Probing and Engineering the Fatty Acyl Substrate Selectivity of Starter Condensation Domains of Nonribosomal Peptide Synthetases in Lipopeptide Biosynthesis

Lipopeptides are produced by nonribosomal peptide synthetases (NRPSs) and contain diverse fatty acyl moieties that are major determinants of antibiotic potency. The lipid chains are incorporated into peptidyl backbones via lipoinitiation, a process comprising free fatty acid activation and the subsequent starter condensation domain (C1)‐catalyzed conjugation of fatty acyl moieties onto the aminoacyl substrates. Thus, a thorough understanding of lipoinitiation biocatalysts would significantly expand their potential to produce novel antibiotics. Here, biochemical assays, in silico analysis, and mutagenesis studies are used to ultimately identify the specific amino acid residues that control the fatty acyl substrate selectivity of C1 in lipopeptide A54145. In silico docking study has identified four candidate amino acids, and subsequent in vitro assays confirmed their functional contribution to the channel that controls substrate selectivity. Two engineered variants with single point mutations in C1 are found to alter the substrate selectivity toward nonnatural fatty acyl substrates. The detailed mechanistic insights into the catalytic contribution of C1 obtained from the present study will facilitate future NPRS biocatalyst efforts     

PDZ/PDZ interaction between PSD‐95 and nNOS neuronal proteins

N‐Methyl‐D‐aspartate (NMDA) receptors are key components in synaptic communication and are highly relevant in central nervous disorders, where they trigger excessive calcium entry into the neuronal cells causing harmful overproduction of nitric oxide by the neuronal nitric oxide synthase (nNOS) protein. Remarkably, NMDA receptor activation is aided by a second protein, postsynaptic density of 95 kDa (PSD95), forming the ternary protein complex NMDA/PSD95/nNOS. To minimize the potential side effects derived from blocking this ternary complex or either of its protein components, a promising approach points to the disruption of the PSD‐95/nNOS interaction which is mediated by a PDZ/PDZ domain complex. Since the rational development of molecules targeting such protein‐protein interaction relies on energetic and structural information herein, we include a thermodynamic and structural analysis of the PSD95‐PDZ2/nNOS‐PDZ. Two energetically relevant events are structurally linked to a “two‐faced” or two areas of recognition between both domains. First, the assembly of a four‐stranded antiparallel β‐sheet between the β hairpins of nNOS and of PSD95‐PDZ2, mainly enthalpic in nature, contributes 80% to the affinity. Second, binding is entropically reinforced by the hydrophobic interaction between side chains of the same nNOS β‐hairpin with the side chains of α2‐helix at the binding site of PSD95‐PDZ2, contributing the remaining 20% of the total affinity. These results suggest strategies for the future rational design of molecules able to disrupt this complex and constitute the first exhaustive thermodynamic analysis of a PDZ/PDZ interaction.     

The mechanism of Single strand binding protein–RecG binding: Implications for SSB interactome function

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes.     

Rational design of cell-permeable cyclic peptides containing a d-Pro-l-Pro motif

Cyclic peptides are capable of binding to challenging targets (e.g., proteins involved in protein-protein interactions) with high affinity and specificity, but generally cannot gain access to intracellular targets because of poor membrane permeability. In this work, we discovered a conformationally constrained cyclic cell-penetrating peptide (CPP) containing a d-Pro-l-Pro motif, cyclo(AFΦrpPRRFQ) (where Φ is l-naphthylalanine, r is d-arginine, and p is d-proline). The structural constraints provided by cyclization and the d-Pro-l-Pro motif permitted the rational design of cell-permeable cyclic peptides of large ring sizes (up to 16 amino acids). This strategy was applied to design a potent, cell-permeable, and biologically active cyclic peptidyl inhibitor, cyclo(Y^pVNFΦrpPRR) (where Y^p is l-phosphotyrosine), against the Grb2 SH2 domain. Multidimensional NMR spectroscopic and circular dichroism analyses revealed that the cyclic CPP as well as the Grb2 SH2 inhibitor assume a predominantly random coil structure but have significant β-hairpin character surrounding the d-Pro-l-Pro motif. These results demonstrate cyclo(AFΦrpPRRFQ) as an effective CPP for endocyclic (insertion of cargo into the CPP ring) or exocyclic delivery of biological cargos (attachment of cargo to the Gln side chain).     

Kunitz型丝氨酸蛋白酶抑制剂研究进展

Kunitz型丝氨酸蛋白酶抑制剂是一类普遍存在的蛋白酶抑制剂,在体内各项生命活动中扮演着重要角色。这类抑制剂结构稳定且富有特色,通常具有一个或几个串联存在的Kunitz结构域,能够以类似底物的方式与丝氨酸蛋白酶结合,从而抑制酶的活性。在功能方面,Kunitz型丝氨酸蛋白酶抑制剂参与凝血和纤维蛋白溶解、肿瘤免疫、炎症调节以及抵抗细菌、真菌感染等过程。文中就Kunitz型丝氨酸蛋白酶抑制剂研究进展作一综述,为新型Kunitz型丝氨酸蛋白酶抑制剂的开发提供研究思路。     

Agglutination Activity of Fasciola gigantica DM9-1, a Mannose-Binding Lectin

The DM9 domain is a protein unit of 60–75 amino acids that has been first detected in the fruit fly Drosophila as a repeated motif of unknown function. Recent research on proteins carrying DM9 domains in the mosquito Anopheles gambiae and the oyster Crassostrea gigas indicated an association with the uptake of microbial organisms. Likewise, in the trematode Fasciola gigantica DM9-1 showed intracellular relocalization following microbial, heat and drug stress. In the present research, we show that FgDM9-1 is a lectin with a novel mannose-binding site that has been recently described for the protein CGL1 of Crassostrea gigas. This property allowed FgDM9-1 to agglutinate gram-positive and -negative bacteria with appropriate cell surface glycosylation patterns. Furthermore, FgDM9-1 caused hemagglutination across all ABO blood group phenotypes. It is speculated that the parenchymal located FgDM9-1 has a role in cellular processes that involve the transport of mannose-carrying molecules in the parenchymal cells of the parasite.     

Identification and Characterization of a Novel Single Domain Antibody Against Ebola Virus

Virologica Sinica - Ebola virus (EBOV) belongs to the Filoviridae family and causes severe illnesses such as hemorrhagic fever with a high mortality rate up to 90%. Now two antibody drugs termed...