding assays and structural analysis, we established this to be a ubiquitin-associated (UBA) domain, unidentified by bioinformatics of the yeast genome. We determined the solution structure of this Swa2p domain and found a characteristic three-helix UBA fold. Comparisons of structures of known UBA folds reveal that the position of the third helix is quite variable. This helix in Swa2p UBA contains a bulkier tyrosine in place of smaller residues found in other UBAs and cannot pack as close to the second helix. The molecular surface of Swa2p UBA has a mostly negative potential, with a single hydrophobic surface patch found also in the UBA domains of human protein, HHR23A. The presence of a UBA domain implicates Swa2p in novel roles involving ubiquitin and ubiquitinated substrates. We propose that Swa2p is a multifunctional protein capable of recognizing several proteins through its protein-protein recognition domains.     

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes

In most of homeodomain–DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1–DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein–DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein–DNA complexes. The order of stability of protein–DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein–DNA complexes. Among specific protein–DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.     

Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen

Replication protein A (RPA) is a three-subunit complex with multiple roles in DNA metabolism. DNA-binding domain A in the large subunit of human RPA (hRPA70A) binds to single-stranded DNA (ssDNA) and is responsible for the species-specific RPA–T antigen (T-ag) interaction required for Simian virus 40 replication. Although Saccharomyces cerevisiae RPA70A (scRPA70A) shares high sequence homology with hRPA70A, the two are not functionally equivalent. To elucidate the similarities and differences between these two homologous proteins, we determined the solution structure of scRPA70A, which closely resembled the structure of hRPA70A. The structure of ssDNA-bound scRPA70A, as simulated by residual dipolar coupling-based homology modeling, suggested that the positioning of the ssDNA is the same for scRPA70A and hRPA70A, although the conformational changes that occur in the two proteins upon ssDNA binding are not identical. NMR titrations of hRPA70A with T-ag showed that the T-ag binding surface is separate from the ssDNA-binding region and is more neutral than the corresponding part of scRPA70A. These differences might account for the species-specific nature of the hRPA70A–T-ag interaction. Our results provide insight into how these two homologous RPA proteins can exhibit functional differences, but still both retain their ability to bind ssDNA.     

Solution structure of the DNA binding domain of AraC protein

We report the solution structure of the DNA binding domain of the Escherichia coli regulatory protein AraC determined in the absence of DNA. The 20 lowest energy structures, determined on the basis of 1507 unambiguous nuclear Overhauser restraints and 180 angle restraints, are well resolved with a pair wise backbone root mean square deviation of 0.7 Å. The protein, free of DNA, is well folded in solution and contains seven helices arranged in two semi‐independent sub domains, each containing one helix‐turn‐helix DNA binding motif, joined by a 19 residue central helix. This solution structure is discussed in the context of extensive biochemical and physiological data on AraC and with respect to the DNA‐bound structures of the MarA and Rob homologs. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Solution structure of GOPC PDZ domain and its interaction with the C-terminal motif of neuroligin

GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein) represents a PDZ domain-containing protein associated with the Golgi apparatus, which plays important roles in vesicular trafficking in secretory and endocytic pathways. GOPC interacts with many other proteins, such as the Wnt receptors Frizzled 8 and neuroligin via its PDZ domain. Neuroligin is a neural cell-adhesion molecule of the post-synapse, which binds to the presynapse molecule neurexin to form a heterotypic intercellular junction. Here we report the solution structure of the GOPC PDZ domain by NMR. Our results show that it is a canonical class I PDZ domain, which contains two alpha-helices and six beta-strands. Using chemical shift perturbation experiments, we further studied the binding properties of the GOPC PDZ domain with the C-terminal motif of neuroligin. The observations showed that the ensemble of the interaction belongs to fast exchange with low affinity. The 3D model of the GOPC PDZ domain/neuroligin C-terminal peptide complex was constructed with the aid of the molecular dynamics simulation method. Our discoveries provide insight into the specific interaction of the GOPC PDZ domain with the C-terminal peptide of Nlg and also provide a general insight about the possible binding mode of the interaction of Nlg with other PDZ domain-containing proteins.     

The crystal structure of the nuclease domain of colicin E7 suggests a mechanism for binding to double-stranded DNA by the H-N-H endonucleases

The bacterial toxin ColE7 contains an H-N-H endonuclease domain (nuclease ColE7) that digests cellular DNA or RNA non-specifically in target cells, leading to cell death. In the host cell, protein Im7 forms a complex with ColE7 to inhibit its nuclease activity. Here, we present the crystal structure of the unbound nuclease ColE7 at a resolution of 2.1A. Structural comparison between the unbound and bound nuclease ColE7 in complex with Im7, suggests that Im7 is not an allosteric inhibitor that induces backbone conformational changes in nuclease ColE7, but rather one that inhibits by blocking the substrate-binding site. There were two nuclease ColE7 molecules in the P1 unit cell in crystals and they appeared as a dimer related to each other by a non-crystallographic dyad symmetry. Gel-filtration and cross-linking experiments confirmed that nuclease ColE7 indeed formed dimers in solution and that the dimeric conformation was more favored in the presence of double-stranded DNA. Structural comparison of nuclease ColE7 with the His-Cys box homing endonuclease I-PpoI further demonstrated that H-N-H motifs in dimeric nuclease ColE7 were oriented in a manner very similar to that of the betabetaalpha-fold of the active sites found in dimeric I-PpoI. A mechanism for the binding of double-stranded DNA by dimeric H-N-H nuclease ColE7 is suggested.