Biochemistry Biochemistry最新文献

To combat the permanent exposure to potential pathogens every organism relies on an immune system. Important factors in innate immunity are antimicrobial peptides (AMPs) that are structurally highly diverse. Some AMPs are known to belong to the saposin-like proteins (SAPLIPs), a group of polypeptides with a broad functional spectrum. The model organism Dictyostelium discoideum possesses a remarkably large arsenal of potential SAPLIPs, which are termed amoebapore-like peptides (Apls), but the knowledge about these proteins is very limited. Here, we report about the biochemical characterization of AplE1, AplE2, AplK1, and AplK2, which are derived from the two precursor proteins AplE and AplK, thereby resembling prosaposins of vertebrates. We produced these Apls as recombinant polypeptides in Escherichia coli using a self-splicing intein to remove an affinity tag used for purification. All recombinant Apls exhibited pore-forming activity in a pH-dependent manner, as evidenced by liposome depolarization, showing higher activities the more acidic the setting was. Lipid preference was detected for negatively charged phospholipids and in particular for cardiolipin. Antimicrobial activity against various bacteria was found to be inferior in classical microdilution assays. However, all of the Apls studied permeabilized the cytoplasmic membrane of live Bacillus subtilis. Collectively, we assume that the selected Apls interact by their cationic charge with negatively charged bacterial membranes in acidic environments such as phagolysosomes and eventually lyse the target cells by pore formation.

An emerging NMR method, MA'AT analysis, has been applied to investigate context effects on the conformational properties of several human milk oligosaccharides (HMOs). The MA'AT model of the β-(1→4) linkage in the disaccharide, methyl β-lactoside (MeL), was compared to those obtained for the same linkage in the HMO trisaccharides, methyl 2'-fucosyllactoside (Me2'FL) and methyl 3-fucosyllactoside (Me3FL), and in the tetrasaccharide, methyl 2',3-difucosyllactoside (Me2',3DFL). MA'AT analysis revealed significant context effects on the mean values and circular standard deviations (CSDs) of the psi (ψ) torsion angles in these linkages. α-Fucosylation at both O2'Gal and O3Glc of MeL to give Me2',3DFL significantly constrained librational motion about ψ (70% reduction in the CSD) and shifted its mean value by ∼18°. α-Fucosylation at the O3Glc of MeL to give Me3FL constrained ψ more than α-fucosylation at the O2Gal to give Me2'FL. These effects can be explained by the expected solution conformation of Me3FL, which closely resembles the Lewis^x trisaccharide. Comparisons of MA'AT models of ψ to those obtained by 1 μs aqueous molecular dynamics simulation (GLYCAM06) revealed identical trends, that is, MA'AT analysis was able to recapitulate molecular behavior in solution that was heretofore only available from MD simulation. The results highlight the capabilities of MA'AT analysis to determine probability distributions of molecular torsion angles in solution as well as degrees of librational averaging of these angles.

KRas4b is a small plasma membrane-bound G-protein that regulates signal transduction pathways. The interaction of KRas4b with the plasma membrane is governed by both its basic C-terminus, which is farnesylated and methylated, and the lipid composition of the membrane itself. The signaling activity of KRas4b is intricately related to its interaction with various binding partners at the plasma membrane, underlining the critical role played by the lipid environment. The calcium-binding protein calmodulin binds farnesylated KRas4b and plays an important role in the dynamic spatial cycle of KRas4b trafficking in the cell. We utilize Biolayer Interferometry to assay the role of lipid headgroup, chain length, and electrostatics in the dissociation kinetics of fully post-translationally modified KRas4b from Nanodisc bilayers with defined lipid compositions. Our results suggest that calmodulin promotes the dissociation of KRas4b from an anionic membrane, with a comparatively slower displacement of KRas4b from PIP2 relative to PS containing bilayers. In addition to this headgroup dependence, KRas4b dissociation appears to be slower from Nanodiscs wherein the lipid composition contains mismatched, unsaturated acyl chains as compared to lipids with a matched acyl chain length. These findings contribute to understanding the role of the lipid composition in the binding of KRas4b and release from lipid bilayers, showing that the overall charge of the bilayer, the identity of the headgroups present, and the length and saturation of the acyl chains play key roles in KRas4b release from the membrane, potentially providing insights in targeting Ras-membrane interactions for therapeutic interventions.

A subunit of factor XIII (FXIII-A) contains a unique activation peptide (AP) that protects the catalytic triad and prevents degradation. In plasma, FXIII is activated proteolytically (FXIII-A*) by thrombin and Ca²⁺ cleaving AP, while in cytoplasm, it is activated nonproteolytically (FXIII-A°) with increased Ca²⁺ concentrations. This study aimed to elucidate the role of individual parts of the FXIII-A AP in protein stability, thrombin activation, and transglutaminase activity. Recombinant FXIII-A AP variants were expressed, and SDS-PAGE was used to monitor thrombin hydrolysis at the AP cleavage sites R37-G38. Transglutaminase activities were assessed by cross-linking lysine mimics to Fbg αC (233-425, glutamine-substrate) and monitoring reactions by mass spectrometry and in-gel fluorescence assays. FXIII-A AP variants, S19P, E23K, and D24V, degraded during purification, indicating their vital role in FXIII-A₂ stability. Mutation of P36 to L36/F36 abolished the proteolytic cleavage of AP and thus prevented activation. FXIII-A N20S and P27L exhibited slower thrombin activation, likely due to the loss of key interdomain H-bonding interactions. Except N20S and P15L/P16L, all activatable FXIII-A* variants (P15L, P16L, S19A, and P27L) showed similar cross-linking activity to WT. By contrast, FXIII-A° P15L, P16L, and P15L/P16L had significantly lower cross-linking activity than FXIII-A° WT, suggesting that loss of these prolines had a greater structural impact. In conclusion, FXIII-A AP residues that play crucial roles in FXIII-A stability, activation, and activity were identified. The interactions between these AP amino acid residues and other domains control the stability and activity of FXIII.

Glycerol 3-phosphate dehydrogenase catalyzes reversible hydride transfer from glycerol 3-phosphate (G3P) to NAD⁺ to form dihydroxyacetone phosphate; from the truncated substrate ethylene glycol to NAD⁺ in a reaction activated by the phosphite dianion substrate fragment; and from G3P to the truncated nicotinamide riboside cofactor in a reaction activated by adenosine 5'-diphosphate, adenosine 5'-monophosphate, and ribose 5-phosphate cofactor fragments. The sum of the stabilization of the transition state for GPDH-catalyzed hydride transfer reactions of the whole substrates by the phosphodianion fragment of G3P and the ADP fragment of NAD⁺ is 25 kcal/mol. Fourteen kcal/mol of this transition state stabilization is recovered as phosphite dianion and AMP activation of the reactions of the substrate and cofactor fragments. X-ray crystal structures for unliganded GPDH, for a binary GPDH·NAD⁺ complex, and for a nonproductive ternary GPDH·NAD⁺·DHAP complex show that the ligand binding energy is utilized to drive an extensive protein conformational change that creates a caged complex for these ligands. The phosphite dianion and AMP fragments are proposed to activate GPDH for the catalysis of hydride transfer by stabilization of this active caged complex. The closure of a conserved loop [292-LNGQKL-297] during substrate binding stabilizes the G3P and NAD⁺ complexes by interactions, respectively, with the Q295 and K296 loop side chains. The appearance and apparent conservation of two side chains that interact with the hydride donor and acceptor to stabilize the active closed enzyme are proposed to represent a significant improvement in the catalytic performance of GPDH.

De novo mutations affecting the pre-mRNA splicing factor U2AF2 are associated with developmental delays and intellectual disabilities, yet the molecular basis is unknown. Here, we demonstrated by fluorescence anisotropy RNA binding assays that recurrent missense mutants (Arg149Trp, Arg150His, or Arg150Cys) decreased the binding affinity of U2AF2 for a consensus splice site RNA. Crystal structures at 1.4 Å resolutions showed that Arg149Trp or Arg150His disrupted hydrogen bonds between U2AF2 and the terminal nucleotides of the RNA site. Reanalysis of publicly available RNaseq data confirmed that U2AF2 depletion altered splicing of transcripts encoding RNA binding proteins (RBPs). These results confirmed that the impaired RNA interactions of Arg149Trp and Arg150His U2AF2 variants could contribute to dysregulating an RBP-governed neurodevelopmental program of alternative splicing.

Some species of the Nocardia genus harbor a highly conserved biosynthetic gene cluster designated as the NOCardiosis-Associated Polyketide (NOCAP) synthase that produces a unique glycolipid natural product. The NOCAP glycolipid is composed of a fully substituted benzaldehyde headgroup linked to a polyfunctional alkyl tail and an O-linked disaccharide composed of 3-α-epimycarose and 2-O-methyl-α-rhamnose. Incorporation of the disaccharide unit is preceded by a critical step involving hydroxylation by NocapM, a flavin monooxygenase. In this study, we employed biochemical, spectroscopic, and kinetic analyses to explore the substrate scope of NocapM. Our findings indicate that NocapM catalyzes hydroxylation of diverse aromatic substrates, although the observed coupling between NADPH oxidation and substrate hydroxylation varies widely from substrate to substrate. Our in-depth biochemical characterization of NocapM provides a solid foundation for future mechanistic studies of this enzyme as well as its utilization as a practical biocatalyst.

Inositol pyrophosphates (PP-InsPs) are eukaryote-specific second messengers that regulate diverse cellular processes, including immunity, nutrient sensing, and hormone signaling pathways in plants. These energy-rich messengers exhibit high sensitivity to the cellular phosphate status, suggesting that the synthesis and degradation of PP-InsPs are tightly controlled within the cells. Notably, the molecular basis of PP-InsP hydrolysis in plants remains largely unexplored. In this study, we report the functional characterization of MpDDP1, a diadenosine and diphosphoinositol polyphosphate phosphohydrolase encoded by the genome of the liverwort, Marchantia polymorpha. We show that MpDDP1 functions as a PP-InsP phosphohydrolase in different heterologous organisms. Consistent with this finding, M. polymorpha plants defective in MpDDP1 exhibit elevated levels of 1/3-InsP₇ and 1/3,5-InsP₈, highlighting the contribution of MpDDP1 in regulating PP-InsP homeostasis in planta. Furthermore, our study reveals that MpDDP1 controls thallus development and vegetative reproduction in M. polymorpha. Collectively, this study provides insights into the regulation of specific PP-InsP messengers by DDP1-type phosphohydrolases in land plants.

In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus, with and without an inactive analog of the farnesyl diphosphate (FPP) substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP structure was solved to 2.65 Å resolution and showed the ligand in an elongated orientation, incapable of undergoing the initial cyclization event to form a C1-C11 bond. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case, well fit by a curled-up tetraethylene glycol molecule recruited, presumably, from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotation of some active-site residues in comparison to the 2FFPP-structure. Most notably, W56 and F183 undergo 90° rotations between the 2FFPP complex and apoenzyme structures, suggesting that these residues provide interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L and F183A variants were observed to be severely restricted in their ability to catalyze C1-C11 cyclization of the FPP substrate and instead produced predominantly acyclic terpene products dominated by farnesol, β-farnesene, and nerolidol.

Antibiotics are essential components of current medical practice, but their effectiveness is being eroded by the increasing emergence of antimicrobial-resistant infections. At the same time, the rate of antibiotic discovery has slowed, and our future ability to treat infections is threatened. Among Christopher T. Walsh's many contributions to science was his early recognition of this threat and the potential of biosynthesis─genes and mechanisms─to contribute solutions. Here, we revisit a 2006 review by Walsh and co-workers that highlighted a major challenge in antibiotic natural product discovery: the daunting odds for identifying new naturally occurring antibiotics. The review described strategies to mitigate the odds challenge. These strategies have been used extensively by the natural product discovery community in the years since and have resulted in some promising discoveries. Despite these advances, the rarity of novel antibiotic natural products remains a barrier to discovery. We compare the challenge of discovering natural product antibiotics to the process of identifying new prime numbers, which are also challenging to find and an essential, if underappreciated, element of modern life. We propose that inclusion of filters for functional compounds early in the discovery pipeline is key to natural product antibiotic discovery, review some recent advances that enable this, and discuss some remaining challenges that need to be addressed to make antibiotic discovery sustainable in the future.