Hydrazidase,a Novel Amidase Signature Enzyme That Hydrolyzes Acylhydrazides

The degradation mechanisms of natural and artificial hydrazides have been elucidated. Here we screened and isolated bacteria that utilize the acylhydrazide 4-hydroxybenzoic acid 1-phenylethylidene hydrazide (HBPH) from soils. Physiological and phylogenetic studies identified one bacterium as Microbacterium sp. strain HM58-2, from which we purified intracellular hydrazidase, cloned its gene, and prepared recombinant hydrazidase using an Escherichia coli expression system. The Microbacterium sp. HM58-2 hydrazidase is a 631-amino-acid monomer that was 31% identical to indoleacetamide hydrolase isolated from Bradyrhizobium japonicum. Phylogenetic studies indicated that the Microbacterium sp. HM58-2 hydrazidase constitutes a novel hydrazidase group among amidase signature proteins that are distributed within proteobacteria, actinobacteria, and firmicutes. The hydrazidase stoichiometrically hydrolyzed the acylhydrazide residue of HBPH to the corresponding acid and hydrazine derivative. Steady-state kinetics showed that the enzyme hydrolyzes structurally related 4-hydrozybenzamide to hydroxybenzoic acid at a lower rate than HBPH, indicating that the hydrazidase prefers hydrazide to amide. The hydrazidase contains the catalytic Ser-Ser-Lys motif that is conserved among members of the amidase signature family; it shares a catalytic mechanism with amidases, according to mutagenesis findings, and another hydrazidase-specific mechanism must exist that compensates for the absence of the catalytic Ser residue. The finding that an environmental bacterium produces hydrazidase implies the existence of a novel bacterial mechanism of hydrazide degradation that impacts its ecological role.     

Structures of dimeric nonstandard nucleotide triphosphate pyrophosphatase from Pyrococcus horikoshii OT3: functional significance of interprotomer conformational changes

Nonstandard nucleotide triphosphate pyrophosphatase (NTPase) can efficiently hydrolyze nonstandard purine nucleotides in the presence of divalent cations. The crystal structures of the NTPase from Pyrococcus horikoshii OT3 (PhNTPase) have been determined in two unliganded forms and in three liganded forms with inosine 5′-monophosphate (IMP), ITP and Mn²⁺, which visualize the recognition of these ligands unambiguously. The overall structure of PhNTPase is similar to that of previously reported crystal structures of the NTPase from Methanococcus jannaschii and the human ITPase. They share a similar protomer folding with two domains and a similar homodimeric quaternary structure. The dimeric interface of NTPase is well conserved, and the dimeric state might be important to constitute the active site of this enzyme. A conformational analysis of the five snapshots of PhNTPase structures using the multiple superposition method reveals that IMP, ITP and Mn²⁺ bind to the active site without inducing large local conformational changes, indicating that a combination of interdomain and interprotomer rigid-body shifts mainly describes the conformational change of PhNTPase. The interdomain and interprotomer conformations of the ITP liganded form are essentially the same as those observed in the unliganded form 1, indicating that ITP binding to PhNTPase in solution may follow the selection mode in which ITP binds to the subunit that happens to be in the conformation observed in the unliganded form 1. In contrast to the human ITPase inducing a large domain closure upon ITP binding, the interdomain active site cleft is generally closed in PhNTPase and only the IMP binding form shows a remarkable domain opening by 14° only in the B subunit. The interprotomer rigid-body rotation of PhNTPase has a tendency to keep the dimeric 2-fold symmetry, which is also true in human ITPase, thereby suggesting its relevance to the positive cooperativity of the dimeric NTPases. The exception of this rule is observed in the IMP liganded form in which the dimeric 2-fold symmetry is broken by a 3° interprotomer rotation in an unusual direction. A combination of the exceptional interdomain and interprotomer relocations is most likely the reason for the observed asymmetric IMP binding that might be necessary for PhNTPase to release the reaction product IMP.     

Structural basis for a disfavored elimination reaction in catalytic antibody 1D4.

Murine antibody 1D4 selectively catalyzes a highly disfavored beta-elimination reaction. Crystal structures of unliganded 1D4 and 1D4 in complex with a transition-state analog (TSA) have elucidated a possible general base mode of catalysis. The structures of the unliganded and liganded Fabs were determined to 1.80 and 1.85 A resolution, respectively. The structure of the complex reveals a binding pocket with high shape complementarity to the TSA, which is recruited to coerce the substrate into the sterically demanding, eclipsed conformation that is required for catalysis. A histidine residue and two water molecules are likely involved in the catalysis. The structure supports either a concerted E2 or stepwise E1cB-like mechanism for elimination. Finally, the liganded 1D4 structure shows minor conformational rearrangements in CDR H2, indicative of induced-fit binding of the hapten. 1D4 has pushed the boundaries of antibody-mediated catalysis into the realm of disfavored reactions and, hence, represents an important milestone in the development of this technology.     

Alteration of functional properties associated with the change in quaternary structure in unliganded haemoglobin.

The preparation of a number of specifically modified haemoglobins lacking various C-terminal residues is described. These haemoglobins can be changed from the unliganded R (or oxy type) quaternary structure to the unliganded T (or deoxy type) on addition of inositol hexaphosphate. This paper shows that this transition is associated with a lowering of the oxygen affinity and an increase in the Hill's coefficient, n, except in the case of des-(Arg141α, Tyr140α) haemoglobin where addition of inositol hexaphosphate lowers the oxygen affinity but does not increase the Hill's coefficient, n. This shows that Tyr140α plays a more important role than Tyr145β in generating co-operativity. The transition between unliganded R and unliganded T is associated with a lowering of the reactivity of the sulphydryl group Cys93β; this is due both to the change in quaternary structure per se and to the formation of the salt bridge between His146β and Asp94β. The Bohr effect associated with the transition from the unliganded to liganded R structure was less than one-tenth of the normal Bohr effect.     

Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t_1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G₀ are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t_1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1⁻ GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.     

Apo and Calcium-Bound Crystal Structures of Cytoskeletal Protein Alpha-14 Giardin (Annexin E1) from the Intestinal Protozoan Parasite Giardia lamblia

Alpha-14 giardin (annexin E1), a member of the alpha giardin family of annexins, has been shown to localize to the flagella of the intestinal protozoan parasite Giardia lamblia. Alpha giardins show a common ancestry with the annexins, a family of proteins most of which bind to phospholipids and cellular membranes in a Ca²⁺-dependent manner and are implicated in numerous membrane-related processes including cytoskeletal rearrangements and membrane organization. It has been proposed that alpha-14 giardin may play a significant role during the cytoskeletal rearrangement during differentiation of Giardia. To gain a better understanding of alpha-14 giardin's mode of action and its biological role, we have determined the three-dimensional structure of alpha-14 giardin and its phospholipid-binding properties. Here, we report the apo crystal structure of alpha-14 giardin determined in two different crystal forms as well as the Ca²⁺-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 Å, respectively. Although the overall fold of alpha-14 giardin is similar to that of alpha-11 giardin, multiwavelength anomalous dispersion phasing was required to solve the alpha-14 giardin structure, indicating significant structural differences between these two members of the alpha giardin family. Unlike most annexin structures, which typically possess N-terminal domains, alpha-14 giardin is composed of only a core domain, followed by a C-terminal extension that may serve as a ligand for binding to cytoskeletal protein partners in Giardia. In the Ca²⁺-bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calcium-binding site not previously observed in annexin structures. This novel high-affinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipid-binding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to phospholipids that coordinate cytoskeletal disassembly/assembly during differentiation of the parasite.     

Hemoglobin tertiary structural change on ligand binding its role in the co-operative mechanism

Analysis of the tertiary structural alterations in hemoglobin induced by ligand binding demonstrates that an allosteric core composed of the heme, histidine F8, the FG corner and part of the F-helix plays an essential role in co-operativity. This conclusion is based on structural and spectroscopic data and theoretical studies of hemoglobin chains. The methodology employed in the calculations is presented with details of the empirical energy function. Energy minimized structures of the unliganded hemoglobin chains, which serve as reference systems for the analysis, are described. To determine the structural changes induced by ligand binding, the effects of FeN bond shortening and of heme translation and tilting perturbations are examined. Energy minimization in the presence of the perturbations serves to provide information concerning the globin structural modifications produced by them. The validity of the results is supported by comparisons with the X-ray data of Anderson, Pulsinelli, Baldwin and Chothia on tertiary changes in the hemoglobin subunits.Internal to the allosteric core, there appear to be two stable positions for its elements: one of these corresponds to the liganded and the other to the unliganded species. The unliganded geometry fits without strain into the deoxy tetramer, while the liganded one fits without strain into the oxy tetramer. On ligation of a subunit in the deoxy tetramer, the structural changes within the allosteric core are in the direction of those found in going from the unliganded deoxy to the liganded oxy system, although they are reduced by the presence of constraints due to the other subunits in the deoxy tetramer. In addition, the quaternary constraints in the deoxy tetramer prevent the large overall displacement of the allosteric core that occurs in the transition to the liganded oxy tetramer. The coupling between the changes internal to the allosteric core, produced on ligation and the overall displacement of the core that accompanies the quaternary transition, is an essential element of the co-operative mechanism. As shown in previous work (Gelin & Karplus, 1977), the proximal histidine serves as the link between the position of the heme and the F-helix; the asymmetric orientation of the histidine in the deoxy structure, coupled with contributions from other heme-protein interactions, appears to initiate the tertiary structural changes induced by ligand binding. The reduced oxygen affinity of hemoglobin results not from tension on the heme in the unliganded structure (there is none) but instead from strain in the liganded subunit of the tetramer within the deoxy quaternary structure. Further, the changes in the allosteric core provide a relatively localized reaction path for transmitting information concerning ligand binding from the heme group to the surface of the subunit; particularly in the α-chain, the residue Val FG5 appears to play an important role in the reaction path.The present analysis has important implications for realistic statistical thermodynamic models of hemoglobin co-operativity. It suggests that the previously formulated model (Szabo & Karplus, 1972) should be generalized by the introduction of two different subunit tertiary structures in the deoxy and in the oxy tetramer; they would be associated with the unliganded and the liganded allosteric core, respectively, and would take account of steric constraints that reduce the ligand affinity of the deoxy tetramer.     

A novel t(4;16)(q25;q23.1) associated with EGF and ELOVL6 deregulation in acute myeloid leukemia

About 50% of acute myeloid leukemia (AML) patients show the occurrence of non-random chromosome rearrangements. Most of the recurrent karyotypic rearrangements in AML have been defined as distinct disease entities in the 2008 World Health Organization (WHO) classification. In this paper we report an AML case showing a novel t(4;16)(q25;q23.1) rearrangement causing the activation of epidermal growth factor (EGF) and elongation of long-chain fatty acids family member 6 (ELOVL6) genes, rather than the generation of a novel fusion gene.     

Ligand-induced conformational changes and a mechanism for domain closure in Aspergillus nidulans dehydroquinate synthase

In order to investigate systematically substrate and cofactor-induced conformational changes in the enzyme dehydroquinate synthase (DHQS), eight structures representing a series of differently liganded states have been determined in a total of six crystal forms. DHQS in the absence of the substrate analogue carbaphosphonate, either unliganded or in the presence of NAD or ADP, is in an open form where a relative rotation of 11-13 degrees between N and C-terminal domains occurs.Analysis of torsion angle difference plots between sets of structures reveals eight rearrangements that appear relevant to domain closure and a further six related to crystal packing. Overlapping 21 different copies of the individual N and C-terminal DHQS domains further reveals a series of pivot points about which these movements occur and illustrates the way in which widely separated secondary structure elements are mechanically inter-linked to form "composite elements", which propagate structural changes across large distances.This analysis has provided insight into the basis of DHQS ligand-initiated domain closure and gives rise to the proposal of an ordered sequence of events involving substrate binding, and local rearrangements within the active site that are propagated to the hinge regions, leading to closure of the active-site cleft.     

ATP-induced structural change of dephosphocoenzyme A kinase from Thermus thermophilus HB8

Dephosphocoenzyme A kinase (DCK) catalyzes phosphorylation in the final step of coenzyme A (CoA) biosynthesis. In this phosphorylation process, domain movements play a very important role. To reveal the structural changes induced by ligand binding, we determined the crystal structure of DCK from Thermus thermophilus HB8 by the multiwavelength anomalous dispersion method at 2.8 A. The crystal structure includes three independent protein molecules in the asymmetric unit: One is a liganded form and the others are unliganded. The topology shows a canonical nucleotide-binding protein possessing the P-loop motif. A structure homology search by DALI revealed the similarity of the DCKs from T. thermophilus HB8, Haemophilus influenzae, and Escherichia coli. Structural comparisons between the liganded and unliganded forms of DCK from T. thermophilus HB8 indicated domain movements induced by adenosine triphosphate (ATP) binding. For the domain movements, proline residues confer flexibility at the domain linkages. In particular, Pro91 plays an important role in moving the CoA domain.     

Slowly interchanging conformers of bovine neurophysin-I in the unliganded dimeric state.

The effect of neurophysin dimerization on Tyr-49, a residue adjacent to the hormone-binding site, was investigated by proton NMR in order to analyze the basis of the dimerization-induced increase in neurophysin hormone affinity. Dimerization-induced changes in Tyr-49 resonances, in two unliganded bovine neurophysins, suggested that Tyr-49 perturbation is an intrinsic consequence of dimerization, although Tyr-49 is distant from the monomer-monomer interface in the crystalline liganded state. To determine whether this perturbation reflects a conformational difference between liganded and unliganded states that places Tyr-49 at the interface in the unliganded state, or a dimerization-induced change in secondary (2 degrees) or tertiary (3 degrees) structure, the more general structural consequences of dimerization were further analyzed. No change in 2 degrees structure upon dimerization was demonstrable by CD. On the other hand, a general similarity of regions involved in dimerization in unliganded and liganded states was indicated by NMR evidence of participation of His-80 and Phe-35 in dimerization in the unliganded state; both residues are at the interface in the crystal structure and distant from Tyr-49. Consistent with a lack of direct participation of Tyr-49 at the monomer-monomer interface, dimerization induced at least two distinct slowly exchanging environmental states for the 3.5 ring protons of Tyr-49 without significantly increased dipolar broadening relative to the monomer. Two environments were also found in the dimer of des-1-8 neurophysin-I for the methyl protons of Thr-9, another residue distant from the monomer-monomer interface and close to the binding site in the liganded state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.     

The oxygen affinity of hemoglobin betaSH chains is concentration dependent.

Oxygen binding to isolated hemoglobin β^SH chains exhibits heterotropic interactions with H⁺, inositol hexaphosphate and CO₂ which implies different structures of the liganded and unliganded β chains. In order to find out if the dissociation behaviour of $\text{β}{}_4{}^{\mathrm{SH}}$ homotetramers is likewise linked to oxygenation, we have measured the oxygen affinity of the pigment as a function of the protein concentration at different pH values. We found that a decrease in protein concentration is associated with a decrease in oxygen affinity. This result accords with predictions reached from studies on the self-association of liganded and unliganded β chains. Furthermore, it was established that both at high and low protein concentrations the oxygen affinity of the β chains is pH dependent.     

Formation of Chromosome Rearrangements by P Factors in Drosophila

We studied a collection of 746 chromosome rearrangements all induced by the activity of members of the P family of transposable elements in Drosophila melanogaster. The chromosomes ranged from simple inversions to complex rearrangements. The distribution of complex rearrangement classes was of the kind expected if each rearrangement came about from a single multibreak event followed by random rejoining of chromosome segments, as opposed to a series of two-break events. Most breakpoints occurred at or very near (within a few hundred nucleotide pairs) the sites of preexisting P elements, but these elements were often lost during the rearrangement event. There were also a few cases of apparent gain of P elements. In cases in which both breakpoints of an inversion retained P elements, that inversion was capable of reverting at high frequencies to the original sequence or something close to it. This reversion occurred with sufficient precision to restore the function of a gene, held-up-b, which had been mutated by the breakpoint. However, some of the reversions had acquired irregularities at the former breakpoints that were detectable either by standard cytology or by molecular methods. The revertants themselves retained the ability to undergo further rearrangements depending on the presence of P elements. We interpret these results to rule out the simplest hypotheses of rearrangement formation that involve cointegrate structures or homologous recombination. The data provide a general picture of the rearrangement process and its possible relationship to transposition.     

Dynamic domains and geometrical properties of HIV-1 gp120 during conformational changes induced by CD4 binding

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by the CD4 and antigen 17b, and the SIV gp120 core pre-bound by the CD4 are known. We have performed dynamic domain studies on the homology models of the CD4-bound and unliganded HIV-1 gp120 with modeled V3 and V4 loops to explore details of conformational changes, hinge axes, and hinge bending regions in the gp120 structures upon CD4 binding. Four dynamic domains were clustered and intricately motional modes for domain pairs were discovered. Together with the detailed comparative analyses of geometrical properties between the unliganded and liganded gp120 models, an induced fit model was proposed to explain events accompanying the CD4 engagement to the gp120, which provided new insight into the dynamics of the molecular induced binding mechanism that complements the molecular dynamics and crystallographic studies.     

Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase

We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.     

Structures and oxygen affinities of crystalline human hemoglobin C (β6 Glu->Lys) in the R and R2 quaternary structures

Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (β6 Glu→Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 Å resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K₄) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2–3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.