Cooperative binding of DNA and CBFbeta to the Runt domain of the CBFalpha studied via MD simulations

The Runt domain (RD) is the DNA-binding region of the Runx genes. A related protein, known as core binding factor β (CBFβ) also binds to the RD to enhance RD–DNA interaction by 6- to 10-fold. Here, we report results from molecular dynamics (MD) simulations of RD alone, as a dimer in complexes with DNA and CBFβ and in a ternary complex with DNA and CBFβ. Consistent with the experimental findings, in the presence of CBFβ the estimated free energy of binding of RD to the DNA is more favorable, which is shown to be due to more favorable intermolecular interactions and desolvation contributions. Also contributing to the enhanced binding are favorable intramolecular interactions between the ‘wing’ residues (RD residues 139–145) and the ‘wing1’ residues (RD residues 104–116). The simulation studies also indicate that the RD–CBFβ binding is more favorable in the presence of DNA due to a more favorable RD–CBFβ interaction energy. In addition, it is predicted that long-range interactions involving ionic residues contribute to binding cooperativity. Results from the MD calculations are used to interpret a variety of experimental mutagenesis data. A novel role for RD Glu116 to the RD–CBFβ interaction is predicted.     

Immunoglobulin motif DNA recognition and heterodimerization of the PEBP2/CBF Runt domain.

The polyomavirus enhancer binding protein 2 (PEBP2) or core binding factor (CBF) is a heterodimeric enhancer binding protein that is associated with genetic regulation of hematopoiesis and osteogenesis. Aberrant forms of PEBP2/CBF are implicated in the cause of the acute human leukemias and in a disorder of bone development known as cleidocranial dysplasia. The common denominator in the natural and mutant forms of this protein is a highly conserved domain of PEBP2/CBF alpha, termed the Runt domain (RD), which is responsible for both DNA binding and heterodimerization with the beta subunit of PEBP2/CBF. The three-dimensional structure of the RD bound to DNA has been determined to be an S-type immunoglobulin fold, establishing a structural relationship between the RD and the core DNA binding domains of NF-kappaB, NFAT1, p53 and the STAT proteins. NMR spectroscopy of a 43.6 kD RD-beta-DNA ternary complex identified the surface of the RD in contact with the beta subunit, suggesting a mechanism for the enhancement of RD DNA binding by beta. Analysis of leukemogenic mutants within the RD provides molecular insights into the role of this factor in leukemogenesis and cleidocranial dysplasia.     

Structural mechanism of DNA recognition by the p204 HIN domain

The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at ∼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway.     

Structural basis for monoubiquitin recognition by the Ede1 UBA domain

Monoubiquitination is a general mechanism for downregulating the activity of cell surface receptors by consigning these proteins for lysosome-mediated degradation through the endocytic pathway. The yeast Ede1 protein functions at the internalization step of endocytosis and binds monoubiquitinated proteins through a ubiquitin associated (UBA) domain. UBA domains are found in a broad range of cellular proteins but previous studies have suggested that the mode of ubiquitin recognition might not be universally conserved. Here we present the solution structure of the Ede1 UBA domain in complex with monoubiquitin. The Ede1 UBA domain forms a three-helix bundle structure and binds ubiquitin through a largely hydrophobic surface in a manner reminiscent of the Dsk2 UBA and the remotely homologous Cue2 CUE domains, for which high-resolution structures have been described. However, the interaction is dissimilar to the molecular models proposed for the hHR23A UBA domains bound to either monoubiquitin or Lys48-linked diubiquitin. Our mutational analyses of the Ede1 UBA domain-ubiquitin interaction reveal several key affinity determinants and, unexpectedly, a negative affinity determinant in the wild-type Ede1 protein, implying that high-affinity interactions may not be the sole criterion for optimal function of monoubiquitin-binding endocytic proteins.     

Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1

The BRCT repeats in BRCA1 are essential for its tumor suppressor activity and interact with phosphorylated protein targets containing the sequence pSer-X-X-Phe, where X indicates any residue. The structure of the tandem BRCA1 BRCT repeats bound to an optimized phosphopeptide reveals that the N-terminal repeat harbors a conserved BRCT phosphoserine-binding pocket, while the interface between the repeats forms a hydrophobic groove that recognizes the phenylalanine. Crystallographic and biochemical data suggest that the structural integrity of both binding sites is essential for peptide recognition. The diminished peptide-binding capacity observed for cancer-associated BRCA1-BRCT variants may explain the enhanced cancer risks associated with these mutations.     

Structural insights into the PIP2 recognition by syntenin-1 PDZ domain

Lipid-mediated regulatory mechanism of the C-terminal ligand binding to PDZ domains is not fully understood, despite their roles in subcellular organization. Here, we provide structural insights into the phosphatidylinositol 4,5-bisphosphate (PIP₂) recognition mode of a PDZ domain, as revealed from the crystal structure of the phosphate-bound PDZ domain. Two adjacent phosphate ions bind to the basic residues close to the amino terminus of the α2 helix in the Tamalin PDZ domain, reflecting an interaction mode of the two phosphate groups of PIP₂. Based on the observed location of the two phosphate molecules within the PDZ domain, we built the docking model of PIP₂ with the PDZ domain of the well-known PIP₂-binding protein, syntenin-1. This model suggests that the hydrophobic diacylglycerol group of PIP₂ could contact the ligand-binding groove of the PDZ domain. These structural features well explain biological phenomena, which were previously reported for the PIP₂-mediated PDZ ligand-binding regulation.     

Towards an understanding of DNA recognition by the methyl-CpG binding domain 1

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations. As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be -3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

Structural basis for APPTPPPLPP peptide recognition by the FBP11WW1 domain

WW domains are small protein-protein interaction modules that recognize proline-rich stretches in proteins. The class II tandem WW domains of the formin binding protein 11 (FBP11) recognize specifically proteins containing PPLPp motifs as present in the formins that are involved in limb and kidney development, and in the methyl-CpG-binding protein 2 (MeCP2), associated with the Rett syndrome. The interaction involves the specific recognition of a leucine side-chain. Here, we report on the novel structure of the complex formed by the FPB11WW1 domain and the formin fragment APPTPPPLPP revealing the specificity determinants of class II WW domains.