Structure-guided DNA-DNA attraction mediated by divalent cations.

Probing the role of surface structure in electrostatic interactions, we report the first observation of sequence-dependent dsDNA condensation by divalent alkaline earth metal cations. Disparate behaviors were found between two repeating sequences with 100% AT content, a poly(A)-poly(T) duplex (AA-TT) and a poly(AT)-poly(TA) duplex (AT-TA). While AT-TA exhibits non-distinguishable behaviors from random-sequence genomic DNA, AA-TT condenses in all alkaline earth metal ions. We characterized these interactions experimentally and investigated the underlying principles using computer simulations. Both experiments and simulations demonstrate that AA-TT condensation is driven by non-specific ion-DNA interactions. Detailed analyses reveal sequence-enhanced major groove binding (SEGB) of point-charged alkali ions as the major difference between AA-TT and AT-TA, which originates from the continuous and close stacking of nucleobase partial charges. These SEGB cations elicit attraction via spatial juxtaposition with the phosphate backbone of neighboring helices, resulting in an azimuthal angular shift between apposing helices. Our study thus presents a distinct mechanism in which, sequence-directed surface motifs act with cations non-specifically to enact sequence-dependent behaviors. This physical insight allows a renewed understanding of the role of repeating sequences in genome organization and regulation and offers a facile approach for DNA technology to control the assembly process of nanostructures.

Calix[4]arene-Based Porous Organic Nanosheets.

Calixarenes are a common motif in supramolecular chemistry but have rarely been incorporated in structurally well-defined covalent 2D materials. Such a task is challenging, especially without a template, because of the nonplanar configuration and conformational flexibility of the calixarene ring. Here, we report the first-of-a-kind solvothermal synthesis of a calix[4]arene-based 2D polymer (CX4-NS) that is porous, covalent, and isolated as few-layer thick (3.52 nm) nanosheets. Experimental and theoretical characterization of the nanosheets is presented. Atomic force microscopy and transmission electron microscopy results are consistent with the calculated lowest energy state of the polymer. In the lowest energy state, parallel layers are tightly packed, and the calixarenes adopt the 1,2-alternate conformation, which gives rise to a two-dimensional pattern and a rhombic unit cell. We tested the material's ability to adsorb I2 vapor and observed a maximum capacity of 114 wt %. Molecular simulations extended to model I2 capture showed excellent agreement with experiments. Furthermore, the material was easily regenerated by mild ethanol washings and could be reused with minimal loss of efficiency.

Characterization of tunnel mutants reveals a catalytic step in ammonia delivery by an aminoacyl-tRNA amidotransferase.

The Helicobacter pylori Asp-tRNA(A) (sn) /Glu-tRNA(G) (ln) amidotransferase (GatCAB) utilizes an uncommonly hydrophilic, ~ 40 Å ammonia tunnel for ammonia/ammonium transport between isolated active sites. Hydrophilicity of this tunnel requires a distinct ammonia transport mechanism, which hypothetically occurs through a series of deprotonation and protonation steps. To explore the initiation of this relay mechanism, the highly conserved tunnel residue D185 (in the GatA subunit) was enzymatically and computationally investigated by comparing D185A, D185N, and D185E mutant enzymes to wild-type GatCAB. Our results indicate that D185 acts as an acid/base residue, participating directly in catalysis. To our knowledge, this is the first example of acid/base chemistry in a glutamine-dependent amidotransferase ammonia tunnel.

Structural and computational dissection of the catalytic mechanism of the inorganic pyrophosphatase from Mycobacterium tuberculosis.

Family I inorganic pyrophosphatases (PPiases) are ubiquitous enzymes that are critical for phosphate metabolism in all domains of life. The detailed catalytic mechanism of these enzymes, including the identity of the general base, is not fully understood. We determined a series of crystal structures of the PPiase from Mycobacterium tuberculosis (Mtb PPiase) bound to catalytic metals, inorganic pyrophosphate (PPi; the reaction substrate) and to one or two inorganic phosphate ions (Pi; the reaction product), ranging in resolution from 1.85 to 3.30Å. These structures represent a set of major kinetic intermediates in the catalytic turnover pathway for this enzyme and suggest an order of association and dissociation of the divalent metals, the substrate and the two products during the catalytic turnover. The active site of Mtb PPiase exhibits significant structural differences from the well characterized Escherichia coli PPiase in the vicinity of the bound PPi substrate. Prompted by these differences, quantum mechanics/molecular mechanics (QM/MM) analysis yielded an atomic description of the hydrolysis step for Mtb PPiase and, unexpectedly, indicated that Asp89, rather than Asp54 that was proposed for E. coli PPiase, can abstract a proton from a water molecule to activate it for a nucleophilic attack on the PPi substrate. Mutagenesis studies of the key Asp residues of Mtb PPiase supported this mechanism. This combination of structural and computational analyses clarifies our understanding of the mechanism of family I PPiases and has potential utility for rational development of drugs targeting this enzyme.

Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain.

Histone deacetylases (HDAC's) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa-specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([18F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed in vitro, in a panel of recombinant HDACs, and in vivo using PET/CT imaging in rats. [18F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [18F]DFAHA and previously reported [18F]FAHA. PET imaging with [18F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo. Furthermore, PET imaging with [18F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.

Computational analysis of ammonia transfer along two intramolecular tunnels in Staphylococcus aureus glutamine-dependent amidotransferase (GatCAB).

Most bacteria and all archaea misacylate the tRNAs corresponding to Asn and Gln with Asp and Glu (Asp-tRNA(Asn) and Glu-tRNA(Gln)).The GatCAB enzyme of most bacteria converts misacylated Glu-tRNA(Gln) to Gln-tRNA(Gln) in order to enable the incorporation of glutamine during protein synthesis. The conversion process involves the intramolecular transfer of ammonia between two spatially separated active sites. This study presents a computational analysis of the two putative intramolecular tunnels that have been suggested to describe the ammonia transfer between the two active sites. Molecular dynamics simulations have been performed for wild-type GatCAB of S. aureus and its mutants: T175(A)V, K88(B)R, E125(B)D, and E125(B)Q. The two tunnels have been analyzed in terms of free energy of ammonia transfer along them. The probability of occurrence of each type of tunnel and the variation of the probability for wild-type GatCAB and its mutants is also discussed.

The kinase activity of the Helicobacter pylori Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase is sensitive to distal mutations in its putative ammonia tunnel.

The Helicobacter pylori (Hp) Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity. AdT has two active sites, in two separate subunits. Kinetic studies have suggested that interdomain communication occurs between these subunits; however, this mechanism is not well understood. To explore domain-domain communication in AdT, we adapted an assay and optimized it to kinetically characterize the kinase activity of Hp AdT. This assay was applied to the analysis of a series of point mutations at conserved positions throughout the putative AdT ammonia tunnel that connects the two active sites. Several mutations that caused significant decreases in AdT's kinase activity (reduced by 55-75%) were identified. Mutations at Thr149 (37 Å distal to the GatB kinase active site) and Lys89 (located at the interface of GatA and GatB) were detrimental to AdT's kinase activity, suggesting that these mutations have disrupted interdomain communication between the two active sites. Models of wild-type AdT, a valine mutation at Thr149, and an arginine mutation at Lys89 were subjected to molecular dynamics simulations. A comparison of wild-type, T149V, and K89R AdT simulation results unmasks 59 common residues that are likely involved in connecting the two active sites.