The crystal structure of NlpI. A prokaryotic tetratricopeptide repeat protein with a globular fold.

There are several different families of repeat proteins. In each, a distinct structural motif is repeated in tandem to generate an elongated structure. The nonglobular, extended structures that result are particularly well suited to present a large surface area and to function as interaction domains. Many repeat proteins have been demonstrated experimentally to fold and function as independent domains. In tetratricopeptide (TPR) repeats, the repeat unit is a helix-turn-helix motif. The majority of TPR motifs occur as three to over 12 tandem repeats in different proteins. The majority of TPR structures in the Protein Data Bank are of isolated domains. Here we present the high-resolution structure of NlpI, the first structure of a complete TPR-containing protein. We show that in this instance the TPR motifs do not fold and function as an independent domain, but are fully integrated into the three-dimensional structure of a globular protein. The NlpI structure is also the first TPR structure from a prokaryote. It is of particular interest because it is a membrane-associated protein, and mutations in it alter septation and virulence.

UCSF Small Molecule Discovery Center: innovation, collaboration and chemical biology in the Bay Area.

The Small Molecule Discovery Center (SMDC) at the University of California, San Francisco, works collaboratively with the scientific community to solve challenging problems in chemical biology and drug discovery. The SMDC includes a high throughput screening facility, medicinal chemistry, and research labs focused on fundamental problems in biochemistry and targeted drug delivery. Here, we outline our HTS program and provide examples of chemical tools developed through SMDC collaborations. We have an active research program in developing quantitative cell-based screens for primary cells and whole organisms; here, we describe whole-organism screens to find drugs against parasites that cause neglected tropical diseases. We are also very interested in target-based approaches for so-called "undruggable", protein classes and fragment-based lead discovery. This expertise has led to several pharmaceutical collaborations; additionally, the SMDC works with start-up companies to enable their early-stage research. The SMDC, located in the biotech-focused Mission Bay neighborhood in San Francisco, is a hub for innovative small-molecule discovery research at UCSF.

Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism.

Identification of protein binding partners is one of the key challenges of proteomics. We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments. Using a library of antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (K(D) approximately 1 mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that the screen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognate from noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experiments demonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guide the further use of this screen in proteomic analysis. Additionally, this work gives insight into the positional inequivalence of stabilizing interactions in antiparallel coiled coils.