Long Range Communication between the Drug-Binding Sites and Nucleotide Binding Domains of the Efflux Transporter ABCB1.

The ABC efflux pump P-glycoprotein (P-gp) transports a wide variety of drugs and is inhibited by others. Some drugs stimulate ATP hydrolysis at the nucleotide binding domains (NBDs) and are transported, others uncouple ATP hydrolysis and transport, and others inhibit ATP hydrolysis. The molecular basis for the different behavior of these drugs is not well understood despite the availability of several structural models of P-gp complexes with ligands bound. Hypothetically, ligands differentially alter the conformational dynamics of peptide segments that mediate the coupling between the drug binding sites and the NBDs. Here, we explore by hydrogen-deuterium exchange mass spectrometry the dynamic consequences of a classic substrate and inhibitor, vinblastine and zosuquidar, binding to mouse P-gp (mdr1a) in lipid nanodiscs. The dynamics of P-gp in nucleotide-free, pre-hydrolysis, and post-hydrolysis states in the presence of each drug reveal distinct mechanisms of ATPase stimulation and implications for transport. For both drugs, there are common regions affected in a similar manner, suggesting that particular networks are the key to stimulating ATP hydrolysis. However, drug binding effects diverge in the post-hydrolysis state, particularly in the intracellular helices (ICHs 3 and 4) and neighboring transmembrane helices. The local dynamics and conformational equilibria in this region are critical for the coupling of drug binding and ATP hydrolysis and are differentially modulated in the catalytic cycle.

Cholesterol Asymmetrically Modulates the Conformational Ensemble of the Nucleotide-Binding Domains of P-Glycoprotein in Lipid Nanodiscs.

P-Glycoprotein (P-gp) is an ATP-dependent efflux pump that clears a wide variety of drugs and toxins from cells. P-gp undergoes large-scale structural changes and demonstrates conformational heterogeneity even within a single catalytic or drug-bound state, although the role of heterogeneity remains unclear. P-gp is found in a variety of cell types that vary in lipid composition, which modulates its activity. An understanding of structural or dynamic changes due to the lipid environment is lacking. We aimed to determine the effects of cholesterol in a membrane on the conformational behavior of P-gp in lipid nanodiscs. The presence of cholesterol stimulates ATP hydrolysis and alters lipid order and fluidity. Hydrogen/deuterium exchange mass spectrometry demonstrates that cholesterol in the membrane induces asymmetric, long-range changes in the distributions and exchange kinetics of conformations of the nucleotide-binding domains, correlating the effects of lipid composition on activity with specific changes in the P-gp conformational landscape.

Dynamics and Mechanism of Binding of Androstenedione to Membrane-Associated Aromatase.

Aromatase (CYP19A1) catalyzes the synthesis of estrogens from androgens and is an invaluable target of pharmacotherapy for estrogen-dependent cancers. CYP19A1 is also one of the most primordial human CYPs and, to the extent that its fundamental dynamics are conserved, is highly relevant to understanding those of the more recently evolved and promiscuous enzymes. A complementary approach employing molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to interrogate the changes in CYP19A1 dynamics coupled to binding androstenedione (ASD). Gaussian-accelerated molecular dynamics and HDX-MS agree that ASD globally suppresses CYP19A1 dynamics. Bimodal HDX patterns of the B'-C loop potentially arising from at least two conformations are present in free 19A1 only, supporting the possibility that conformational selection is operative. Random-acceleration molecular dynamics and adaptive biasing force simulations illuminate ASD's binding pathway, predicting ASD capture in the lipid headgroups and a pathway to the active site shielded from solvent. Intriguingly, the predicted access channel in 19A1 aligns well with the steroid binding sites of other human sterol-oxidizing CYPs.

Interplay of disordered and ordered regions of a human small heat shock protein yields an ensemble of 'quasi-ordered' states.

Small heat shock proteins (sHSPs) are nature's 'first responders' to cellular stress, interacting with affected proteins to prevent their aggregation. Little is known about sHSP structure beyond its structured α-crystallin domain (ACD), which is flanked by disordered regions. In the human sHSP HSPB1, the disordered N-terminal region (NTR) represents nearly 50% of the sequence. Here, we present a hybrid approach involving NMR, hydrogen-deuterium exchange mass spectrometry, and modeling to provide the first residue-level characterization of the NTR. The results support a model in which multiple grooves on the ACD interact with specific NTR regions, creating an ensemble of 'quasi-ordered' NTR states that can give rise to the known heterogeneity and plasticity of HSPB1. Phosphorylation-dependent interactions inform a mechanism by which HSPB1 is activated under stress conditions. Additionally, we examine the effects of disease-associated NTR mutations on HSPB1 structure and dynamics, leveraging our emerging structural insights.

Hydrogen-deuterium exchange mass spectrometry of membrane proteins in lipid nanodiscs.

Hydrogen deuterium exchange mass spectrometry (H/DX MS) provides a quantitative comparison of the relative rates of exchange of amide protons for solvent deuterons. In turn, the rate of amide exchange depends on a complex combination of the stability of local secondary structure, solvent accessibility, and dynamics. H/DX MS has, therefore, been widely used to probe structure and function of soluble proteins, but its application to membrane proteins was limited previously to detergent solubilized samples. The large excess of lipids from model membranes, or from membrane fractions derived from in vivo samples, presents challenges with mass spectrometry. The lipid nanodisc platform, consisting of apolipoprotein A-derived membrane scaffold proteins, provides a native like membrane environment in which to capture analyte membrane proteins with a well defined, and low, ratio of lipid to protein. Membrane proteins in lipid nanodiscs are amenable to H/DX MS, and this is expected to lead to a rapid increase in the number of membrane proteins subjected to this analysis. Here we review the few literature examples of the application of H/DX MS to membrane proteins in nanodiscs. The incremental improvements in the experimental workflow of the H/DX MS are described and potential applications of this approach to study membrane proteins are described.

HspB1 and Hsc70 chaperones engage distinct tau species and have different inhibitory effects on amyloid formation.

The microtubule-associated protein tau forms insoluble, amyloid-type aggregates in various dementias, most notably Alzheimer's disease. Cellular chaperone proteins play important roles in maintaining protein solubility and preventing aggregation in the crowded cellular environment. Although tau is known to interact with numerous chaperones, it remains unclear how these chaperones function mechanistically to prevent tau aggregation and how chaperones from different classes compare in terms of mechanism. Here, we focused on the small heat shock protein HspB1 (also known as Hsp27) and the constitutive chaperone Hsc70 (also known as HspA8) and report how each chaperone interacts with tau to prevent its fibril formation. Using fluorescence and NMR spectroscopy, we show that the two chaperones inhibit tau fibril formation by distinct mechanisms. HspB1 delayed tau fibril formation by weakly interacting with early species in the aggregation process, whereas Hsc70 was highly efficient at preventing tau fibril elongation, possibly by capping the ends of tau fibrils. Both chaperones recognized aggregation-prone motifs within the microtubule-binding repeat region of tau. However, HspB1 binding remained transient in both aggregation-promoting and non-aggregating conditions, whereas Hsc70 binding was significantly tighter under aggregation-promoting conditions. These differences highlight the fact that chaperones from different families play distinct but complementary roles in the prevention of pathological protein aggregation.

pH-dependent structural modulation is conserved in the human small heat shock protein HSBP1.

The holdase activity and oligomeric propensity of human small heat shock proteins (sHSPs) are regulated by environmental factors. However, atomic-level details are lacking for the mechanisms by which stressors alter sHSP responses. We previously demonstrated that regulation of HSPB5 is mediated by a single conserved histidine over a physiologically relevant pH range of 6.5-7.5. Here, we demonstrate that HSPB1 responds to pH via a similar mechanism through pH-dependent structural changes that are induced via protonation of the structurally analogous histidine. Results presented here show that acquisition of a positive charge, either by protonation of His124 or its substitution by lysine, reduces the stability of the dimer interface of the α-crystallin domain, increases oligomeric size, and modestly increases chaperone activity. Our results suggest a conserved mechanism of pH-dependent structural regulation among the human sHSPs that possess the conserved histidine, although the functional consequences of the structural modulations vary for different sHSPs.

Control over overall shape and size in de novo designed proteins.

We recently described general principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions. The principles relate secondary structure patterns to tertiary packing motifs and enable design of different protein topologies. To achieve fine control over protein shape and size within a particular topology, we have extended the design rules by systematically analyzing the codependencies between the lengths and packing geometry of successive secondary structure elements and the backbone torsion angles of the loop linking them. We demonstrate the control afforded by the resulting extended rule set by designing a series of proteins with the same fold but considerable variation in secondary structure length, loop geometry, ß-strand registry, and overall shape. Solution NMR structures of four designed proteins for two different folds show that protein shape and size can be precisely controlled within a given protein fold. These extended design principles provide the foundation for custom design of protein structures performing desired functions.