Kinetics and thermodynamics of metal‐binding to histone deacetylase 8

Histone deacetylase 8 (HDAC8) was originally classified as a Zn(II)‐dependent deacetylase on the basis of Zn(II)‐dependent HDAC8 activity in vitro and illumination of a Zn(II) bound to the active site. However, in vitro measurements demonstrated that HDAC8 has higher activity with a bound Fe(II) than Zn(II), although Fe(II)‐HDAC8 rapidly loses activity under aerobic conditions. These data suggest that in the cell HDAC8 could be activated by either Zn(II) or Fe(II). Here we detail the kinetics, thermodynamics, and selectivity of Zn(II) and Fe(II) binding to HDAC8. To this end, we have developed a fluorescence anisotropy assay using fluorescein‐labeled suberoylanilide hydroxamic acid (fl‐SAHA). fl‐SAHA binds specifically to metal‐bound HDAC8 with affinities comparable to SAHA. To measure the metal affinity of HDAC, metal binding was coupled to fl‐SAHA and assayed from the observed change in anisotropy. The metal KD values for HDAC8 are significantly different, ranging from picomolar to micromolar for Zn(II) and Fe(II), respectively. Unexpectedly, the Fe(II) and Zn(II) dissociation rate constants from HDAC8 are comparable, koff ∼0.0006 s−1, suggesting that the apparent association rate constant for Fe(II) is slow (∼3 × 103 M−1 s−1). Furthermore, monovalent cations (K+ or Na+) that bind to HDAC8 decrease the dissociation rate constant of Zn(II) by ≥100‐fold for K+ and ≥10‐fold for Na+, suggesting a possible mechanism for regulating metal exchange in vivo. The HDAC8 metal affinities are comparable to the readily exchangeable Zn(II) and Fe(II) concentrations in cells, consistent with either or both metal cofactors activating HDAC8.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110703/1/pro2623.pd

Fluorescence-based biosensing of zinc using carbonic anhydrase

Measurement of free zinc levels and imaging of zinc fluxes remains technically difficult due to low levels and the presence of interfering cations such as Mg and Ca. We have developed a series of fluorescent zinc indicators based on the superb sensitivity and selectivity of a protein, human apo-carbonic anhydrase II, for Zn(II). These indicators transduce the level of free zinc as changes in intensity, wavelength ratio, lifetime, and/or anisotropy; the latter three approaches permit quantitative imaging of zinc levels in the microscope. A unique attribute of sensors incorporating biological macromolecules as transducers is their capability for modification by site-directed mutagenesis. Thus we have produced variants of carbonic anhydrase with improved affinity for zinc, altered selectivity, and enhanced binding kinetics, all of which are difficult to modify in small molecule indicators.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44187/1/10534_2004_Article_355471.pd

Discovering RNA-Protein Interactome by Using Chemical Context Profiling of the RNA-Protein Interface

SummaryRNA-protein (RNP) interactions generally are required for RNA function. At least 5% of human genes code for RNA-binding proteins. Whereas many approaches can identify the RNA partners for a specific protein, finding the protein partners for a specific RNA is difficult. We present a machine-learning method that scores a protein’s binding potential for an RNA structure by utilizing the chemical context profiles of the interface from known RNP structures. Our approach is applicable even when only a single RNP structure is available. We examined 801 mammalian proteins and find that 37 (4.6%) potentially bind transfer RNA (tRNA). Most are enzymes involved in cellular processes unrelated to translation and were not known to interact with RNA. We experimentally tested six positive and three negative predictions for tRNA binding in vivo, and all nine predictions were correct. Our computational approach provides a powerful complement to experiments in discovering new RNPs

Identification of a Novel Class of Farnesylation Targets by Structure-Based Modeling of Binding Specificity

Farnesylation is an important post-translational modification catalyzed by farnesyltransferase (FTase). Until recently it was believed that a C-terminal CaaX motif is required for farnesylation, but recent experiments have revealed larger substrate diversity. In this study, we propose a general structural modeling scheme to account for peptide binding specificity and recapitulate the experimentally derived selectivity profile of FTase in vitro. In addition to highly accurate recovery of known FTase targets, we also identify a range of novel potential targets in the human genome, including a new substrate class with an acidic C-terminal residue (CxxD/E). In vitro experiments verified farnesylation of 26/29 tested peptides, including both novel human targets, as well as peptides predicted to tightly bind FTase. This study extends the putative range of biological farnesylation substrates. Moreover, it suggests that the ability of a peptide to bind FTase is a main determinant for the farnesylation reaction. Finally, simple adaptation of our approach can contribute to more accurate and complete elucidation of peptide-mediated interactions and modifications in the cell

Ionic interactions between PRNA and P protein in Bacillus subtilis RNase P characterized using a magnetocapture-based assay

Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the cleavage of the 5′ end of precursor tRNA. To characterize the interface between the Bacillus subtilis RNA (PRNA) and protein (P protein) components, the intraholoenzyme K(D) is determined as a function of ionic strength using a magnetocapture-based assay. Three distinct phases are evident. At low ionic strength, the affinity of PRNA for P protein is enhanced as the ionic strength increases mainly due to stabilization of the PRNA structure by cations. Lithium substitution in lieu of potassium enhances the affinity at low ionic strength, whereas the addition of ATP, known to stabilize the structure of P protein, does not affect the affinity. At high ionic strength, the observed affinity decreases as the ionic strength increases, consistent with disruption of ionic interactions. These data indicate that three to four ions are released on formation of holoenzyme, reflecting the number of ion pairs that occur between the P protein and PRNA. At moderate ionic strength, the two effects balance so that the apparent K(D) is not dependent on the ionic strength. The K(D) between the catalytic domain (C domain) and P protein has a similar triphasic dependence on ionic strength. Furthermore, the intraholoenzyme K(D) is identical to or tighter than that of full-length PRNA, demonstrating that the P protein binds solely to the C domain. Finally, pre-tRNA(asp) (but not tRNA(asp)) stabilizes the PRNA•P protein complex, as predicted by the direct interaction between the P protein and pre-tRNA leader