Hemant P. Yennawar

Structure of RCC1 chromatin factor bound to the nucleosome core particle.

The small GTPase Ran enzyme regulates critical eukaryotic cellular functions including nuclear transport and mitosis through the creation of a RanGTP gradient around the chromosomes. This concentration gradient is created by the chromatin-bound RCC1 (regulator of chromosome condensation) protein, which recruits Ran to nucleosomes and activates Ran’s nucleotide exchange activity. Although RCC1 has been shown to bind directly with the nucleosome, the molecular details of this interaction were not known. Here we determine the crystal structure of a complex of Drosophila RCC1 and the nucleosome core particle at 2.9 Å resolution, providing an atomic view of how a chromatin protein interacts with the histone and DNA components of the nucleosome. Our structure also suggests that the Widom 601 DNA positioning sequence present in the nucleosomes forms a 145-base-pair nucleosome core particle, not the expected canonical 147-base-pair particle.

Intermolecular N–H Oxidative Addition of Ammonia, Alkylamines, and Arylamines to a Planar σ3-Phosphorus Compound via an Entropy-Controlled Electrophilic Mechanism

Ammonia, alkyl amines, and aryl amines are found to undergo rapid intermolecular N-H oxidative addition to a planar mononuclear σ(3)-phosphorus compound (1). The pentacoordinate phosphorane products (1·[H][NHR]) are structurally robust, permitting full characterization by multinuclear NMR spectroscopy and single-crystal X-ray diffraction. Isothermal titration calorimetry was employed to quantify the enthalpy of the N-H oxidative addition of n-propylamine to 1 ((n)PrNH2 + 1 → 1·[H][NH(n)Pr], ΔHrxn(298) = -10.6 kcal/mol). The kinetics of n-propylamine N-H oxidative addition were monitored by in situ UV absorption spectroscopy and determination of the rate law showed an unusually large molecularity (ν = k[1][(n)PrNH2](3)). Kinetic experiments conducted over the temperature range of 10-70 °C revealed that the reaction rate decreased with increasing temperature. Activation parameters extracted from an Eyring analysis (ΔH(⧧) = -0.8 ± 0.4 kcal/mol, ΔS(⧧) = -72 ± 2 cal/(mol·K)) indicate that the cleavage of strong N-H bonds by 1 is entropy controlled due to a highly ordered, high molecularity transition state. Density functional calculations indicate that a concerted oxidative addition via a classical three-center transition structure is energetically inaccessible. Rather, a stepwise heterolytic pathway is preferred, proceeding by initial amine-assisted N-H heterolysis upon complexation to the electrophilic phosphorus center followed by rate-controlling N → P proton transfer.

Two-component signaling in the AAA + ATPase DctD: binding Mg2+ and BeF3− selects between alternate dimeric states of the receiver domain

A crystal structure is described for the Mg2+‐BeF3‐‐bound receiver domain of Sinorhizobium meliloti DctD bearing amino acid substitution E121K. Differences between the apo‐ and ligandbound active sites are similar to those reported for other receiver domains. However, the off and on states of the DctD receiver domain are characterized by dramatically different dimeric structures, which supports the following hypothesis of signal transduction. In the off state, the receiver domain and coiled‐coil linker form a dimer that inhibits oligomerization of the AAA+ ATPase domain. In this conformation, the receiver domain cannot be phosphorylated or bind Mg2+ and BeF3‐. Instead, these modifications stabilize an alternative dimeric conformation that repositions the subunits by approximately 20 Å, thus replacing the α4‐β5‐α5 interface with an α4‐β5 interface. Reoriented receiver domains permit the ATPase domain to oligomerize and stimulate open complex formation by the ς54 form of RNA polymerase. NtrC, which shares 38% sequence identity with DctD, works differently. Its activated receiver domain must facilitate oligomerization of its ATPase domain. Significant differences exist in the signaling surfaces of the DctD and NtrC receiver domains that may help explain how triggering the common two‐component switch can variously regulate assembly of a AAA+ ATPase domain.

Reversible Intermolecular E–H Oxidative Addition to a Geometrically Deformed and Structurally Dynamic Phosphorous Triamide

The synthesis and reactivity of geometrically constrained tricoordinate phosphorus (σ(3)-P) compounds supported by tridentate triamide chelates (N[o-NR-C6H4]2(3-); R = Me or (i)Pr) are reported. Studies indicate that 2 (P{N[o-NMe-C6H4]2}) adopts a Cs-symmetric structure in the solid state. Variable-temperature NMR studies demonstrate a low-energy inversion at phosphorus in solution (ΔG(‡)(exptl)(298) = 10.7(5) kcal/mol), for which DFT calculations implicate an edge-inversion mechanism via a metastable C2-symmetric intermediate. In terms of reactivity, compound 2 exhibits poor nucleophilicity, but undergoes oxidative addition at ambient temperature of diverse O-H- and N-H-containing compounds (including alcohols, phenols, carboxylic acids, amines, and anilines). The resulting pentacoordinate adducts 2·[H][OR] and 2·[H][NHR] are characterized by multinuclear NMR spectroscopy and X-ray crystallography, and their structures (which span the pseudorotation coordinate between trigonal bipyramidal and square planar) are evaluated in terms of negative hyperconjugation. At elevated temperatures, the oxidative addition is shown to be reversible for volatile alcohols and amines.

A dimeric two-component receiver domain inhibits the sigma54-dependent ATPase in DctD.

We report the crystal structure of a fragment of Sinorhizobium meliloti DctD, a bacterial enhancer binding protein, at 1.7 Å. The fragment contains the proteins two‐component receiver module and adjacent linker, which in the native protein joins the receiver domain to a σ54‐dependent ATPase domain. The structure reveals a novel dimerization surface, which sequence analysis indicates is common to 4.5% of the known two‐component receiver domains. Genetic, biochemical, and structural data for amino acid substitution variants indicate that the dimer is necessary to inhibit the basal activity of the ATPase domain. The dimerization element is thus needed to maintain the “off” state, and changes within it may signal activation. Analytical ultracentrifugation data for the phosphorylated fragment of DctD appear to rule out the simple model that signaling is mediated via monomerization of the receiver domain.

Refinement of protein crystal structures using energy restraints derived from linear-scaling quantum mechanics

A novel method is proposed in which combined restraints derived from linear-scaling semiempirical quantum-mechanical (QM) calculations and X-ray diffraction data are combined to refine crystal structures of proteins. Its performance has been tested on a small protein molecule, bovine pancreatic trypsin inhibitor (BPTI). The refinement involves minimization of the sum of a geometric energy function and an X-ray target function based on either the least-squares residual or the maximum-likelihood formalism. For comparison, similar refinement runs have also been performed using energy restraints derived from the force field available in the Crystallography & NMR System (CNS) program. The QM refinements were carried out with weights that were varied by several orders of magnitude and the optimal weights were identified by observing the trend in the final free R values, QM heats of formation and coordinate root-mean-square deviations (r.m.s.d.s) from the crystal structure. It is found that the QM weights are typically smaller but generally on the same scale as the molecular-mechanics (MM) weights for the respective X-ray target functions. The crystallographic R, free R, real-space R values and correlation coefficients based on the structures refined with the energy restraints derived from our QM calculations and Engh and Huber parameters are comparable, suggesting that the QM restraints are capable of maintaining reasonable stereochemistry to a similar degree as the force-field parameters. A detailed inspection of the structures refined with the QM and MM energy restraints reveals that one of the common differences between them and the crystal structure is that the strained bond angles in the crystal structure are corrected after energetically restrained refinements. Systematic differences in certain bond lengths between the QM-refined structures and the statistical averages of experimental structures have also been observed and discussed.

Orientation of Diamagnetic Layered Transition Metal Oxide Particles in 1-Tesla Magnetic Fields

The magnetic field-driven orientation of microcrystals of six diamagnetic layered transition metal oxides (HLaNb(2)O(7), HCa(2)Nb(3)O(10)·0.5H(2)O, KNaCa(2)Nb(4)O(13), KTiTaO(5), KTiNbO(5), and H(2.2)K(1.8)Nb(6)O(17)·nH(2)O) suspended in epoxy resins was studied by X-ray diffraction using permanent magnets producing a 0.8 T field. Although the degree of orientation, quantified as the Hermans order parameter, was strongly affected by the particle size distribution, in all cases microcrystals with ∼1-2 μm lateral dimensions were found to orient with the magnetic field vector in the layer plane. Control of the orientation of ionically conducting layered oxides is of interest for practical applications in batteries and fuel cells. The consistent direction of orientation of the lamellar oxides studied can be rationalized in the framework of a quantitative bond anisotropy model developed by Uyeda (Phys. Chem. Miner.1993, 20, 77-80). The asymmetry of metal-oxygen bonding at the faces of the octahedral layers results in long and short M-O bonds perpendicular to the plane of the sheets. This distortion of the M-O octahedra, which is a structural feature of almost all layered materials that contain octahedral bonding frameworks, gives rise to the diamagnetic anisotropy and results in an easy axis or plane of magnetization in the plane of the sheets.

P–N Cooperative Borane Activation and Catalytic Hydroboration by a Distorted Phosphorous Triamide Platform

Studies of the stoichiometric and catalytic reactivity of a geometrically constrained phosphorous triamide 1 with pinacolborane (HBpin) are reported. The addition of HBpin to phosphorous triamide 1 results in cleavage of the B-H bond of pinacolborane through addition across the electrophilic phosphorus and nucleophilic N-methylanilide sites in a cooperative fashion. The kinetics of this process of were investigated by NMR spectroscopy, with the determined overall second-order empirical rate law given by ν = -k[1][HBpin], where k = 4.76 × 10-5 M-1 s-1 at 25 °C. The B-H bond activation process produces P-hydrido-1,3,2-diazaphospholene intermediate 2, which exhibits hydridic reactivity capable of reacting with imines to give phosphorous triamide intermediates, as confirmed by independent synthesis. These phosphorous triamide intermediates are typically short lived, evolving with elimination of the N-borylamine product of imine hydroboration with regeneration of the deformed phosphorous triamide 1. The kinetics of this latter process are shown to be first-order, indicative of a unimolecular mechanism. Consequently, catalytic hydroboration of a variety of imine substrates can be realized with 1 as the catalyst and HBpin as the terminal reagent. A mechanistic proposal implicating a P-N cooperative mechanism for catalysis that incorporates the various independently verified stoichiometric steps is presented, and a comparison to related phosphorus-based systems is offered.

2,3-Diphenyl-2,3,5,6-tetra­hydro-4H-1,3-thia­zin-4-one

The six-membered thiazine ring in the title compound, C16H15NOS, adopts a half-chair conformation, with the S atom forming the back of the chair. The base of the chair has a slight twist reflected in the r.m.s. deviation (0.0756 Å) of those five atoms from the plane defined by them. The phenyl substituents are almost perpendicular to each other [dihedral angle 87.06 (9)°]. In the crystal, molecules are linked into chains parallel to the c axis through C—H⋯O interactions.

X-ray crystal structure and small-angle X-ray scattering of sheep liver sorbitol dehydrogenase.

The X-ray crystal structure of sheep liver sorbitol dehydrogenase (slSDH) has been determined using the crystal structure of human sorbitol dehydrogenase (hSDH) as a molecular-replacement model. slSDH crystallized in space group I222 with one monomer in the asymmetric unit. A conserved tetramer that superposes well with that seen in hSDH (despite belonging to a different space group) and obeying the 222 crystal symmetry is seen in slSDH. An acetate molecule is bound in the active site, coordinating to the active-site zinc through a water molecule. Glycerol, a substrate of slSDH, also occupies the substrate-binding pocket together with the acetate designed by nature to fit large polyol substrates. The substrate-binding pocket is seen to be in close proximity to the tetramer interface, which explains the need for the structural integrity of the tetramer for enzyme activity. Small-angle X-ray scattering was also used to identify the quaternary structure of the tetramer of slSDH in solution.