Two structures of fructose 6-phosphate aldolase, the wild-type and an engineered variant containing five active-site mutations, have been solved by cryoelectron microscopy (cryo-EM). The engineered variant affords production of aldols from aryl substituted ketones and aldehydes. This structure was solved to a resolution of 3.1 A & ring; and contains the critical iminium reaction intermediate trapped in the active site. This provides new information that rationalizes the acquired substrate scope and aids in formulating hypotheses of the chemical mechanism. A Tyr residue (Y131) is positioned for a role as catalytic acid/base during the aldol reaction and the different structures demonstrate mobility of this amino acid residue. Further engineering of this fructose 6-phosphate aldolase (FSA) variant, guided by this new structure, identified additional FSA variants that display improved carboligation activities with 2-hydroxyacetophenone and phenylacetaldehyde.

Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid–extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2’s MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought.

Huntington's disease (HD) is a late-onset neurological disorder for which therapeutics are not available. Its key pathological mechanism involves the proteolysis of polyglutamine-expanded (polyQ-expanded) mutant huntingtin (mHTT), which generates N-terminal fragments containing polyQ, a key contributor to HD pathogenesis. Interestingly, a naturally occurring spliced form of HTT mRNA with truncated exon 12 encodes an HTT (HTT & UDelta;12) with a deletion near the caspase-6 cleavage site. In this study, we used a multidisciplinary approach to characterize the therapeutic potential of targeting HTT exon 12. We show that HTT & UDelta;12 was resistant to caspase-6 cleavage in both cell-free and tissue lysate assays. However, HTT & UDelta;12 retained overall biochemical and structural properties similar to those of wt-HTT. We generated mice in which HTT exon 12 was truncated and found that the canonical exon 12 was dispensable for the main physiological functions of HTT, including embryonic development and intracellular trafficking. Finally, we pharmacologically induced HTT & UDelta;12 using the antisense oligonucleotide (ASO) QRX-704. QRX-704 showed predictable pharmacology and efficient biodistribution. In addition, it was stable for several months and inhibited pathogenic proteolysis. Furthermore, QRX-704 treatments resulted in a reduction of HTT aggregation and an increase in dendritic spine count. Thus, ASO-induced HTT exon 12 splice switching from HTT may provide an alternative therapeutic strategy for HD.

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed biochemical and biophysical analysis on CHD7 chromatin remodeler and uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome and contains a high conserved arginine stretch, which is reminiscent of arginine anchor. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. Furthermore, smFRET analysis shows the mutations in the N-CRD causes the defects in remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LOin vitro.

Natural products have played a dominant role in the discovery of lead compounds for the development of drugs aimed at the treatment of human diseases. This electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)—based study demonstrates that dietary antioxidants, isolated components from the stigmas of saffron (Crocus sativus L.) may be effective in inhibiting Aβ fibrillogenesis, a neuropathological hallmark of Alzheimer’s Disease (AD). This study reveals a substantial alteration in the monomer/oligomer distribution of Aβ_1-40, concomitant with re-direction of fibril formation, induced by the natural product interaction. These alterations on the Aβ_1-40 aggregation pathway are most prominent for trans-crocin-4 (TC4). Use of ESI-IMS-MS, electron microscopy alongside Thioflavin-T kinetics, and the interpretation of 3-dimensional Driftscope plots indicate a correlation of these monomer/oligomer distribution changes with alterations to Aβ_1-40 amyloid formation. The latter could prove instrumental in the development of novel aggregation inhibitors for the prevention, or treatment of AD.

Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide 17. The fusion protein NT*-17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that 17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, 17 adopts a -sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.

Increased intracellular water content defined as cytotoxic brain tissue edema is a serious secondary clinical complication to traumatic brain injury (TBI) and stroke and without knowledge to the etiology. Recently a hypothesis to the nervous tissue edema was presented suggesting that external dynamic and internal mechanical static impact forces caused protein unfolding resulting in an increased brain tissue water content. The hypothesis was confirmed by computer simulation tests. In this laboratory study we further evaluated the hypothesis by using the mature protein laminin LN521 upon the effects of both dynamic as well as static impact forces, respectively. Laminin was chosen as a representative protein due to it´s general and abundance presence in the cells. The treated laminin solutions were then analyzed with denatured electrophoresis and Electron Microscopy showing aggregation and fragmentation of the laminin structures. The present results confirm earlier hypothesis and computer simulation suggesting for the first time that dynamic impact force in an accident and increased mechanical static force in stroke unfold mature proteins having the potential to increase the intracellular water content defined as cytotoxic brain tissue edema. The clinical condition resembles the phenomenon when elasmobranchs including white sharks prevent their cells from too high hydrostatic pressure in the deep sea. Thus, the present laboratory study results and knowledge from marine physics may be considered to improve the clinical treatment and outcome of TBI and stroke patients.

Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.

Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.