Solution Synthesis,Conformational Analysis,and Antimicrobial Activity of Three Alamethicin F50/5 Analogs Bearing a Trifluoroacetyl Label

We prepared, by solution‐phase methods, and fully characterized three analogs of the membrane‐active peptaibiotic alamethicin F50/5, bearing a single trifluoroacetyl (Tfa) label at the N‐terminus, at position 9 (central region) or at position 19 (C‐terminus), and with the three Gln at positions 7, 18, and 19 replaced by Glu(OMe) residues. To add the Tfa label at position 9 or 19, a γ‐trifluoroacetylated α,γ‐diaminobutyric acid (Dab) residue was incorporated as a replacement for the original Val⁹ or Glu(OMe)¹⁹ amino acid. We performed a detailed conformational analysis of the three analogs (using FT‐IR absorption, CD, 2D‐NMR, and X‐ray diffraction), which clearly showed that Tfa labeling does not introduce any dramatic backbone modification in the predominantly α‐helical structure of the parent peptaibiotic. The results of an initial solid‐state ¹⁹F‐NMR study on one of the analogs favor the conclusion that the Tfa group is a very promising reporter for the analysis of peptaibiotic? membrane interactions. Finally, we found that the antimicrobial activities of the three newly synthesized analogs depend on the position of the Tfa label in the peptide sequence.     

Synthesis,GluN2B affinity and selectivity of benzo[7]annulen-7-amines

Due to their beneficial side effect profile, NMDA receptor antagonists interacting selectively with the allosteric ifenprodil binding site of the GluN2B subunit are of major interest for the treatment of neurological and neurodegenerative disorders. A series of benzo[7]annulen-7-amines 6 was designed by conformational restriction of ifenprodil (1). At first the benzo[7]annulen-7-one 11 was prepared in a three-step synthesis comprising of a double Knoevenagel condensation of phthalaldehyde (7) with dimethyl 3-oxoglutarate (8), hydrogenation of 9 and saponification/decarboxylation of 10. Reductive amination of the ketone 11 with primary amines and NaBH(OAc)₃ led to the secondary amines 6a–d, cis-6h and trans-6i. The tertiary amines 6e–g were obtained by S_N2-substitution of the nosylate 13. Although H-bond forming substituents in 2- and 5-position are missing, the amines 6 exhibit high affinity towards GluN2B containing NMDA receptors. A distance of four to five bond lengths between the basic amino moiety and the phenyl ring in the side chain appears to be optimal for high GluN2B affinity. The phenylcyclohexylamine cis-6h and the 4-benzylpiperidine 6g show the highest GluN2B affinities (K_i = 2.3 nM and 2.9 nM, respectively). With respect to selectivity against the PCP binding site, σ₁ and σ₂ receptors the phenylpiperazine 6f is the most promising GluN2B antagonist.     

Cyclic side-chain-linked opioid analogs utilizing cis- and trans-4-aminocyclohexyl-d-alanine

Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generating analogs with improved bioactivities. Cyclization can be achieved through various bridging bonds between peptide ends or side-chains. In our earlier paper we have reported the synthesis and biological activity of a cyclic peptide, Tyr-c[d-Lys-Phe-Phe-Asp]NH₂ (1), which can be viewed as an analog of endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH₂). Cyclization was achieved through an amide bond between side-chains of d-Lys and Asp residues. Here, to increase rigidity of the cyclic structure, we replaced d-Lys with cis- or trans-4-aminocyclohexyl-d-alanine (d-ACAla). Two sets of analogs incorporating either Tyr or Dmt (2′,6′-dimethyltyrosine) residues in position 1 were synthesized. In the binding studies the analog incorporating Dmt and trans-d-ACAla showed high affinity for both, μ- and δ-opioid receptors (MOR and DOR, respectively) and moderate affinity for the κ-opioid receptor (KOR), while analog with Dmt and cis-d-ACAla was exceptionally MOR-selective. Conformational analyses by NMR and molecular docking studies have been performed to investigate the molecular structural features responsible for the noteworthy MOR selectivity.     

Conformational Heterogeneity in Antibody-Protein Antigen Recognition: IMPLICATIONS FOR HIGH AFFINITY PROTEIN COMPLEX FORMATION

Specific, high affinity protein-protein interactions lie at the heart of many essential biological processes, including the recognition of an apparently limitless range of foreign proteins by natural antibodies, which has been exploited to develop therapeutic antibodies. To mediate biological processes, high affinity protein complexes need to form on appropriate, relatively rapid timescales, which presents a challenge for the productive engagement of complexes with large and complex contact surfaces (∼600–1800 Ų). We have obtained comprehensive backbone NMR assignments for two distinct, high affinity antibody fragments (single chain variable and antigen-binding (Fab) fragments), which recognize the structurally diverse cytokines interleukin-1β (IL-1β, β-sheet) and interleukin-6 (IL-6, α-helical). NMR studies have revealed that the hearts of the antigen binding sites in both free anti-IL-1β Fab and anti-IL-6 single chain variable exist in multiple conformations, which interconvert on a timescale comparable with the rates of antibody-antigen complex formation. In addition, we have identified a conserved antigen binding-induced change in the orientation of the two variable domains. The observed conformational heterogeneity and slow dynamics at protein antigen binding sites appears to be a conserved feature of many high affinity protein-protein interfaces structurally characterized by NMR, suggesting an essential role in protein complex formation. We propose that this behavior may reflect a soft capture, protein-protein docking mechanism, facilitating formation of high affinity protein complexes on a timescale consistent with biological processes.     

Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design

At the first glance CK2α, the catalytic subunit of protein kinase CK2, is a rigid molecule: in contrast to many eukaryotic protein kinases in CK2α the canonical regulatory key elements like the activation segment occur exclusively in their typical active conformations. This observation fits well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2α structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2α structures. In the latter a surprising plasticity of important ATP-binding elements – the interdomain hinge region and the glycine-rich loop – was discovered. In fully active CK2α the hinge region is open and does not anchor the ATP ribose, but alternatively it can adopt a closed conformation, form hydrogen bonds to the ribose moiety and thus retract the γ-phospho group from its functional position. In addition to this partially inactive state human CK2α was recently found in a fully inactive conformation. It is incompatible with ATP-binding due to a combination of a closed hinge and a collapse of the glycine-rich loop into the ATP cavity. These conformational transitions are apparently correlated with the occupation state of a remote docking site located at the interface to the non-catalytic subunit CK2β: if CK2β blocks this site, the fully active conformation of CK2α is stabilized, while the binding of certain small molecule seems to favour the partially and fully inactive states. This observation may be exploited to design effective and selective CK2 inhibitors.     

Conformational study of the preferred conformations of the peptide sequence VP3(110-121) of HAV by circular dichroism and molecular mechanics

Summary A conformational analysis of the fragment 110–121 of VP3 coating protein of the hepatitis A virus was carried out using circular dichroism spectroscopy and computational studies. The latter studies indicate the tendency of the peptide to adopt hairpin-type structures. Circular dichroism experiments indicate that, in spite of the fact that the isolated peptide exhibits no structure under different experimental conditions, negatively charged liposomes induce a secondary structure that agrees with the results of the computational study.     

用组合模型综合比较的方法分析气候变化对朱鹮潜在生境的影响

气候变化的不确定性和物种与环境关系的不确定性使气候变化生物学的研究充满变数。为了降低不确定性,人们开始用组合模型综合比较的方法研究物种对气候变化的响应。以朱鹮(Nipponia nippon)为研究对象,介绍组合模型综合比较方法的特点。朱鹮曾经高度濒危,目前种群大小在迅速恢复中;然而其分布区依旧狭小,气候变化可能是朱鹮面临的新威胁。应用BIOMOD模型中的9种模型,选择了每年的最低温和最高温、温度的季节性变异、每年的总降水量和降水的季节性变异共5个气候因子,依据WorldClim气候数据的CGCM2气候模型的A2a排放情形,计算了朱鹮当前(1950—2000年)的适宜生境和2020年、2050年、2080年3个阶段的潜在生境范围。结果表明朱鹮潜在生境将逐渐北移,生境中心脱离现在的保护区。因此,制定朱鹮的长期保护策略是必要的。9个模型在预测结果上、变量权重上和拟合优度的指标上都有差异,反映了模型本身的不确定性。气候变化的生物学效应比较复杂,应用多个模型进行综合比较,可以尽可能地减少模型所导致的误差。     

Molecular basis of ligand recognition by OASS from E. histolytica: Insights from structural and molecular dynamics simulation studies

Background

O-acetyl serine sulfhydrylase (OASS) is a pyridoxal phosphate (PLP) dependent enzyme catalyzing the last step of the cysteine biosynthetic pathway. Here we analyze and investigate the factors responsible for recognition and different conformational changes accompanying the binding of various ligands to OASS.

Methods

X ray crystallography was used to determine the structures of OASS from Entamoeba histolytica in complex with methionine (substrate analog), isoleucine (inhibitor) and an inhibitory tetra-peptide to 2.00 Å, 2.03 Å and 1.87 Å resolutions, respectively. Molecular dynamics simulations were used to investigate the reasons responsible for the extent of domain movement and cleft closure of the enzyme in presence of different ligands.

Results

Here we report for the first time an OASS-methionine structure with an unmutated catalytic lysine at the active site. This is also the first OASS structure with a closed active site lacking external aldimine formation. The OASS-isoleucine structure shows the active site cleft in open state. Molecular dynamics studies indicate that cofactor PLP, N88 and G192 form a triad of energy contributors to close the active site upon ligand binding and orientation of the Schiff base forming nitrogen of the ligand is critical for this interaction.

Conclusions

Methionine proves to be a better binder to OASS than isoleucine. The β branching of isoleucine does not allow it to reorient itself in suitable conformation near PLP to cause active site closure.

General significance

Our findings have important implications in designing better inhibitors against OASS across all pathogenic microbial species.     

Habitat assessment of Marco Polo sheep (Ovis ammon polii) in Eastern Tajikistan: Modeling the effects of climate change

Identifying the factors predicting the high‐elevation suitable habitats of Central Asian argali wild sheep and how these suitable habitats are affected by the changing climate regimes could help address conservation and management efforts and identify future critical habitat for the species in eastern Tajikistan. This study used environmental niche models (ENMs) to map and compare potential present and future distributions of suitable environmental conditions for Marco Polo argali. Argali occurrence points were collected during field surveys conducted from 2009 to 2016. Our models showed that terrain ruggedness and annual mean temperature had strong correlations on argali distribution. We then used two greenhouse gas concentration trajectories (RCP 4.5 and RCP 8.5) for two future time periods (2050 and 2070) to model the impacts of climate change on Marco Polo argali habitat. Results indicated a decline of suitable habitat with majority of losses observed at lower elevations (3,300–4,300 m). Models that considered all variables (climatic and nonclimatic) predicted losses of present suitable areas of 60.6% (6,928 km²) and 63.2% (7,219 km²) by 2050 and 2070, respectively. Results also showed averaged habitat gains of 46.2% (6,106 km²) at much higher elevations (4,500–6,900 m) and that elevational shifts of habitat use could occur in the future. Our results could provide information for conservation planning for this near threatened species in the region.