Structure of the APPL1 BAR-PH domain and characterization of its interaction with Rab5

APPL1 is an effector of the small GTPase Rab5. Together, they mediate a signal transduction pathway initiated by ligand binding to cell surface receptors. Interaction with Rab5 is confined to the amino (N)-terminal region of APPL1. We report the crystal structures of human APPL1 N-terminal BAR-PH domain motif. The BAR and PH domains, together with a novel linker helix, form an integrated, crescent-shaped, symmetrical dimer. This BAR–PH interaction is likely conserved in the class of BAR-PH containing proteins. Biochemical analyses indicate two independent Rab-binding sites located at the opposite ends of the dimer, where the PH domain directly interacts with Rab5 and Rab21. Besides structurally supporting the PH domain, the BAR domain also contributes to Rab binding through a small surface region in the vicinity of the PH domain. In stark contrast to the helix-dominated, Rab-binding domains previously reported, APPL1 PH domain employs β-strands to interact with Rab5. On the Rab5 side, both switch regions are involved in the interaction. Thus we identified a new binding mode between PH domains and small GTPases

The 2009 BFA Graduating Class Department of Visual Arts

The bachelor of fine arts (visual arts) program is a professional program designed to educate and train students in the history, theory and practice of the visual arts. The curriculum has been devised to produce well-rounded generalists with a solid grounding in all aspects of visual arts. The curriculum illustrates the philosophy that artistic freedom and creative expression require technical skill, intellectual awareness and a personal vision, acquired through a disciplined application of effort and a critical understanding of artistic issues, past and present

The Structure and Competitive Substrate Inhibition of Dihydrofolate Reductase from <i>Enterococcus faecalis</i> Reveal Restrictions to Cofactor Docking

We are addressing bacterial resistance to antibiotics by repurposing a well-established classic antimicrobial target, the dihydrofolate reductase (DHFR) enzyme. In this work, we have focused on <i>Enterococcus faecalis</i>, a nosocomial pathogen that frequently harbors antibiotic resistance determinants leading to complicated and difficult-to-treat infections. An inhibitor series with a hydrophobic dihydrophthalazine heterocycle was designed from the anti-folate trimethoprim. We have examined the potency of this inhibitor series based on inhibition of DHFR enzyme activity and bacterial growth, including in the presence of the exogenous product analogue folinic acid. The resulting preferences were rationalized using a cocrystal structure of the DHFR from this organism with a propyl-bearing series member (RAB-propyl). In a companion apo structure, we identify four buried waters that act as placeholders for a conserved hydrogen-bonding network to the substrate and indicate an important role in protein stability during catalytic cycling. In these structures, the nicotinamide of the nicotinamide adenine dinucleotide phosphate cofactor is visualized outside of its binding pocket, which is exacerbated by RAB-propyl binding. Finally, homology models of the TMP^R sequences <i>dfrK</i> and <i>dfrF</i> were constructed. While the <i>dfrK-</i>encoded protein shows clear sequence changes that would be detrimental to inhibitor binding, the <i>dfrF</i>-encoded protein model suggests the protein would be relatively unstable. These data suggest a utility for anti-DHFR compounds for treating infections arising from <i>E. faecalis</i>. They also highlight a role for water in stabilizing the DHFR substrate pocket and for competitive substrate inhibitors that may gain advantages in potency by the perturbation of cofactor dynamics