Previously Uncharacterized Variants, OCF-E-OCF-J, of the Antifungal Occidiofungin Produced by Burkholderia contaminans MS14.

The rise of multidrug resistant fungal infections highlights the need to identify and develop novel antifungal agents. Occidiofungin is a nonribosomally synthesized glycolipopeptide that has a unique mechanism of action, disrupting actin-mediated functions and inducing cellular apoptosis. Antifungal activity has been observed in vitro against various fungal species, including multidrug resistant Candida auris, and in vivo efficacy has been demonstrated in a murine vulvovaginal candidiasis model. Occidiofungin, a cyclic glycolipopeptide, is composed of eight amino acids and in previous studies, an asparagine residue was assigned at position 7 (ASN7). In this study, new structural variants of occidiofungin have been characterized which have aspartic acid (ASP7), glutamine (GLN7), or glutamic acid (GLU7) at position 7. The side chain of the ASP7 variant contains the only terminal carboxylic acid in the peptide and provides a useful site for selective chemical modifications. Analogues were synthesized at the ASP7 position and tested for antifungal activity. These analogues were shown to be more active as compared to the ASP7 variant against a panel of Candida species. The naturally occurring variants of occidiofungin with a side chain containing a carboxylic acid at the seventh amino acid position can be used to develop semisynthetic analogues with enhanced therapeutic properties.

Synthesis and characterization of semisynthetic analogs of the antifungal occidiofungin.

Occidiofungin is a broad-spectrum antifungal compound produced by Burkholderia contaminans MS14. It is a cyclic glycol-lipopeptide with a novel beta-amino acid (NAA2) containing a hydroxylated C18 fatty acid chain with a xylose sugar. This study reports a strategy to produce semisynthetic analogs of occidiofungin to further explore the structure activity relationships of this class of compounds. Oxidative cleavage of the diol present on carbons five C(5) and six C(6) removes the xylose and twelve carbons of the fatty acid chain. The resulting cyclic peptide product, occidiofungin aldehyde, is devoid of antifungal activity. However, the free aldehyde group on this product can be subjected to reductive amination reactions to provide interesting semisynthetic analogs. This chemistry allows the quick generation of analogs to study the structure activity relationships of this class of compounds. Despite restoring the length of the aliphatic side chain by reductive amination addition with undecylamine or dodecylamine to the free aldehyde group, the obtained analogs did not demonstrate any antifungal activity. The antifungal activity was partially restored by the addition of a DL-dihydrosphingosine. The dodecylamine analog was demonstrated to still bind to the cellular target actin, suggesting that the diol on the side chain of native occidiofungin is important for entry into the cell enabling access to cellular target F-actin. These results show that the alkyl side chain on NAA2 along with the diol present on this side chain is important for occidiofungin's antifungal activity.

HIV-1 Virus Interactions With Host Proteins: Interaction of the N-terminal Domain of the HIV-1 Capsid Protein With Human Calmodulin.

The human immunodeficiency virus (HIV-1 virus) exploits several host factors for assembly, infection, and replication within the infected cells. In this work, we describe the evidence for an interaction of the N-terminal domain of the HIV-1 capsid protein with human calmodulin. The precise role of this interaction within the life cycle of the HIV-1 virus is yet to be defined. Potential roles for this interaction in the viral capsid uncoating are discussed.

Covalent Structure and Bioactivity of the Type AII Lantibiotic Salivaricin A2.

Lantibiotics are a class of lanthionine-containing, ribosomally synthesized, and posttranslationally modified peptides (RiPPs) produced by Gram-positive bacteria. Salivaricin A2 belongs to the type AII lantibiotics, which are generally considered to kill Gram-positive bacteria by binding to the cell wall precursor lipid II via a conserved ring A structure. Salivaricin A2 was first reported to be isolated from a probiotic strain, Streptococcus salivarius K12, but the structural and bioactivity characterizations of the antibiotic have remained limited. In this study, salivaricin A2 was purified and its covalent structure was characterized. N-terminal analogues of salivaricin A2 were generated to study the importance for bioactivity of the length and charge of the N-terminal amino acids. Analogue salivaricin A2(3-22) has no antibacterial activity and does not have an antagonistic effect on the native compound. The truncated analogue also lost its ability to bind to lipid II in a thin-layer chromatography (TLC) assay, suggesting that the N-terminal amino acids are important for binding to lipid II. The creation of N-terminal analogues of salivaricin A2 promoted a better understanding of the bioactivity of this antibiotic and further elucidated the structural importance of the N-terminal leader peptide. The antibacterial activity of salivaricin A2 is due not only to the presence of the positively charged N-terminal amino acid residues, but to the length of the N-terminal linear peptide.IMPORTANCE The amino acid composition of the N-terminal linear peptide of salivaricin A2 is crucial for function. Our study shows that the length of the amino acid residues in the linear peptide is crucial for salivaricin A2 antimicrobial activity. Very few type AII lantibiotic covalent structures have been confirmed. The characterization of the covalent structure of salivaricin A2 provides additional support for the predicted lanthionine and methyl-lanthionine ring formations present in this structural class of lantibiotics. Removal of the N-terminal Lys1 and Arg2 residues from the peptide causes a dramatic shift in the chemical shift values of amino acid residues 7 through 9, suggesting that the N-terminal amino acids contribute to a distinct structural conformer for the linear peptide region. The demonstration that the bioactivity could be partially restored with the substitution of N-terminal alanine residues supports further studies aimed at determining whether new analogues of salivaricin A2 for novel applications can be synthesized.

The Siderophore Product Ornibactin Is Required for the Bactericidal Activity of Burkholderia contaminans MS14.

Burkholderia contaminans MS14 was isolated from soil in Mississippi. When it is cultivated on nutrient broth-yeast extract agar, the colonies exhibit bactericidal activity against a wide range of plant-pathogenic bacteria. A bacteriostatic compound with siderophore activity was successfully purified and was determined by nuclear magnetic resonance spectroscopy to be ornibactin. Isolation of the bactericidal compound has not yet been achieved; therefore, the exact nature of the bactericidal compound is still unknown. During an attempt to isolate the bactericidal compound, an interesting relationship between the production of ornibactin and the bactericidal activity of MS14 was characterized. Transposon mutagenesis resulted in two strains that lost bactericidal activity, with insertional mutations in a nonribosomal peptide synthetase (NRPS) gene for ornibactin biosynthesis and a luxR family transcriptional regulatory gene. Coculture of these two mutant strains resulted in restoration of the bactericidal activity. Furthermore, the addition of ornibactin to the NRPS mutant restored the bactericidal phenotype. It has been demonstrated that, in MS14, ornibactin has an alternative function, aside from iron sequestration. Comparison of the ornibactin biosynthesis genes in Burkholderia species shows diversity among the regulatory elements, while the gene products for ornibactin synthesis are conserved. This is an interesting observation, given that ornibactin is thought to have the same defined function within Burkholderia species. Ornibactin is produced by most Burkholderia species, and its role in regulating the production of secondary metabolites should be investigated.IMPORTANCE Identification of the antibacterial product from strain MS14 is not the key feature of this study. We present a series of experiments that demonstrate that ornibactin is directly involved in the bactericidal phenotype of MS14. This observation provides evidence for an alternative function for ornibactin, aside from iron sequestration. Ornibactin should be further evaluated for its role in regulating the biosynthesis of secondary metabolites in other Burkholderia species.

Identification of initial leads directed at the calmodulin-binding region on the Src-SH2 domain that exhibit anti-proliferation activity against pancreatic cancer.

Cellular calmodulin binds to the SH2 domain of Src kinase, and upon Fas activation it recruits Src into the death-inducing signaling complex. This results in Src-ERK activation of cell survival pathway through which pancreatic cancer cells survive and proliferate. We had proposed that the inhibition of the interaction of calmodulin with Src-SH2 domain is an attractive strategy to inhibit the proliferation of pancreatic cancer. Thus we have performed screening of compound libraries by a combination of methods and identified some compounds (initial leads) that target the calmodulin-binding region on the SH2 domain and inhibit the proliferation of pancreatic cancer cells in in vitro assays. Most of these compounds also exhibited varying degrees of cytotoxicity when tested against immortalized breast epithelial cell line (MCF10A). These initial leads are likely candidates for development in targeted delivery of compounds to cancer cells without affecting normal cells.

Identification of the binding site of an allosteric ligand using STD-NMR, docking, and CORCEMA-ST calculations.

Singling out the truth: A combined application of STD-NMR, molecular docking, and CORCEMA-ST calculations is described as an attractive, easily applicable tool for the identification and validation of the binding site for allosteric ligands, with potential application as an aid in drug discovery research.

Sidedness of interfacial arginine residues and anti-atherogenicity of apolipoprotein A-I mimetic peptides.

To test the hypothesis that sidedness of interfacial arginine (Arg) in apoA-I mimetic peptides, similar to that observed in apoA-I (Bashtovyy, D. et al. 2011. Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function. J. Lipid Res. 52: 435-450.), may be important for biological activity, we compared properties of 4F and analogs, [K4,¹5>R]4F and [K9,¹³>R]4F, with Lys>Arg substitutions on the right and left side, respectively, of the 4F amphipathic helix. Intraperitoneal administration of these peptides into female apoE null mice (n = 13 in each group) reduced en face lesions significantly compared with controls; 4F and [K4,¹5>R]4F were equally effective whereas [K9,¹³>R]4F was less effective. Turnover experiments indicated that [K4,¹5>R]4F reached the highest, whereas [K9,¹³>R]4F had the lowest, plasma peak levels with a similar half life as the [K4,¹5>R]4F analog. The half life of 4F was two times longer than the other two peptides. The order in their abilities to associate with HDL in human plasma, generation of apoA-I particles with pre-ß mobility from isolated HDL, lipid associating ability, and sensitivity of lipid complexes to trypsin digestion was: 4F>[K4,¹5,>R]4F>[K9,¹³>R]4F. These studies support our hypothesis that the sidedness of interfacial Arg residues in the polar face of apoA-I mimetics results in differential biological properties.

1H, 15N, and 13C resonance assignments for a monomeric mutant of the HIV-1 capsid protein.

The mature fullerene cone-shaped capsid of the human immunodeficiency virus 1 is composed of about 1,500 copies of the capsid protein (CA). The CA is 231 residues long, and consists of two distinct structural domains, the N-terminal domain and the C-terminal domain (CTD), joined by a flexible linker. The wild type CA exhibits monomer-dimer equilibrium in solution through the CTD-CTD dimerization. This CTD-CTD interaction, together with other intermolecular interdomain interactions, plays significant roles during the assembly of the mature capsid. In addition, CA-CA interactions also play a role in the assembly of the immature virion. The CA also interacts with some host cell proteins within the viral replication cycle. Thus, the capsid protein has been of significant interest as a target for designing inhibitors of assembly of immature virions and mature capsids and inhibitors of its interactions with host cell proteins. However, the equilibrium exhibited by the wild-type CA protein between the monomeric and dimeric states, along with the inherent flexibility from the interdomain linker, have hindered attempts at structural determination by solution NMR and X-ray crystallography methods. In this study, we have utilized a CA protein with W184A and M185A mutations that abolish the dimerization of CA protein as well as its infectivity, but preserve most of the remaining properties of the wild type CA. We have determined the detailed solution structure of the monomeric W184A/M185A-CA protein using 3D-NMR spectroscopy. Here, we present the detailed sequence-specific NMR assignments for this protein.

Structure of a monomeric mutant of the HIV-1 capsid protein.

The capsid protein (CA) of HIV-1 plays a significant role in the assembly of the immature virion and is the critical building block of its mature capsid. Thus, there has been significant interest in the CA protein as a target in the design of inhibitors of early and late stage events in the HIV-1 replication cycle. However, because of its inherent flexibility from the interdomain linker and the monomer-dimer equilibrium in solution, the HIV-1 wild-type CA monomer has defied structural determinations by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here we report the detailed solution structure of full-length HIV-1 CA using a monomeric mutant that, though noninfective, preserves many of the critical properties of the wild-type protein. The structure shows independently folded N-terminal (NTD) and C-terminal domains (CTD) joined by a flexible linker. The CTD shows some differences from that of the dimeric wild-type CTD structures. This study provides insights into the molecular mechanism of the wild-type CA dimerization critical for capsid assembly. The monomeric mutant allows investigation of interactions of CA with human cellular proteins exploited by HIV-1, directly in solution without the complications associated with the monomer-dimer equilibrium of the wild-type protein. This structure also permits the design of inhibitors directed at a novel target, viz., interdomain flexibility, as well as inhibitors that target multiple interdomain interactions critical for assembly and interactions of CA with host cellular proteins that play significant roles within the replication cycle of HIV-1.

Structure and lipid interactions of an anti-inflammatory and anti-atherogenic 10-residue class G(*) apolipoprotein J peptide using solution NMR.

The surprising observation that a 10-residue class G(⁎) peptide from apolipoprotein J, [113-122]apoJ, possesses anti-inflammatory and anti-atherogenic properties prompted us to delineate its structural characteristics in the presence of normal and oxidized lipid. Towards this, we have determined high-resolution structure of [113-122]apoJ in solution using nuclear magnetic resonance (NMR) spectroscopy and studied its interaction with lipids, including oxidized lipids, using a number of biophysical methods. Circular dichroism and NMR studies established that in the presence of dodecylphosphocholine (DPC) micelle, this peptide adopts amphipathic α-helical structure. The observed Nuclear Overhauser effects indicate that the amphipathic helical structure of the peptide is stabilized by the N-terminal acetyl and C-terminal amide blocking groups. We used isothermal titration calorimetry to measure binding enthalpy of the peptide with DPC micelle, an oxidized lipid, 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), and the mixture of these two lipids (5mol% KOdiA-PC in DPC micelle). We find that the peptide binding with DPC micelle is associated with an enthalpy change (-16.75±0.16 Kcal/mol) much larger than that resulting from the binding with KodiA-PC (-3.67±0.13 Kcal/mol). Incorporation of a small amount of KOdiA-PC (5mol%) in DPC micelle also results in the lowering of peptide binding enthalpy (-13.43±0.18 Kcal/mol). These results are consistent with overall negative charge and altered conformational properties of oxidized sn-2 chain of KOdiA-PC. Our results have unambiguously established the amphipathic α-helical structure of [113-122]apoJ peptide in the presence of DPC micelle as well as its ability to bind oxidized lipid. These in vitro results help explain the previously observed anti-inflammatory and anti-atherosclerotic properties of this peptide.

Nuclear magnetic resonance and circular dichroism study of metastin (Kisspeptin-54) structure in solution.

KISS1 was first discovered as a metastasis suppressor, but also plays crucial roles in the onset of puberty. The KISS1 gene encodes a secreted protein of 145 amino acids that exhibits no sequence similarity with any known proteins. KISS1 protein is proteolytically processed to generate a number of so-called kisspeptins (KP), the most well characterized is known as KP-54 or metastin. KP-54 is carboxy-terminally amidated and binds to and activates the KISS1 receptor (KISS1R). The current studies were undertaken in order to determine structure of KP-54 using nuclear magnetic resonance and circular dichroism. KP-54 is mostly disordered both in water and in trifluoroethanol/water mixed solvent, with no structural motifs. In sodium dodecyl sulfate micelles, KP-54 remains mostly disordered except for a small increase in helical propensity (from 3.7% in water to 9.9% in micelles). Despite this apparent lack of structure, KP-54 is biologically active. The intrinsic disorder of KP-54 may confer advantages in its ability to recognize and bind a wide range of target proteins.

Solution structure of a double mutant of the carboxy-terminal dimerization domain of the HIV-1 capsid protein.

As in other retroviruses, the HIV-1 capsid (CA) protein is composed of two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), joined by a flexible linker. The dimerization of the CTD is thought to be a critical step in the assembly of the immature and mature viral capsids. The precise nature of the functional form of CTD dimerization interface has been a subject of considerable interest. Previously, the CTD dimer was thought to involve a face-to-face dimerization observed in the early crystallographic studies. Recently, the crystallographic structure for a domain-swapped CTD dimer has been determined. This dimer, with an entirely different interface that includes the major homology region (MHR) has been suggested as the functional form during the Gag assembly. The structure determination of the monomeric wt CTD of HIV-1 has not been possible because of the monomer-dimer equilibrium in solution. We report the NMR structure of the [W184A/M185A]-CTD mutant in its monomeric form. These mutations interfere with dimerization without abrogating the assembly activity of Gag and CA. The NMR structure shows some important differences compared to the CTD structure in the face-to-face dimer. Notably, the helix-2 is much shorter, and the kink seen in the crystal structure of the wt CTD in the face-to-face dimer is absent. These NMR studies suggest that dimerization-induced conformational changes may be present in the two crystal structures of the CTD dimers and also suggest a mechanism that can simultaneously accommodate both of the distinctly different dimer models playing functional roles during the Gag assembly of the immature capsids.

Streptozotocin inhibits O-GlcNAcase via the production of a transition state analog.

Streptozotocin (STZ) is a 2-deoxy-d-glucopyranose derivative of a class of drugs known as alkylnitrosoureas, and is an established diabetogenic agent whose cytotoxic affects on pancreatic beta-cells has been partially explained by the presence of its N-methyl-N-nitrosourea side chain, which has the ability to release nitric oxide as well as donate methyl groups to nucleotides in DNA. It has also been observed that STZ administration results in a rise in the level of O-GlcNAcylated proteins within beta-cells. Not coincidentally, STZ has also been shown to directly inhibit the O-GlcNAcase activity of the enzyme NCOAT in vitro, which is the only enzyme that possesses the ability to remove O-GlcNAc modifications on proteins in the nucleus and cytosol. Since O-GlcNAc modification plays a role on a number of proteins in a vast amount of cellular processes, this shift in whole-cell protein O-GlcNAcylation state affords another source of cell death. We set about to find the exact mechanism by which STZ inhibits O-GlcNAcase activity. Inhibition is achievable because the GlcNAc analog STZ targets the active site of the enzyme whereby it is catalyzed. During this process, the enzyme converts STZ to a compound that closely resembles the natural ligand transition state, but is distinctly more stable energetically. As a result, this analog is catalyzed to completion at a much slower rate, thereby out-competing GlcNAc substrate for the active site, and inhibiting the enzyme.