SNARE selectivity of COPII and AP-1 vesicle coat proteins, and, Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation

2005 2005

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Abstract (summary)

The COPII vesicle coat—comprising the Sar1 GTPase, Sec23/24 subcomplex, and Sec13/31 subcomplex—coordinates budding of transport vesicles from the endoplasmic reticulum in the initial step of the secretory pathway. The coat orchestrates a sequence of events, including self-assembly on the membrane, cargo and SNARE molecule selection, and deformation of the membrane into a bud to drive vesicle fission. The molecular basis for concentrative sorting of cargo into transport vesicles has been a longstanding question. Chapter 1 of this dissertation discusses identification of motifs recognized by the COPE coat within the SNARE proteins responsible for vesicle fusion during ER-to-Golgi transport. Crystallographic analysis of COPII-SNARE complexes reveals two distinct binding sites on the Sec24 subunit, one of which also recognizes common di-acidic cargo sorting motifs. The accessibility of these SNARE motifs is regulated by the assembly state of the SNARES (free monomers opposed to three- or four-helix bundles of t- or v-/t-SNARE Complexes), suggesting that the COPII coat selects fusogenic forms of these SNAREs in readiness for vesicle fusion during the ER-to-Golgi step. Chapter 2 describes similar experiments performed with the clathrin adaptor proteins, AP-1A and AP-1B, thought to mediate polarized traffic to the plasma membrane of epithelial cells; so far, however, no interactions with SNAREs have been detected.

Current drug discovery efforts focus primarily on proteins with defined enzymatic or small molecule binding sites. Autoregulatory domains represent attractive alternative targets for small molecule inhibitors, since they also occur in non-catalytic proteins and may avoid specificity problems inherent in active site-directed inhibitors. Chapter 3 reports the identification of wiskostatin, a chemical inhibitor of Wiskott-Aldrich syndrome family proteins (WASP/N-WASP), which are key elements in signaling pathways controlling polymerization of the actin cytoskeleton. Wiskostatin interacts with the regulatory GTPase-binding domain (GBD) of WASP in the NMR structure of the complex. The compound induces folding of isolated, unstructured GBD into its autoinhibited conformation, suggesting that wiskostatin inhibits N-WASP by stabilizing its autoinhibited state. Use of small molecules to bias conformational equilibria represents a potentially general strategy for chemical inhibition of autoinhibited proteins, even where allosteric sites have not evolved naturally in a target.

Indexing (details)

Cellular biology
0487: Biochemistry
0379: Cellular biology
Identifier / keyword
Pure sciences; Biological sciences; AP-1; Autoinhibited; COPII; SNARE; Vesicle coat proteins; WASP
SNARE selectivity of COPII and AP-1 vesicle coat proteins, and, Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation
Bickford, Lincoln Charles
Number of pages
Publication year
Degree date
School code
DAI-B 66/03, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
9780542053528, 0542053527
Goldberg, Jonathan
Weill Medical College of Cornell University
University location
United States -- New York
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
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