A Hierarchical Graph for Nucleotide Binding Domain 2

One of the most prevalent inherited diseases is cystic fibrosis. This disease is caused by a mutation in a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is known to function as a chloride channel that regulates the viscosity of mucus that lines the ducts of a number of organs. Generally, most of the prevalent mutations of CFTR are located in one of two nucleotide binding domains, namely, the nucleotide binding domain 1 (NBD1). However, some mutations in nucleotide binding domain 2 (NBD2) can equally cause cystic fibrosis. In this work, a hierarchical graph is built for NBD2. Using this model for NBD2, we examine the consequence of single point mutations on NBD2. We collate the wildtype structure with eight of the most prevalent mutations and observe how the NBD2 is affected by each of these mutations

CFTR Gating II: Effects of Nucleotide Binding on the Stability of Open States

Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADP. To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of ΔR/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. ΔR/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of ∼100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Single-channel kinetic analysis for ΔR/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the NH(2)-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation

Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase.

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle

Dynamics of ABC Transporter P-glycoprotein in Three Conformational States.

We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to obtain a comprehensive view of transporter dynamics (85.8% sequence coverage) occurring throughout the multidrug efflux transporter P-glycoprotein (P-gp) in three distinct conformational states: predominantly inward-facing apo P-gp, pre-hydrolytic (E552Q/E1197Q) P-gp bound to Mg+2-ATP, and outward-facing P-gp bound to Mg+2-ADP-VO4-3. Nucleotide affinity was measured with bio-layer interferometry (BLI), which yielded kinetics data that fit a two Mg+2-ATP binding-site model. This model has one high affinity site (3.2 ± 0.3 µM) and one low affinity site (209 ± 25 µM). Comparison of deuterium incorporation profiles revealed asymmetry between the changes undergone at the critical interfaces where nucleotide binding domains (NBDs) contact intracellular helices (ICHs). In the pre-hydrolytic state, both interfaces between ICHs and NBDs decreased exchange to similar extents relative to inward-facing P-gp. In the outward-facing state, the ICH-NBD1 interface showed decreased exchange, while the ICH-NBD2 interface showed less of an effect. The extracellular loops (ECLs) showed reduced deuterium uptake in the pre-hydrolytic state, consistent with an occluded conformation. While in the outward-facing state, increased ECL exchange corresponding to EC domain opening was observed. These findings point toward asymmetry between both NBDs, and they suggest that pre-hydrolytic P-gp occupies an occluded conformation

Mining Disaggregase Sequence Space to Safely Counter TDP-43, FUS, and α-Synuclein Proteotoxicity.

Hsp104 is an AAA+ protein disaggregase, which can be potentiated via diverse mutations in its autoregulatory middle domain (MD) to mitigate toxic misfolding of TDP-43, FUS, and α-synuclein implicated in fatal neurodegenerative disorders. Problematically, potentiated MD variants can exhibit off-target toxicity. Here, we mine disaggregase sequence space to safely enhance Hsp104 activity via single mutations in nucleotide-binding domain 1 (NBD1) or NBD2. Like MD variants, NBD variants counter TDP-43, FUS, and α-synuclein toxicity and exhibit elevated ATPase and disaggregase activity. Unlike MD variants, non-toxic NBD1 and NBD2 variants emerge that rescue TDP-43, FUS, and α-synuclein toxicity. Potentiating substitutions alter NBD1 residues that contact ATP, ATP-binding residues, or the MD. Mutating the NBD2 protomer interface can also safely ameliorate Hsp104. Thus, we disambiguate allosteric regulation of Hsp104 by several tunable structural contacts, which can be engineered to spawn enhanced therapeutic disaggregases with minimal off-target toxicity

Mutations in the Arabidopsis Peroxisomal ABC Transporter COMATOSE Allow Differentiation between Multiple Functions In Planta: Insights from an Allelic Series

COMATOSE (CTS), the Arabidopsis homologue of human Adrenoleukodystrophy protein (ALDP), is required for import of substrates for peroxisomal β-oxidation. A new allelic series and a homology model based on the bacterial ABC transporter, Sav1866, provide novel insights into structure-function relations of ABC subfamily D proteins. In contrast to ALDP, where the majority of mutations result in protein absence from the peroxisomal membrane, all CTS mutants produced stable protein. Mutation of conserved residues in the Walker A and B motifs in CTS nucleotide-binding domain (NBD) 1 resulted in a null phenotype but had little effect in NBD2, indicating that the NBDs are functionally distinct in vivo. Two alleles containing mutations in NBD1 outside the Walker motifs (E617K and C631Y) exhibited resistance to auxin precursors 2,4-dichlorophenoxybutyric acid (2,4-DB) and indole butyric acid (IBA) but were wild type in all other tests. The homology model predicted that the transmission interfaces are domain-swapped in CTS, and the differential effects of mutations in the conserved "EAA motif" of coupling helix 2 supported this prediction, consistent with distinct roles for each NBD. Our findings demonstrate that CTS functions can be separated by mutagenesis and the structural model provides a framework for interpretation of phenotypic data

Nanomolar-potency 'co-potentiator' therapy for cystic fibrosis caused by a defined subset of minimal function CFTR mutants.

Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of 'co-potentiators' (combination-potentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC50 down to ~300 nM following initial structure-activity studies. Increased CFTR chloride conductance by up to 8-fold was observed when a co-potentiator (termed 'Class II potentiator') was used with a classical potentiator ('Class I potentiator') such as VX-770 or GLPG1837. To investigate the range of CFTR mutations benefitted by co-potentiators, 14 CF-associated CFTR mutations were studied in transfected cell models. Co-potentiator efficacy was found for CFTR missense, deletion and nonsense mutations in nucleotide binding domain-2 (NBD2), including W1282X, N1303K, c.3700A > G and Q1313X (with corrector for some mutations). In contrast, CFTR mutations G85E, R334W, R347P, V520F, R560T, A561E, M1101K and R1162X showed no co-potentiator activity, even with corrector. Co-potentiator efficacy was confirmed in primary human bronchial epithelial cell cultures generated from a N1303K homozygous CF subject. The Class II potentiators identified here may have clinical benefit for CF caused by mutations in the NBD2 domain of CFTR

Structural and Mechanistic Insights into the Yeast Disaggregase Hsp104

Hsp104 is a hexameric, AAA+ disaggregase from yeast, which couples ATP hydrolysis to remodeling diverse substrates ranging from amorphous aggregates to amyloid fibers. A mechanistic understanding of Hsp104\u27s substrate remodeling activities remains poorly defined. The hexamer undergoes large conformational changes upon ATP hydrolysis, but the details of these changes and how they are coupled to substrate remodeling are unresolved. The goals of this thesis were to elucidate low and high-resolution structural information about the Hsp104 hexamer and to discover new details of the mechanism of substrate remodeling. We used the in solution structural techniques small angle x-ray scattering and synchrotron x-ray footprinting, complemented by several biochemical assays, to elucidate novel roles for several Hsp104 domains, and to develop a comprehensive model for how the Hsp104 hexamer engages substrate and unleashes its remodeling capabilities. We discovered that the N-terminal domain (NTD) is involved in substrate binding, productive interactions with Hsp70, and an interface with nucleotide binding domain 1 (NBD1) and the middle domain (MD). We discovered a loop in NBD1 that may engage substrate in the ADP bound state to prevent premature substrate release, identified the region of the MD (helix 2) responsible and the mechanism of signal transmission between NBD1 and NBD2, and confirmed the validity of a hexameric model of the NBD2 domain. Hsp104 engages substrate in the ATP-bound state. We have found that in this state Hsp104 displays an increase in rigidity, which we propose as a pre-payment of the entropic cost of substrate binding. Initial substrate engagement in the NTD and NBD1, as well as Hsp70 interactions at the NTD:NBD1:MD interface, serve to `prime the pump\u27. These interactions result in large conformational changes of the MD, specifically in helix 2, which spans the entirety of the domain. These conformational changes increase MD dynamics, partially releasing MD:NBD2 contacts, and allow signal transmission between NBD1 and NBD2. As NBD2 responds to these signals, a positive feedback loop is created. Further nucleotide binding in NBD2 stimulates ATP hydrolysis in NBD1, and substrate is remodeled by iterative binding events and peristaltic motions of the Hsp104 hexamer channel

Rustless translation

ATP binding cassette proteins are a large and diverse family of molecular machines and include transmembrane transporter, chromosome maintenance and DNA repair proteins, and translation factors. However, the function of the ABCE1, the only member of subfamily E of ABC proteins, remained mysterious for over a decade, even though it is perhaps the most conserved ABC protein in eukaryotes and archaea. Recent results have now identified ABCE1 as the ribosome-recycling factor of eukaryotes and archaea. Thus, two iron-sulfur clusters - the hallmark feature of ABCE1 - help catalyze an integral step of the translational cycle at the core of the protein synthesis machinery

Subunit interactions influence the biochemical and biological properties of Hsp104

Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization. We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold, changing V(max) with little effect on K(m). In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, lowering V(max) with little effect on K(m). ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104. Thus, interactions between subunits influence the ATPase activity of Hsp104, play a vital role in its biological functions, and provide a mechanism for conditionally inactivating Hsp104 function in vivo