The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.

The broad-spectrum phosphonate antibiotic fosfomycin is currently in use for clinical treatment of infections caused by both Gram-positive and Gram-negative uropathogens. The antibiotic is biosynthesized by various streptomycetes, as well as by pseudomonads. Notably, the biosynthetic strategies used by the two genera share only two steps: the first step in which the primary metabolite phosphoenolpyruvate (PEP) is converted to phosphonopyruvate (PnPy), and the terminal step in which 2-hydroxypropylphosphonate (2-HPP) is converted to fosfomycin. Otherwise, distinct enzymatic paths are employed. Here, we biochemically confirm the last two steps in the fosfomycin biosynthetic pathway of Pseudomonas syringae PB-5123, showing that Psf3 carries out the reduction of 2-oxopropylphosphonate (2-OPP) to (S)-2-HPP, followed by the Psf4-catalyzed epoxidation of (S)-2-HPP to fosfomycin. Psf4 can also accept (R)-2-HPP as a substrate, but instead performs an oxidation to make 2-OPP. We show that the combined activities of Psf3 and Psf4 can be used to convert racemic 2-HPP to fosfomycin in an enantioconvergent process. X-ray structures of each enzyme with bound substrates provide insights into the stereospecificity of each conversion. These studies shed light into the reaction mechanisms of the terminal two enzymes in a distinct pathway employed by pseudomonads for the production of a potent antimicrobial agent.

Alkaline phytase isolated from pollen grains of Lilium longiflorum (LlALP) possesses unique catalytic and thermal stability properties that suggest it has the potential to be used as a feed supplement. However, substantial amounts of active enzymes are needed for animal feed studies and endogenous levels of LlALP in lily pollen are too low to provide the required amounts. Active rLlALP2 (coded by LlAlp2, one of two isoforms of alkaline phytase cDNA identified in lily pollen) has been successfully expressed in intracellular compartments of Pichia pastoris, however enzyme yields have been modest (25–30 mg/L) and purification of the enzyme has been challenging. Expression of foreign proteins to the extracellular medium of P. pastoris greatly simplifies protein purification because low levels of endogenous proteins are secreted by the yeast. In this paper, we first describe the generation of P. pastoris strains that will secrete rLlALP2 to the extracellular medium. Data presented here indicates that deletion of native signal peptides at the N- and C-termini of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion by four-fold; chicken egg white lysozyme signal peptide was ineffective in the extracellular secretion of rLlALP2. Second, we describe our efforts to increase expression levels by employing a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) in place of the strong, tightly controlled promoter of alcohol oxidase 1 gene (PAOX1). PGAP enhanced the extracellular expression levels of rLlALP2 compared to PAOX1. Finally, we report on the optimization of the culture medium to enhance yields of rLlALP2. The strength of PGAP varies depending on the carbon source available for cell growth; secreted expression of rLlALP2 was highest when glycerol was the carbon source. The addition of histidine and Triton X-100 also enhanced extracellular expression. Taken together, the employment of PGAP under optimized culture conditions resulted in approximately eight-fold (75–80 mg/L) increase in extracellular activity compared to PAOXI (8–10 mg/L). The P. pastoris expression system can be employed as a source of active alkaline phytase for animal feed studies.

Background Deficiency in blood coagulation factor VIII (FVIII) results in life‐threating bleeding (hemophilia A) treated by infusions of FVIII concentrates. To improve disease treatment, FVIII has been modified to increase its plasma half‐life, which requires understanding mechanisms of FVIII catabolism. An important catabolic actor is hepatic low density lipoprotein receptor‐related protein 1 (LRP1), which also regulates many other clinically significant processes. Previous studies showed complexity of FVIII site for binding LRP1. Objectives To characterize binding sites between FVIII and LRP1 and suggest a model of the interaction. Methods A series of recombinant ligand‐binding complement‐type repeat (CR) fragments of LRP1 including mutated variants was generated in a baculovirus system and tested for FVIII interaction using surface plasmon resonance, tissue culture model, hydrogen–deuterium exchange mass spectrometry, and in silico. Results Multiple CR doublets within LRP1 clusters II and IV were identified as alternative FVIII‐binding sites. These interactions follow the canonical binding mode providing major binding energy, and additional weak interactions are contributed by adjacent CR domains. A representative CR doublet was shown to have multiple contact sites on FVIII. Conclusions FVIII and LRP1 interact via formation of multiple complex contacts involving both canonical and non‐canonical binding combinations. We propose that FVIII‐LRP1 interaction occurs via switching such alternative binding combinations in a dynamic mode, and that this mechanism is relevant to other ligand interactions of the low‐density lipoprotein receptor family members including LRP1.