Structural insights into the mechanism of protein O-fucosylation

Protein O-fucosylation is an essential post-translational modification, involved in the folding of target proteins and in the role of these target proteins during embryonic development and adult tissue homeostasis, among other things. Two different enzymes are responsible for this modification, Protein O-fucosyltransferase 1 and 2 (POFUT1 and POFUT2, respectively). Both proteins have been characterised biologically and enzymatically but nothing is known at the molecular or structural level. Here we describe the first crystal structure of a catalytically functional POFUT1 in an apo-form and in complex with GDP-fucose and GDP. The enzyme belongs to the GT-B family and is not dependent on manganese for activity. GDP-fucose/GDP is localised in a conserved cavity connected to a large solvent exposed pocket, which we show is the binding site of epidermal growth factor (EGF) repeats in the extracellular domain of the Notch Receptor. Through both mutational and kinetic studies we have identified which residues are involved in binding and catalysis and have determined that the Arg240 residue is a key catalytic residue. We also propose a novel S(N)1-like catalytic mechanism with formation of an intimate ion pair, in which the glycosidic bond is cleaved before the nucleophilic attack; and theoretical calculations at a DFT (B3LYP/6-31+G(d,p) support this mechanism. Thus, the crystal structure together with our mutagenesis studies explain the molecular mechanism of POFUT1 and provide a new starting point for the design of functional inhibitors to this critical enzyme in the future.     

BRing it on: new insights into the mechanism of brassinosteroid action

Several recent breakthroughs have filled in key details of the brassinosteroid (BR) response. Identification of BAK1, a BRI1 interacting protein, the negative regulator BIN2, as well as direct targets of BIN2, BZR1 and BES1, provide a link between BR perception at the cell surface and regulation of gene expression in the nucleus. Global expression studies further defined the downstream events in this pathway, confirming the role of several factors acting in negative feedback regulation on BR levels. New links to the plant hormone, auxin, were also uncovered.     

Capping protein: new insights into mechanism and regulation

Temporal and spatial control of the actin cytoskeleton are crucial for a range of eukaryotic cellular processes. Capping protein (CP), a ubiquitous highly conserved heterodimer, tightly caps the barbed (fast-growing) end of the actin filament and is an important component in the assembly of various actin structures, including the dynamic branched filament network at the leading edge of motile cells. New research into the molecular mechanism of how CP interacts with the actin filament in vitro and the function of CP in vivo, including discoveries of novel interactions of CP with other proteins, has greatly enhanced our understanding of the role of CP in regulating the actin cytoskeleton.     

When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124I

Using a combination of single molecule and bulk solution measurements, we have examined the DNA translocation activity of a helicase, the Type I restriction modification enzyme EcoR124I. We find that EcoR124I can translocate past covalent interstrand crosslinks, inconsistent with an obligatory unwinding mechanism. Instead, translocation of the intact dsDNA occurs principally via contacts to the sugar-phosphate backbone and bases of the 3'-5' strand; contacts to the 5'-3' strand are not essential for motion but do play a key role in stabilising the motor on the DNA. A model for dsDNA translocation is presented that could be applicable to a wide range of other enzyme complexes that are also labelled as helicases but which do not have actual unwinding activity.     

Novel insights into the mechanism of chaperone-assisted protein disaggregation

Cell survival under severe thermal stress requires the activity of a bi-chaperone system, consisting of the ring-forming AAA+ chaperone ClpB (Hsp104) and the DnaK (Hsp70) chaperone system, which acts to solubilize and reactivate aggregated proteins. Recent studies have provided novel insight into the mechanism of protein disaggregation, demonstrating that ClpB/Hsp104 extracts unfolded polypeptides from an aggregate by threading them through its central pore. This translocation activity is necessary but not sufficient for aggregate solubilization. In addition, the middle (M) domain of ClpB and the DnaK system have essential roles, possibly by providing an unfolding force, which facilitates the extraction of misfolded proteins from aggregates.     

Structural insights into the protein splicing mechanism of PI-SceI

PI-SceI is a member of a class of proteins (inteins) that excise themselves from a precursor protein and in the process ligate the flanking protein sequences (exteins). We report here the 2.1-A resolution crystal structure of a PI-SceI miniprecursor (VMA29) containing 10 N-terminal extein residues and 4 C-terminal extein residues. Mutations at the N- and C-terminal splicing junctions, blocking in vivo protein splicing, allowed the miniprecursor to be purified and crystallized. The structure reveals both the N- and C-terminal scissile peptide bonds to be in distorted trans conformations (tau approximately 100 degrees ). Modeling of the wild-type PI-SceI based on the VMA29 structure indicates a large conformational change (movement of >9 A) must occur to allow transesterification to be completed. A zinc atom was discovered at the C-terminal splicing junction. Residues Cys(455), His(453), and Glu(80) along with a water molecule (Wat(53)) chelate the zinc atom. The crystal structure of VMA29 has captured the intein in its pre-spliced state.     

Cryo-EM analysis reveals new insights into the mechanism of action of pyruvate carboxylase

Pyruvate carboxylase (PC) is a conserved multifunctional enzyme linked to important metabolic diseases. PC homotetramer is arranged in two layers with two opposing monomers per layer. Cryo-EM explores the conformational variability of PC in the presence of different substrates. The results demonstrate that the biotin-carboxyl carrier protein (BCCP) domain localizes near the biotin carboxylase (BC) domain of its own monomer and travels to the carboxyltransferase (CT) domain of the opposite monomer. All density maps show noticeable conformational differences between layers, mainly for the BCCP and BC domains. This asymmetry may be indicative of a coordination mechanism where monomers from different layers catalyze the BC and CT reactions consecutively. A conformational change of the PC tetramerization (PT) domain suggests a new functional role in communication. A long-range communication pathway between subunits in different layers, via interacting PT-PT and BC-BC domains, may be responsible for the cooperativity of PC from Staphylococcus aureus.     

New insights into motor cortex

An exciting new experiment on the motor cortex of monkeys, by Shenoy and colleagues, begins to elucidate how the neuronal ensemble travels in a systematic fashion through state space. This trajectory through state space may help to explain how the motor cortex sets up and then triggers arm movements.     

Molecular mechanism of divalent-metal-induced activation of NS3 helicase and insights into Zika virus inhibitor design

Zika virus has attracted increasing attention because of its potential for causing human neural disorders, including microcephaly in infants and Guillain–Barré syndrome. Its NS3 helicase domain plays critical roles in NTP-dependent RNA unwinding and translocation during viral replication. Our structural analysis revealed a pre-activation state of NS3 helicase in complex with GTPγS, in which the triphosphate adopts a compact conformation in the absence of any divalent metal ions. In contrast, in the presence of a divalent cation, GTPγS adopts an extended conformation, and the Walker A motif undergoes substantial conformational changes. Both features contribute to more extensive interactions between the GTPγS and the enzyme. Thus, this study provides structural evidence on the allosteric modulation of MgNTP²⁻ on the NS3 helicase activity. Furthermore, the compact conformation of inhibitory NTP identified in this study provides precise information for the rational drug design of small molecule inhibitors for the treatment of ZIKV infection.     

The yeast prion protein Ure2: insights into the mechanism of amyloid formation

Ure2, a regulator of nitrogen metabolism, is the protein determinant of the [URE3] prion state in Saccharomyces cerevisiae. Upon conversion into the prion form, Ure2 undergoes a heritable conformational change to an amyloid-like aggregated state and loses its regulatory function. A number of molecular chaperones have been found to affect the prion properties of Ure2. The studies carried out in our laboratory have been aimed at elucidating the structure of Ure2 fibrils, the mechanism of amyloid formation and the effect of chaperones on the fibril formation of Ure2.     

Structural insights into the autoactivation mechanism of p21-activated protein kinase

p21-activated kinases (PAKs) play an important role in diverse cellular processes. Full activation of PAKs requires autophosphorylation of a critical threonine/serine located in the activation loop of the kinase domain. Here we report crystal structures of the phosphorylated and unphosphorylated PAK1 kinase domain. The phosphorylated PAK1 kinase domain has a conformation typical of all active protein kinases. Interestingly, the structure of the unphosphorylated PAK1 kinase domain reveals an unusual dimeric arrangement expected in an authentic enzyme-substrate complex, in which the activation loop of the putative "substrate" is projected into the active site of the "enzyme." The enzyme is bound to AMP-PNP and has an active conformation, whereas the substrate is empty and adopts an inactive conformation. Thus, the structure of the asymmetric homodimer mimics a trans-autophosphorylation complex, and suggests that unphosphorylated PAK1 could dynamically adopt both the active and inactive conformations in solution.     

New insights into maitotoxin action

Maitotoxin (3 ng/mol) induced a massive uptake of 45Ca2+ into BC3H1 cells. This effect exhibits a lag phase of 3 min. Inositol diphosphate formation occurred concomittantly with the 45Ca2+ uptake but inositol monophosphate formation was found only after a 5-min delay following toxin addition. Maitotoxin-induced 45Ca2+ influxes could not be blocked by either 1 microM verapamil, 1 microM nifedipine or 1 mM La3+ but was blocked by Zn2+ (IC50 = 41 microM). In addition to inositol phosphate formation and 45Ca2+ uptake, maitotoxin stimulated a large uptake of Na+ and a great loss of K+ in BC3H1 cells. In the absence of Ca2+ (1 mM EGTA) none of the four maitotoxin effects could be detected. After restoration of Ca2+, the maitotoxin effects reappeared even when the toxin itself was no longer present. The divalent cation, Co2+ (1 mM), inhibited ion movements induced by maitotoxin and also digitonin (8.1 microM). The toxin action showed a very pronounced pH dependence. At low pH, maitotoxin was inactive. The dose-response curves for H+ ion inhibition of maitotoxin-induced Ca2+ uptake showed a shift to the right when determined in the absence of HCO3- and HCO3-/Cl- ions. It was concluded that the primary action of maitotoxin in BC3H1 cells was a pore-forming or channel-forming activity of a non-classical type. Some properties of maitotoxin resemble those of alpha-latrotoxin, others those of pore-forming agents such as melittin or alpha-toxin of Staphylococcus aureus.     

Structural and functional insights into the human Upf1 helicase core

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway that recognizes and degrades aberrant mRNAs containing premature stop codons. A critical protein in NMD is Upf1p, which belongs to the helicase super family 1 (SF1), and is thought to utilize the energy of ATP hydrolysis to promote transitions in the structure of RNA or RNA-protein complexes. The crystal structure of the catalytic core of human Upf1p determined in three states (phosphate-, AMPPNP- and ADP-bound forms) reveals an overall structure containing two RecA-like domains with two additional domains protruding from the N-terminal RecA-like domain. Structural comparison combined with mutational analysis identifies a likely single-stranded RNA (ssRNA)-binding channel, and a cycle of conformational change coupled to ATP binding and hydrolysis. These conformational changes alter the likely ssRNA-binding channel in a manner that can explain how ATP binding destabilizes ssRNA binding to Upf1p.     

Structural insights into the SNARE mechanism

Eukaryotic cells distribute materials among intracellular organelles and secrete into the extracellular space through cargo-loaded vesicles. A concluding step during vesicular transport is the fusion of a transport vesicle with a target membrane. SNARE proteins are essential for all vesicular fusion steps, thus they possibly comprise a conserved membrane fusion machinery. According to the "zipper" model, they assemble into stable membrane-bridging complexes that gradually bring membranes in juxtaposition. Hence, complex formation may provide the necessary energy for overcoming the repulsive forces between two membranes. During the last years, detailed structural and functional studies have extended the evidence that SNAREs are mostly in accord with the zipper model. Nevertheless, it remains unclear whether SNARE assembly between membranes directly leads to the merger of lipid bilayers.     

Crystal structure of a cAMP-dependent protein kinase mutant at 1.26A: new insights into the catalytic mechanism

The catalytic subunit of cAMP-dependent protein kinase has served as a paradigm for the entire kinase family. In the course of studying the structure-function relationship of the P+1 loop (Leu198-Leu205) of the kinase, we have solved the crystal structure of the Tyr204 to Ala mutant in complexes with Mg.ATP and an inhibitory peptide at 1.26A, with overall structure very similar to that of the wild-type protein. However, at the nucleotide binding site, ATP was found largely hydrolyzed, with the products ADP-PO(4) retained in the structure. High-resolution refinement suggests that 26% of the molecules contain the intact ATP, whereas 74% have the hydrolyzed products. The observation of the substrate and product states in the same structure adds significant information to our understanding of the phosphoryl transfer process. Structural examination of the mutation site substantiates and extends the emerging concept that the hydrophobic core in the large lobe of the kinase might serve as a stable platform for anchoring key segments involved in catalysis. We propose that Tyr204 is critical for anchoring the P+1 loop to the core. Further analysis has highlighted two major connections between the P+1 loop and the catalytic loop (Arg165-Asn171). One emphasizes the hydrophobic packing of Tyr204 and Leu167 mediated through residues from the alphaF-helix, recently recognized as a signal integration motif, which together with the alphaE-helix forms the center of the hydrophobic core network. The other connection is mediated by the hydrogen bond interaction between Thr201 and Asp166, in a substrate-dependent manner. We speculate that the latter interaction may be important for the kinase to sense the presence of substrate and prepare itself for the catalytic reaction. Thus, the P+1 loop is not merely involved in substrate binding; it mediates the communication between substrate and catalytic residues.     

FUScinating insights into motor neuron degeneration

Point mutations in FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease—but do they do that by a loss of the protein's normal function, or by endowing it with novel toxic functions, or both? In this issue of The EMBO Journal, Scekic‐Zahirovic et al ( 2016 ) report that mutant FUS, but not the complete loss of FUS, triggers motor neuron degeneration in mice, arguing for a toxic gain‐of‐function mechanism.     

Mental motor imagery: a window into the representational stages of action

The physiological basis of mental states can be effectively studied by combining cognitive psychology with human neuroscience. Recent research has employed mental motor imagery in normal and brain-damaged subjects to decipher the content and the structure of covert processes preceding the execution of action. The mapping of brain activity during motor imagery discloses a pattern of activation similar to that of an executed action.