Protein and Peptide Letters - Volume 24, Issue 6, 2017

Background: Bioluminescence in Ca2+-binding photoproteins is an intramolecular reaction triggered by the addition of Ca2+. A comparative study has been done on Ca2+-depleted and Ca2+-loaded apo-mnemiopsin to understand the structural transition of the photoprotein by Ca2+ binding. Ca2+ is removed by TCA (trichloroacetic acid) precipitation to obtain Ca2+-depleted apomnemiopsin. Method: UV–visible, CD and fluorescence spectroscopic studies demonstrate that the addition of Ca2+ is brought about by the overall structure of apo-mnemiopsin becomes more open in a concentration- dependent manner without significantly influencing the secondary structure and indicate that the Ca2+-depleted form of apo-mnemiopsin, in contrast to most other EF-hand calcium binding proteins, adopt a closed conformation when compared to the Ca2+-loaded form. On the other hand, dynamic quenching and limited proteolysis analysis revealed that Ca2+-loaded apo-mnemiopsin became much more flexible than Ca2+ free apo-mnemiopsin. Results: It seems that increased flexibility of the protein, which occurs due to calcium binding, is a critical factor in oxidative decarboxylation reaction on coelenterazine and consequently light emission.

Background: Antimicrobial peptides could be used in several fields of application, and large quantities of antimicrobial peptides would be required. However, their production is very expensive; this is why a suitable production method, alternative to traditional chemical synthesis is necessary. Production of recombinant antimicrobial peptides in prokaryotic systems has demonstrated the viability of this approach. Nevertheless, expression of antimicrobial peptides in Escherichia coli an others microorganisms is potentially limited due to their toxicity to host cells and susceptibility to proteolytic degradation. As an alternative, we describe a successful antimicrobial peptide production system in Streptomyces lividans which showed to be effective for the secretion of large quantities of cationic antimicrobial peptides. Objective: Therefore, as a solution to the difficulties for heterologous expression of CAP we demonstrate efficient production by S. lividans. Method: In this study, a strategy for CAP overexpression is presented based on the construction of an expression cassette for Streptomyces lividans TK24. For the construction of this cassette, the peptide of interest was fused to the vsi promoter and signal sequence (vsi-ss) of the subtilisin inhibitor from Streptomyces venezuelae CBS762.70, which is a signal peptide with a proven high secretion efficiency. The cloning vector used was pIJ486, which includes a transcription terminator sequence and a thiostrepton resistance marker. This system contains elements that allow the increase of the efficiency of the peptide's expression. Results: The production system allows the efficient secretion of the peptide to the growth medium, thereby simplifying its recovery and avoiding its toxic effect on the producing organism. The production obtained demonstrated the system's efficiency by achieving a peptide concentration of 11.61 mg/ml. This represents at least a 10-fold increase compared to previously established strategies. Conclusion: The expression system constructed may facilitate the production of large amounts of peptides with antimicrobial activity.

Background: BAFF and APRIL are members of TNF superfamily. They play vital roles in the pathogenesis of autoimmune diseases. BCMA, a receptor, shows higher affinity for APRIL than for BAFF. Previous studies found that ligand binding specificity of BCMA may be determined by sequence outside DxL motif. Objective: Investigate the contribution of a segment outside the DxL motif of BCMA for binding with ligands. Method: In this study, the conservative region of BCMA was divided into two segments: BCMA1 (NEYFDSLLHACIPC), a segment of the DXL motif and BCMA2 (QLRCSSNTPPLT), a segment outside of the DXL motif. Two peptides corresponding to the two segments were synthesized and their contribution to the ligands binding were detected by competitive ELISA. BCMA1-Fc fusion protein was also constructed, purified and analyzed by indirect and competitive ELISA. Results: BCMA2 had no inhibiting effect on the interaction of BCMA-Fc and BCMA1-Fc with BAFF, but, it inhibited 22.5% and 15.2% of the interaction of BCMA-Fc and BCMA1-Fc with mAPRIL respectively. The binding rates of BCMA1-Fc for BAFF were 91.7%, but 80.6% for mAPRIL, suggesting that BCMA1-Fc without BCMA2, bound BAFF well and less efficiently to mAPRIL. Conclusion: These results suggest that BCMA2 outside of the conservative DxL motif of BCMA may play an important role in the binding selectivity to its ligands.

Background: Depiction of protein structures as networks of interacting residues has enabled us to understand the structure and function of the protein. Previous investigations on closeness centrality have identified protein functional sites from three- dimensional structures. It is well recognized that ligand binding to a receptor protein induces a wide range of structural changes. Objective: An interesting question is how central residues function during conformational changes triggered during ligand binding? The aim of this study is to comprehend at what extent central residues change during ligand binding to receptor proteins. Method: To determine this, we examined 37 pairs of protein structures consisting of ligand-bound and ligand-free forms. These protein structures were modelled as an undirected network and significant central residues were obtained using residue centrality measures. In addition to these, the basic network parameters were also analysed. Results: On analysing the residue centrality measures, we observed that 60% of central residues were common in both the ligand-bound and ligand-free states. The geometry of the central residues revealed that they were situated closer to the protein center of the mass. Finally, we demonstrated the effectiveness of central residues in amino acids substitutions and in the evolution itself. The closeness centrality was also analyzed among different protein domain sizes and the values gradually declined from single-domains to multi-domain proteins suggesting that the network has potential for hierarchical organization. Betweenness centrality measure was also used to determine the central residues and 31% of these residues were common between the holo/apo states. Conclusion: Findings reveal that central residues play a significant role in determining the functional properties of proteins. These results have implications in predicting binding/active site residues, specifically in the context of drug designing, if additional information concerning ligand binding is exploited.

Background: Peptidyl-prolyl cis-trans isomerase (PPIase) accelerates the intrinsically slow conversion between cis- and trans- configurations of proline, thus affecting backbone conformation and altering the direction of peptide chains. PPIase from Pseudomonas syringae pv. Tomato (PSPTO) DC3000 (PSPTO-PPIase) is considered to belong to the FKBP subfamily of PPIase. Objective: To solve the high resolution structure of the PSPTO-PPIase, and to explore its potential function in plants pathogen PSPTO DC3000. Method: The PSPTO-PPIase was expressed in E.coli and purified through ion exchange and size exclusion chromatography. While only the C-terminal domain of PSPTO-PPIase was successfully crystalized, and its structure was solved to 1.6 Å resolution by molecular replacement method. Results: Structural comparison showed that PSPTO-PPIase adopts a similar overall fold with microphage infectivity potentiators (MIPs), which also belong to the FKBP subfamily of PPIase. In addition, the BIAcore result confirmed that PSPTO-PPIase can bind an immunosuppressive drug FK506 as some other FKBP subfamily members do. Conclusion: Our results suggested that PSPTO-PPIase may function in a similar manner to virulent factor MIPs during pathogenesis. And the immunosuppressive drugs FK506 and rapamycin binding to PSPTO-PPIase potentially interferes and inhibits the plant pathogen PSPTO DC3000. In addition, the amino acids with short side chains in the fourth loop (L4) of PSPTO-PPIase may account for its variable roles in the respective pathogen.