Mitofusins (Mfn) promote fusion-mediated mitochondrial content exchange and subcellular trafficking. Mutations in Mfn2 cause neurodegenerative Charcot Marie Tooth disease type 2A (CMT2A). Here we show that Mfn2 activity can be determined by Met376 and His380 interactions with Asp725 and Leu727 and controlled by PINK1 kinase-mediated phosphorylation of adjacent Mfn2 Ser378. Small molecule mimics of the peptide-peptide interface of Mfn2 disrupted this interaction, allosterically activating Mfn2 and promoting mitochondrial fusion. These first-in-class mitofusin agonists overcame dominant mitochondrial defects provoked in cultured neurons by CMT2A mutants Mfn2 Arg94Gln and Thr105Met, as evidenced by improved mitochondrial dysmotility, fragmentation, depolarization, and clumping. A mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of Mfn2 Thr105Met mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism/trafficking.

The structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple-stranded antiparallel @-sheet and a single helix covering one face of the 0-sheet. The cysteine side chains connecting the &sheet and the helix form the core of the molecule. One edge of the 0-sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface-exposed residues, ArgZ4, Lys", and Arg3' of the &sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K + channel. The sequential and spatial locations of ArgZ4 and Arg3' are not conserved among the homologous toxins. Knowledge on the details of the channel-binding sites of agitoxin 2 formed a basis for site-directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel-toxin interactions.Keywords: agitoxin; K+ channel; NMR; protein; scorpion toxin Scorpion venoms contain a large number of closely related peptide inhibitors of voltage-and Ca'+-dependent K+ channels. These peptides interact directly with the central catalytic region of K + channels-the ion conduction pore. They occlude the pore and prevent ion conduction by binding with one-to-one stoichiometry to the extracellular entryway of the channel (Miller et al., 1985;MacKinnon & Miller, 1988, 1989Miller, 1988;Giangiacomo et al., 1992). Knowledge of the precise structure and dimensions of scorpion toxins has been most valuable for probing the structure of K+ channels. The toxin molecules A.M. Krezel and C. Kasibhatla contributed equally to this work. Abbreviations: AgTxl, AgTx2, AgTx3, agitoxin variants 1, 2, and 3; ChTx, charybdotoxin; Lq2, Leiurus quinquestriatus variant 2 toxin; IbTx, iberiotoxin; KTx, kaliotoxin; MgTx, margatoxin; NxTx, noxiustoxin; Stv3, scorpion toxin variant-3; NOESY, NOE spectroscopy; HSQC, heteronuclear single-quantum coherence; TOCSY, total correlation spectroscopy; DQF-COSY, double quantum filtered correlated spectroscopy; 2D, two dimensional; 3D, three dimensional; HNHA and HNHB, ISN-separated quantitative J-correlation experiments; RMSD, RMS deviation. are used as calipers to measure the dimensions and geometry of the ion conduction pore regions of K+ channels.The known scorpion toxin K + channel inhibitors are 37-39 amino acids in length and contain six cysteine residues. They fall into three subclasses (Garcia et al., 1994). Within each subclass, toxins show greater than 70% amino a...

The structure of the leech protein decorsin, a potent 39-residue antagonist of glycoprotein IIb-IIIa and inhibitor of platelet aggregation, was determined by nuclear magnetic resonance. In contrast to other disintegrins, the Arg-Gly-Asp (RGD)-containing region of decorsin is well defined. The three-dimensional structure of decorsin is similar to that of hirudin, an anticoagulant leech protein that potently inhibits thrombin. Amino acid sequence comparisons suggest that ornatin, another glycoprotein IIb-IIIa antagonist, and antistasin, a potent Factor Xa inhibitor and anticoagulant found in leeches, share the same structural motif. Although decorsin, hirudin, and antistasin all affect the blood clotting process and appear similar in structure, their mechanisms of action and epitopes important for binding to their respective targets are distinct.

The crystal structure of the heterodimer formed by the basic leucine zipper (bZIP) domains of activating transcription factor-4 (ATF4) and CCAAT box/enhancerbinding protein ␤ (C/EBP␤), from two different bZIP transcription factor families, has been determined and refined to 2.6 Å. The structure shows that the heterodimer forms an asymmetric coiled-coil. Even in the absence of DNA, the basic region of ATF4 forms a continuous ␣-helix, but the basic region of C/EBP␤ is disordered. Proteolysis, electrophoretic mobility shift assay, circular dichroism, and NMR analyses indicated that (i) the bZIP domain of ATF4 is a disordered monomer and forms a homodimer upon binding to the DNA target; (ii) the bZIP domain of ATF4 forms a heterodimer with the bZIP domain of C/EBP␤ that binds the cAMP response element, but not CCAAT box DNA, with high affinity; and (iii) the basic region of ATF4 has a higher ␣-helical propensity than that of C/EBP␤. These results suggest that the degree of ordering of the basic region and the fork and the dimerization properties of the leucine zipper combine to distinguish the structurally similar bZIP domains of ATF4 and C/EBP␤ with respect to DNA target sequence. This study provides insight into the mechanism by which dimeric bZIP transcription factors discriminate between closely related but distinct DNA targets.Protein-protein interactions are involved in a number of different cellular processes, including the regulation of transcription. Many, if not most, transcription factors, including the bZIP 1 proteins, bind their cognate DNA elements as dimers. Dimerization between transcription factors from the same or different families can generate considerable functional diversity using only a relatively small number of components. By binding with different specificity to distinct DNA sites within promoters, heterodimeric transcription factors facilitate the positioning and orientation of proteins on DNA, thus providing distinct surfaces for interaction with other transcriptional regulatory proteins bound to adjacent DNA sites. Among coiled-coil proteins, bZIP family members form dimers via their characteristic leucine zipper helices, which consist of seven-residue (heptad) repeats, (abcdefg) n . Dimer formation by bZIP proteins is selective: some form only homodimers, some form only heterodimers, and others form both homo-and heterodimers. Hydrophobic interactions by leucine and other hydrophobic amino acids in positions 3 and 4 in the helix (the a and d positions of the helical wheel) form the hydrophobic core of the bZIP dimer. Leucine residues occupy most of the d positions in the leucine zipper helix. At the same time, hydrophilic amino acids located just outside of the hydrophobic core (the e and g positions of the helical wheel) also participate in stabilizing the dimer. A highly conserved asparagine amino acid in the a position in the middle of the leucine zipper helix has been shown to destabilize oligomer formation in several bZIP dimers (1). Thus, depending on the precise distribution of h...

Vitamin K antagonists are widely used anticoagulants targeting vitamin K epoxide reductases (VKOR), a family of integral membrane enzymes. To elucidate their catalytic cycle and inhibitory mechanism, here we report eleven x-ray crystal structures of human VKOR and pufferfish VKOR-like with substrates and antagonists in different redox states. Substrates entering the active site in a partially oxidized state form a cysteine adduct that induces an open-to-closed conformational change, triggering reduction. Binding and catalysis is facilitated by hydrogen-bonding interactions in a hydrophobic pocket. The antagonists bind specifically to the same hydrogen-bonding residues and induce a similar closed conformation. Thus, vitamin K antagonists act through mimicking the key interactions and conformational changes required for the VKOR catalytic cycle.