The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the (1)H, (15)N, and (13)C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N(2)-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal p K(a) whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.
The bis-salicylhydrazides class of HIV-1 integrase (IN) inhibitors has been postulated to function by metal chelation. However, members of this series exhibit potent inhibition only when Mn2+ is used as cofactor. The current study found that bis-aroylhydrazides could acquire inhibitory potency in Mg2+ using dihydroxybenzoyl substituents as both the right and left components of the hydrazide moiety. Employing a 2,3-dihydro-6,7-dihydroxy-1 H-isoindol-1-one ring system as a conformationally constrained 2,3-dihydroxybenzoyl equivalent provided good selectivity for IN-catalyzed strand transfer versus the 3'-processing reactions as well as antiviral efficacy in cells using HIV-1 based vectors.
When treated with dithionite at neutral pH, the recombinant hemoglobin from Synechocystis sp. PCC 6803 reconstituted with ferric heme undergoes a rapid chemical reaction resulting in the attachment of the heme group to the polypeptide chain. The nature of the cross-linked species was studied by NMR and mass spectral methods. 1H NMR data indicated that the 2-vinyl group was the reacting moiety of the heme. Mass spectrometry of pepsin digests located the site of attachment within a 12-mer at the C-terminal end of the protein. Homonuclear and 1H-15N NMR data identified the modified residue as His117, which underwent addition to the vinyl Calpha through the imidazole Nepsilon. Dithionite treatment of the globin reconstituted with Zn protoporphyrin IX sample did not lead to 2-vinyl group modification, suggesting that the chemical reduction of the heme iron facilitated the attachment.
The truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803 is a bis-histidyl hexacoordinate complex in the absence of exogenous ligands. This protein can form a covalent cross-link between His117 in the H-helix and the heme 2-vinyl group. Cross-linking, the physiological importance of which has not been established, is avoided with the His117Ala substitution. In the present work, H117A Hb was used to explore exogenous ligand binding to the heme group. NMR and thermal denaturation data showed that the replacement was of little consequence to the structural and thermodynamic properties of ferric Synechocystis Hb. It did, however, decelerate the association of cyanide ions with the heme iron. Full complexation required hours, instead of minutes, of incubation at optical and NMR concentrations. At neutral pH and in the presence of excess cyanide, binding occurred with a first-order dependence on cyanide concentration, eliminating distal histidine decoordination as the rate-limiting step. The cyanide complex of the H117A variant was characterized for the conformational changes occurring as the histidine on the distal side, His46 (E10), was displaced. Extensive rearrangement allowed Tyr22 (B10) to insert in the heme pocket and Gln43 (E7) and Gln47 (E11) to come in contact with it. H-bond formation to the bound cyanide was identified in solution with the use of 1H2O/2H2O mixtures. Cyanide binding also resulted in a change in the ratio of heme orientational isomers, in a likely manifestation of heme environment reshaping. Similar observations were made with the related Synechococcus sp. PCC 7002 H117A Hb, except that cyanide binding was rapid in this protein. In both cases, the 15N chemical shift of bound cyanide was reminiscent of that in peroxidases and the orientation of the proximal histidine was as in other truncated Hbs. The ensemble of the data provided insight into the structural cooperativity of the heme pocket scaffold and pointed to the reactive 117 site of Synechocystis Hb as a potential determinant of biophysical and, perhaps, functional properties.
The water-soluble domain of rat hepatic cytochrome b(5) undergoes marked structural changes upon heme removal. The solution structure of apocytochrome b(5) shows that the protein is partially folded in the absence of the heme group, exhibiting a stable module and a disordered heme-binding loop. The quality of the apoprotein structure in solution was improved with the use of heteronuclear NMR data. Backbone amide hydrogen exchange was studied to characterize cooperative units in the protein. It was found that this criterion distinguished the folded module from the heme-binding loop in the apoprotein, in contrast to the holoprotein. The osmolyte trimethylamine N-oxide (TMAO) did not affect the structure of the apoprotein in the disordered region. TMAO imparted a small stabilization consistent with an unfolded state effect correlating with the extent of buried surface area in the folded region of the native apoprotein. The failure of the osmolyte to cause large conformational shifts in the disordered loop supported the view that the specificity of the local sequence for the holoprotein fold was best developed with the stabilization of the native state through heme binding. To dissect the role of the heme prosthetic group in forcing the disordered region into the holoprotein conformation, the axial histidine belonging to the flexible loop (His63) was replaced with an alanine, and the structural properties of the protein with carbon-monoxide-ligated reduced iron were studied. The His63Ala substitution resulted in a protein with lower heme affinity but nevertheless capable of complete refolding. This indicated that the coordination bond was not necessary to establish the structural features of the holoprotein. In addition, the weak binding of the heme in this protein resulted in conformational shifts at a location distant from the binding site. The data suggested an uneven distribution of cooperative elements in the structure of the cytochrome.
HIV-1 integrase (IN) is a validated therapeutic target for the treatment of AIDS. However, the emergence of resistance to raltegravir, the sole marketed FDA-approved IN inhibitor, emphasizes the need to develop second-generation inhibitors that retain efficacy against clinically relevant IN mutants. We report herein bicyclic hydroxy-1H-pyrrolopyridine-triones as a new family of HIV-1 integrase inhibitors that were efficiently prepared using a key 'Pummerer cyclization deprotonation cycloaddition' cascade of imidosulfoxides. In in vitro HIV-1 integrase assays, the analogs showed low micromolar inhibitory potencies with selectivity for strand transfer reactions as compared with 3¢-processing inhibition. A representative inhibitor (5e) retained most of its inhibitory potency against the three major raltegravir-resistant IN mutant enzymes, G140S ⁄ Q148H, Y143R, and N155H. In antiviral assays employing viral vectors coding these IN mutants, compound 5e was approximately 200-and 20-fold less affected than raltegravir against the G140S ⁄ Q148H and Y143R mutations, respectively. Against the N155H mutation, 5e was approximately 10-fold less affected than raltegravir. Thus, our new compounds represent a novel structural class that may be further developed to overcome resistance to raltegravir, particularly in the case of the G140S ⁄ Q148H mutations.
Using 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one and 4,5-dihydroxy-1H-isoindole-1,3(2H)-dione based HIV-1 integrase inhibitors as display platforms, we undertook a thorough examination of the effects of modifying the halogen substituents on a key benzyl ring that is hypothesized to bind in a hydrophobic pocket of the integrase•DNA complex. Data from this study suggest that in general dihalo -substituted analogues have higher potency than monohalo -substituted compounds, but that further addition of halogens is not beneficial.Integrase (IN) is a key enzyme in the life cycle of human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). Approximately 30 drugs that have been approved by the FDA for treatment of HIV-1 infection, 1 Raltegravir [Merck & Co.,] 2 is the most recently approved drug (October 2007) and the only integrase inhibitor. Another IN inhibitor, Elvitegravir, [Gilead Sciences, 3 is currently undergoing phase III clinical trials in HIV-1-infected patients. These two inhibitors show high potency against IN -catalyzed "strand transfer" (ST) reactions, while being less effective against the IN 3′ -processing (3′-P) step. 4 This combination of characteristics is characteristic of a broad range of IN inhibitors, many of which contain the elements typified by General Structure I (Figure 1). A key component of these inhibitors is an array of heteroatoms that are hypothesized to chelate two divalent metal ions associated with catalytically essential IN residues Asp64, Asp116 and Glu152 ("DDE" motif). 5 Aromatic functionality, frequently in the form of a benzyl group linked to the chelating portion of the inhibitor, can make a significant contribution to overall binding affinity. The empirical observation that halogen substituents on this aryl ring can enhance potency has led to the hypothesis that this aryl ring may bind in one or more hydrophobic pockets of the IN•DNA complex. 6,7Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A diversity of halogen-substituted benzyl groups has been introduced into IN inhibitors ( Figure 1). [8][9][10][11][12][13][14][15][16] We recently reported a series of 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-ones (general structure A , Table 1) that exhibit potent ST inhibition in extracellular assays and antiviral efficacies in HIV-1 infected cells. 14 The closely related 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones (general structure B , Table 1) represent phthalimide -based analogues that can be prepared by the chemistry described in our earlier report. 14 For series A we had observ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.