The recognition of the widespread biological importance of allostery-the transduction of a signal from one site to effect a change in biochemical properties, i.e. catalytic rate or cooperative binding, at a remote site-has led to the hope that drugs targeting allosteric networks will deliver subtle and precise control over enzyme behavior. Such control will enable the development of novel therapeutic strategies against a host of pathologies, including HIV and cancer. To design these strategies for a wide class of enzymes, it is critical to understand the detailed biophysical mechanisms of regulation through "action at a distance." Ubiquitination, the primary means for targeting proteins for degradation in eukaryotic cells, is accomplished by several enzymes that act in tandem to identify, bind, and tag target molecules. Biochemical evidence suggests that a crucial step in ubiquitination, transfer of the ubiquitin from the E2 to the E3-bound substrate, is allosterically activated, as the E3-binding site is spatially removed from the active site cysteine. However, the mechanism of E2 activation by E3 binding remains quite unclear. We propose to apply complementary computational and experimental approaches to test the hypotheses that allosteric networks in these systems (1) consist of a diffuse network of residues positioned to ensure the flexibility required for activation and (2) transmit information from the E3 binding site to the active site through semi-rigid secondary structural elements. Molecular dynamics-based information theoretic methods will identify key correlations defining the allosteric network. Dynamic NMR measurements, including residual dipolar couplings, of wild type enzymes and allosterically compromised mutants will assess changes in flexibility of key residues and the reorientation of torque-transmitting helices in the presence or absence of the allosteric activator. It is tempting to generalize descriptions of allosteric mechanisms from studies performed on a single archetypal enzyme. However, the degree to which similar enzymes utilize the same or different mechanisms of regulation has not been widely explored. This question is of utmost importance for the application of allosteric therapeutics, as the similarity of the mechanism will reflect the ability to specifically target an individual member of an enzyme family without compromising the function of others. The formation of K48-linked polyubiquitin chains by the anaphase promoting complex (APC/C) in the ubiquitin-proteasome pathway requires the sequential action of two E2s, Ubc1 and Ubc4, each with a unique role in assembling the chain topology. We propose to test the hypothesis that divergence of the allosteric network is related to specialization of the E2s with respect to ubiquitination targets and polyubiquitin chain topologies by using computational methods to compare the dynamics of the complete set of E2s from sacchromyces cerevisiae and by performing in vitro assays of ubiquitination activity with a number of dynamically distinct E2s and E2/E3 complexes.

Public Health Relevance

Allosteric protein sites are regarded as promising targets for pharmaceutical development due to the sensitive and specific control they offer over a host of biochemical pathways. Establishing a detailed mechanistic picture of how the information from the allosteric site is transmitted to the active site will inform efforts to modulate the activity of the ubiquitin-proteasome system, a critical regulatory pathway for protein degradation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM096643-02
Application #
8208280
Study Section
Special Emphasis Panel (ZRG1-F04B-B (20))
Program Officer
Flicker, Paula F
Project Start
2010-12-02
Project End
2012-12-01
Budget Start
2011-12-02
Budget End
2012-12-01
Support Year
2
Fiscal Year
2012
Total Cost
$52,190
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143