Notch signaling mediates communication between cells and is essential for proper embryonic patterning and development. CSL is a DNA binding transcription factor that regulates transcription of Notch target genes by interacting with coregulators. Transcriptional activation requires the displacement of corepressors from CSL by the intracellular portion of the receptor Notch (NotchIC) and the recruitment of the coactivator protein Mastermind to the complex. Here we report the 3.1 A structure of the ternary complex formed by CSL, NotchIC, and Mastermind bound to DNA. As expected, the RAM domain of Notch interacts with the beta trefoil domain of CSL; however, the C-terminal domain of CSL has an unanticipated central role in the interface formed with the Notch ankyrin repeats and Mastermind. Ternary complex formation induces a substantial conformational change within CSL, suggesting a molecular mechanism for the conversion of CSL from a repressor to an activator.

Staphylococcal bacteria, including Staphylococcus epidermidis and Staphylococcus aureus, cause chronic biofilm-related infections. The homologous proteins Aap and SasG mediate biofilm formation in S. epidermidis and S. aureus, respectively. The self-association of these proteins in the presence of Zn 2+ leads to the formation of extensive adhesive contacts between cells. This study reports the crystal structure of a Zn 2+ -bound construct from the self-associating region of Aap. Several unusual structural features include elongated β-sheets that are solvent-exposed on both faces and the lack of a canonical hydrophobic core. Zn 2+ -dependent dimers are observed in three distinct crystal forms, formed via pleomorphic coordination of Zn 2+ in trans across the dimer interface. These structures illustrate how a long, flexible surface protein is able to form tight intercellular adhesion sites under adverse environmental conditions.H ealthcare-associated infections affect an estimated 1.5-2 million patients annually in the United States, with ∼99,000 annual fatalities (1). Staphylococci represent the most commonly isolated genus in healthcare-associated infections (2) and are the most common cause of infections on implanted devices (3). The key pathogenic mechanism in these infections is formation of a biofilm. A biofilm is a specialized bacterial colony with higherorder organization analogous to that of a tissue in multicellular organisms, in which there is concerted regulation of metabolic activity and gene expression (3). The entire colony is encased in an extensive extracellular matrix that can comprise polysaccharide, protein, nucleic acids, or combinations thereof (4). The extracellular matrix is important for mediating adhesion among neighboring bacteria as well as to diverse surfaces (3). Bacteria within a biofilm are resistant to antibiotics (5) and to host immune defenses (6), reducing the efficacy of available antimicrobials. Understanding the mechanisms of biofilm formation will allow us to combat the significant pathogenic advantages of biofilm-based infectious diseases.There are species-and strain-specific differences that affect staphylococcal biofilm formation (7), but certain key similarities have been identified. An important group of adhesive proteins includes the Staphylococcus epidermidis protein Aap and its Staphylococcus aureus homologs SasG and Pls. These are multidomain, multifunctional proteins with significant roles in biofilm formation. Exogenous expression of Aap or SasG in non-biofilmforming cocci is sufficient to mediate adhesion to host cells (8,9) and to initiate biofilm formation (10, 11). Aap knockout ablates biofilm formation (12).Aap, SasG, and Pls all have similar domain arrangements. In Aap, the N-terminal portion of the protein is comprised of an A-repeat region, with short (∼16-residue), imperfect sequence repeats, followed by a putative globular (α/β) domain with predicted α-helical and β-sheet content (Fig. 1A). This region of Aap mediates attachment to host cells via interact...

The Notch pathway is a conserved cell-to-cell signaling mechanism, in which extracellular signals are transduced into transcriptional outputs through the nuclear effector CSL. CSL is converted from a repressor to an activator through the formation of the CSL-NotchIC-Mastermind ternary complex. The RAM (RBP-J associated molecule) domain of NotchIC avidly interacts with CSL; however, its role in assembly of the CSLNotchIC-Mastermind ternary complex is not understood. Here we provide a comprehensive thermodynamic, structural, and biochemical analysis of the RAM-CSL interaction for components from both mouse and worm. Our binding data show that RAM and CSL form a high affinity complex in the presence or absence of DNA. Our structural studies reveal a striking distal conformational change in CSL upon RAM binding, which creates a docking site for Mastermind to bind to the complex. Finally, we show that the addition of a RAM peptide in trans facilitates formation of the CSL-NotchIC-Mastermind ternary complex in vitro.

The crystal structure of a collagen-binding domain (CBD) with an N-terminal domain linker from Clostridium histolyticum class I collagenase was determined at 1.00 A resolution in the absence of calcium (1NQJ) and at 1.65 A resolution in the presence of calcium (1NQD). The mature enzyme is composed of four domains: a metalloprotease domain, a spacing domain and two CBDs. A 12-residue-long linker is found at the N-terminus of each CBD. In the absence of calcium, the CBD reveals a beta-sheet sandwich fold with the linker adopting an alpha-helix. The addition of calcium unwinds the linker and anchors it to the distal side of the sandwich as a new beta-strand. The conformational change of the linker upon calcium binding is confirmed by changes in the Stokes and hydrodynamic radii as measured by size exclusion chromatography and by dynamic light scattering with and without calcium. Furthermore, extensive mutagenesis of conserved surface residues and collagen-binding studies allow us to identify the collagen-binding surface of the protein and propose likely collagen-protein binding models.

The development of new ion activation techniques continues to be a dynamic area of scientific discovery, in part to complement the tremendous innovations in ionization methods that have allowed the mass spectrometric analysis of an enormous array of molecules. Ion activation/dissociation provides key information about ion structures, binding energies, and differentiation of isomers, as well as affording a primary means of identifying compounds in mixtures. Numerous new activation methods have emerged over the past two decades in an effort to develop alternatives to collisional activated dissociation, the gold standard for providing structurally diagnostic fragmentation patterns. Collisional activated dissociation does not always offer sufficiently high or controllable energy deposition, thus rendering it less useful for certain classes of molecules, such as large proteins or macromolecular complexes. Photodissociation is one of the most promising alternatives and is readily implemented in ion trapping and time-of-flight mass spectrometers. Photodissociation generally entails using a laser to irradiate ions with UV, visible, or IR photons, thus resulting in internal energy deposition based on the number and wavelengths of the photons. The activation process can be extremely rapid and efficient, as well as having the potential for high total energy deposition. This review describes infrared multiphoton dissociation in quadrupole ion trap mass spectrometry. A comparison of photodissociation and collisional activated dissociation is covered, in addition to some of the methods to increase photodissociation efficiency. Numerous applications of IRMPD are discussed as well, including ones related to the analysis of drugs, peptides, nucleic acids, and oligosaccharides.