Characterization of the Escherichia coli pyridoxal 5'-phosphate homeostasis protein (YggS): Role of lysine residues in PLP binding and protein stability.

The pyridoxal 5'-phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP-dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP-dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.

An Investigation of Structure-Activity Relationships of Azolylacryloyl Derivatives Yielded Potent and Long-Acting Hemoglobin Modulators for Reversing Erythrocyte Sickling.

Aromatic aldehydes that bind to sickle hemoglobin (HbS) to increase the protein oxygen affinity and/or directly inhibit HbS polymer formation to prevent the pathological hypoxia-induced HbS polymerization and the subsequent erythrocyte sickling have for several years been studied for the treatment of sickle cell disease (SCD). With the exception of Voxelotor, which was recently approved by the U.S. Food and Drug Administration (FDA) to treat the disease, several other promising antisickling aromatic aldehydes have not fared well in the clinic because of metabolic instability of the aldehyde moiety, which is critical for the pharmacologic activity of these compounds. Over the years, our group has rationally developed analogs of aromatic aldehydes that incorporate a stable Michael addition reactive center that we hypothesized would form covalent interactions with Hb to increase the protein affinity for oxygen and prevent erythrocyte sickling. Although, these compounds have proven to be metabolically stable, unfortunately they showed weak to no antisickling activity. In this study, through additional targeted modifications of our lead Michael addition compounds, we have discovered other novel antisickling agents. These compounds, designated MMA, bind to the α-globin and/or ß-globin to increase Hb affinity for oxygen and concomitantly inhibit erythrocyte sickling with significantly enhanced and sustained pharmacologic activities in vitro.

Enhancing the membrane activity of Piscidin 1 through peptide metallation and the presence of oxidized lipid species: Implications for the unification of host defense mechanisms at lipid membranes.

Piscidins are host-defense peptides (HDPs) from fish that exhibit antimicrobial, antiviral, anti-cancer, anti-inflammatory, and wound-healing properties. They are distinctively rich in histidine and contain an amino terminal copper and nickel (ATCUN) binding motif due to the presence of a conserved histidine at position 3. Metallation lowers their total charge and provides a redox center for the formation of radicals that can convert unsaturated fatty acids (UFAs) into membrane-destabilizing oxidized phospholipids (OxPLs). Here, we focus on P1, a particularly membrane-active isoform, and investigate how metallating it and making OxPL available influence its membrane activity. First, we quantify through dye leakage experiments the permeabilization of the apo- and holo-forms of P1 on model membranes containing a fixed ratio of anionic phosphatidylglycerol (PG) and zwitterionic phosphatidylcholine (PC) but varying amounts of Aldo-PC, an OxPL derived from the degradation of several UFAs. Remarkably, metallating P1 increases membranolysis by a factor of five in each lipid system. Conversely, making Aldo-PC available improves permeabilization by a factor of two for each peptide form. Second, we demonstrate through CD-monitored titrations that the strength of the peptide-membrane interactions is similar in PC/PG and PC/PG/Aldo-PC. Thus, peptide-induced membrane activity is boosted by properties intrinsic to the peptide (e.g., charge and structural changes associated with metallation) and bilayer (e.g., reversal of sn-2 chain due to oxidation). Third, we show using oriented-sample 15N solid-state NMR that the helical portion of P1 lies parallel to the bilayer surface in both lipid systems. 31P NMR experiments show that both the apo- and holo-states interact more readily with PC in PC/PG. However, the presence of Aldo-PC renders the holo-, but not the apo-state, more specific to PG. Hence, the membrane disruptive effects of P1 and its specificity for the anionic lipids found on pathogenic cell membrane surfaces are simultaneously optimized when it is metallated and the OxPL is present. Overall, this study deepens our insights into how OxPLs affect peptide-lipid interactions and how host defense metallopeptides could help integrate the effects of antimicrobial agents.