H. Raghuraman

The Deacetylase SIRT1 Promotes Membrane Localization and Activation of Akt and PDK1 During Tumorigenesis and Cardiac Hypertrophy

Deacetylation of Akt and its activating kinase PDK1 promotes cell growth in physiological and pathological settings. Deacetylation for Activation Cell growth can be physiological (such as when heart cells expand in size in response to exercise, a process called cardiac hypertrophy) or pathological (such as in cancer) and is promoted by the kinase Akt. Sundaresan et al. showed that acetylation blocked the activity of Akt and its activating kinase PDK1 by interfering with the lipid-binding sites of these proteins, whereas deacetylation enhanced their activities. Mice injected with cells containing a mutant Akt that mimicked acetylated Akt formed smaller tumors, and the extent of cardiac hypertrophy was decreased in mice that lacked SIRT1, the protein that deacetylated Akt. These results provide insight into understanding the mechanisms that regulate the activity of Akt and may enable the development of new ways to promote or inhibit cell growth. Signaling through the kinase Akt regulates many biological functions. Akt is activated during growth factor stimulation through a process that requires binding of Akt to phosphatidylinositol 3,4,5-trisphosphate (PIP3), which promotes membrane localization and phosphorylation of Akt by the upstream kinase PDK1 (phosphoinositide-dependent protein kinase 1). We show that Akt and PDK1 are acetylated at lysine residues in their pleckstrin homology domains, which mediate PIP3 binding. Acetylation blocked binding of Akt and PDK1 to PIP3, thereby preventing membrane localization and phosphorylation of Akt. Deacetylation by SIRT1 enhanced binding of Akt and PDK1 to PIP3 and promoted their activation. Mice injected with cells expressing a mutant that mimicked a constitutively acetylated form of Akt developed smaller tumors than those injected with cells expressing wild-type Akt. Furthermore, impaired Akt activation in the hearts of SIRT1-deficient mice was associated with reduced cardiac hypertrophy in response to physical exercise and angiotensin II. These findings uncover a key posttranslational modification of Akt that is important for its oncogenic and hypertrophic activities.

Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial Sirt3

Honokiol (HKL) is a natural biphenolic compound derived from the bark of magnolia trees with anti-inflammatory, anti-oxidative, anti-tumor and neuroprotective properties. Here we show that HKL blocks agonist-induced and pressure overload-mediated, cardiac hypertrophic responses, and ameliorates pre-existing cardiac hypertrophy, in mice. Our data suggest that the anti-hypertrophic effects of HKL depend on activation of the deacetylase SIRT3. We demonstrate that HKL is present in mitochondria, enhances SIRT3 expression nearly two-fold and suggest that HKL may bind to SIRT3 to further increase its activity. Increased SIRT3 activity is associated with reduced acetylation of mitochondrial SIRT3 substrates, MnSOD and OSCP. HKL-treatment increases mitochondrial rate of oxygen consumption and reduces ROS synthesis in wild-type, but not in SIRT3-KO cells. Moreover, HKL-treatment blocks cardiac fibroblast proliferation and differentiation to myofibroblasts in SIRT3-dependent manner. These results suggest that HKL is a pharmacological activator of SIRT3 capable of blocking, and even reversing, the cardiac hypertrophic response.

Protein conformational dynamics in the mechanism of HIV-1 protease catalysis

We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the “flap” structures (residues 37–61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis.

Instantaneous ion configurations in the K+ ion channel selectivity filter revealed by 2D IR spectroscopy

Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose four K+ binding sites. Here, we report on the two-dimensional infrared (2D IR) spectra of a semisynthetic KcsA channel with site-specific heavy (13C18O) isotope labels in the selectivity filter. The ultrafast time resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by molecular dynamics simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating

Significance C-type inactivation gating in K+ channels plays an important role in controlling the firing patterns of excitable cells and is fundamental in determining the length and frequency of the cardiac action potential. At a molecular level, toxins, blockers, and metal ions bind to the outer vestibule and modulate the functional behavior of K+ channels. Using KcsA, we show that the shuttling between the inactivated and conductive states of K+ channels is accompanied by changes in local outer vestibule dynamics, in the absence of large conformational changes. The altered structural and hydration dynamics of the outer vestibule appear to be likely and important modulators of selectivity filter gating transitions in K+ channels. In K+ channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K+ channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K+ channels.

Mechanism of Cd2+ Coordination during Slow Inactivation in Potassium Channels

In K+ channels, rearrangements of the pore outer vestibule have been associated with C-type inactivation gating. Paradoxically, the crystal structure of Open/C-type inactivated KcsA suggests these movements to be modest in magnitude. In this study, we show that under physiological conditions, the KcsA outer vestibule undergoes relatively large dynamic rearrangements upon inactivation. External Cd2+ enhances the rate of C-type inactivation in an cysteine mutant (Y82C) via metal-bridge formation. This effect is not present in a non-inactivating mutant (E71A/Y82C). Tandem dimer and tandem tetramer constructs of equivalent cysteine mutants in KcsA and Shaker K+ channels demonstrate that these Cd2+ metal bridges are formed only between adjacent subunits. This is well supported by molecular dynamics simulations. Based on the crystal structure of Cd2+ -bound Y82C-KcsA in the closed state, together with electron paramagnetic resonance distance measurements in the KcsA outer vestibule, we suggest that subunits must dynamically come in close proximity as the channels undergo inactivation.

Binding of the CYK-4 Subunit of the Centralspindlin Complex Induces a Large Scale Conformational Change in the Kinesin Subunit

Background: Cytokinesis requires formation of the centralspindlin complex. Results: The neck linker regions in ZEN-4 are highly mobile when free, but their mobility is greatly restricted by CYK-4 binding. Conclusion: Central spindle assembly requires a binding event that modulates the structure of the kinesin ZEN-4. Significance: Structural changes in the centralspindlin complex contribute to cell division. Centralspindlin is a critical regulator of cytokinesis in animal cells. It is a tetramer consisting of ZEN-4/MKLP1, a kinesin-6 motor, and CYK-4/MgcRacGAP, a Rho GTPase-activating protein. At anaphase, centralspindlin localizes to a narrow region of antiparallel microtubule overlap and initiates central spindle assembly. Central spindle assembly requires complex formation between ZEN-4 and CYK-4. However, the structural consequences of CYK-4 binding to ZEN-4 are unclear as are the mechanisms of microtubule bundling. Here we investigate whether CYK-4 binding induces a conformational change in ZEN-4. Characterization of the structure and conformational dynamics of the minimal interacting regions between ZEN-4 and CYK-4 by continuous wave EPR and double electron-electron resonance (DEER) spectroscopy reveals that CYK-4 binding dramatically stabilizes the relative positions of the neck linker regions of ZEN-4. Additionally, our data indicate that each neck linker is similarly structured in the bound and unbound states. CYK-4 binding decreases the rate of ZEN-4-mediated microtubule gliding. These results constrain models for the molecular organization of centralspindlin.