The conformational landscape of a serpin N-terminal subdomain facilitates folding and in-cell quality control.

Many multi-domain proteins including the serpin family of serine protease inhibitors contain non-sequential domains composed of regions that are far apart in sequence. Because proteins are translated vectorially from N- to C-terminus, such domains pose a particular challenge: how to balance the conformational lability necessary to form productive interactions between early and late translated regions while avoiding aggregation. This balance is mediated by the protein sequence properties and the interactions of the folding protein with the cellular quality control machinery. For serpins, particularly α1-antitrypsin (AAT), mutations often lead to polymer accumulation in cells and consequent disease suggesting that the lability/aggregation balance is especially precarious. Therefore, we investigated the properties of progressively longer AAT N-terminal fragments in solution and in cells. The N-terminal subdomain, residues 1-190 (AAT190), is monomeric in solution and efficiently degraded in cells. More ß-rich fragments, 1-290 and 1-323, form small oligomers in solution, but are still efficiently degraded, and even the polymerization promoting Siiyama (S53F) mutation did not significantly affect fragment degradation. In vitro, the AAT190 region is among the last regions incorporated into the final structure. Hydrogen-deuterium exchange mass spectrometry and enhanced sampling molecular dynamics simulations show that AAT190 has a broad, dynamic conformational ensemble that helps protect one particularly aggregation prone ß-strand from solvent. These AAT190 dynamics result in transient exposure of sequences that are buried in folded, full-length AAT, which may provide important recognition sites for the cellular quality control machinery and facilitate degradation and, under favorable conditions, reduce the likelihood of polymerization.

Modeling the native ensemble of PhuS using enhanced sampling MD and HDX-ensemble reweighting.

The cytoplasmic heme binding protein from Pseudomonas aeruginosa, PhuS, plays two essential roles in regulating heme uptake and iron homeostasis. First, PhuS shuttles exogenous heme to heme oxygenase (HemO) for degradation and iron release. Second, PhuS binds DNA and modulates the transcription of the prrF/H small RNAs (sRNAs) involved in the iron-sparing response. Heme binding to PhuS regulates this dual function, as the unliganded form binds DNA, whereas the heme-bound form binds HemO. Crystallographic studies revealed nearly identical structures for apo- and holo-PhuS, and yet numerous solution-based measurements indicate that heme binding is accompanied by large conformational rearrangements. In particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of apo- versus holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7, and 8 (α6,7,8), which contribute to the heme binding pocket. These helices were mostly labile in apo-PhuS but largely protected in holo-PhuS. In contrast, in silico-predicted deuterium uptake levels of α6,7,8 from molecular dynamics (MD) simulations of the apo- and holo-PhuS structures are highly similar, consistent only with the holo-PhuS HDX-MS data. To rationalize this discrepancy between crystal structures, simulations, and observed HDX-MS, we exploit a recently developed computational approach (HDXer) that fits the relative weights of conformational populations within an ensemble of structures to conform to a target set of HDX-MS data. Here, a combination of enhanced sampling MD, HDXer, and dimensionality reduction analysis reveals an apo-PhuS conformational landscape in which α6, 7, and 8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 toward the HemO binding interface. Circular dichroism analysis confirms the loss of secondary structure, and the extracted ensembles of apo-PhuS and of heme-transfer-impaired H212R mutant, are consistent with known heme binding and transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.

Evaluation of the Physicochemical Properties of the Iron Nanoparticle Drug Products: Brand and Generic Sodium Ferric Gluconate.

Complex iron nanoparticle-based drugs are one of the oldest and most frequently administered classes of nanomedicines. In the US, there are seven FDA-approved iron nanoparticle reference drug products, of which one also has an approved generic drug product (i.e., sodium ferric gluconate (SFG)). These products are indicated for the treatment of iron deficiency anemia and are administered intravenously. On the molecular level, iron nanomedicines are colloids composed of an iron oxide core with a carbohydrate coating. This formulation makes nanomedicines more complex than conventional small molecule drugs. As such, these products are often referred to as nonbiological complex drugs (e.g., by the nonbiological complex drugs (NBCD) working group) or complex drug products (e.g., by the FDA). Herein, we report a comprehensive study of the physiochemical properties of the iron nanoparticle product SFG. SFG is the single drug for which both an innovator (Ferrlecit) and generic product are available in the US, allowing for comparative studies to be performed. Measurements focused on the iron core of SFG included optical spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), X-ray powder diffraction (XRPD), 57Fe Mössbauer spectroscopy, and X-ray absorbance spectroscopy (XAS). The analysis revealed similar ferric-iron-oxide structures. Measurements focused on the carbohydrate shell comprised of the gluconate ligands included forced acid degradation, dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and gel permeation chromatography (GPC). Such analysis revealed differences in composition for the innovator versus the generic SFG. These studies have the potential to contribute to future quality assessment of iron complex products and will inform on a pharmacokinetic study of two therapeutically equivalent iron gluconate products.