BgK is a K؉ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 ؎ 1.0 Å. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K ؉ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the threedimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.Functional properties of proteins are frequently associated with a small number of important residues. For example, enzyme activities depend on a few residues that are essential for catalysis. Also, protein-protein recognition processes have been predicted (1) and recently demonstrated (2) to be energetically driven by a small proportion of the residues forming the contacting areas in protein-protein complexes, as identified by x-ray studies (3, 4). Among the proteins whose major functions require protein-protein interactions are animal toxins, which bind to various molecular targets, such as receptors or ion channels, using a small number of binding residues (5-8). As has been shown for enzymes (9), toxins with different architectures are capable of exerting similar functions (10). However, in contrast to enzymes, the molecular basis associated with the conservation of the function in structurally unrelated toxins remains unknown. In this paper, we show that two families of animal toxins with different folding patterns but a comparable capacity to bind to potassium channels include similar functional diads, composed of a critical lysine and an aromatic amino acid separated from each other by 6.6 Ϯ 1.0 Å. MATERIALS AND METHODS Synthesis of Toxin and Mutants-The amino acid sequence of BgK 1 was proposed a few years ago (11). However, chemical synthesis attempts, based on these data, systematically failed. The proposed amino acid sequence was therefore questioned, re-examined, and ultimately corrected.2 The revised amino acid sequence of BgK from Bunodosoma granulifera is: VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTC-ELC. BgK and each alanine-substituted analog were synthesized by solid phase synthesis using an Applied Biosystems model 431A peptide synthesizer, starting from 0.1 mmol of Rink-resin (4-(2Ј,4Ј-dimethoxyphenylhydroxymethylphenoxy resin; 0.48 mmol/g). A 10-fold excess (1 mmol) of Fmoc (N-(9-fluorenyl)methoxycarbonyl)-protected amino acid was used and coupled in N-methylpyrrolidone in the presence of N,NЈ-dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The following side chain protections wer...

We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V-X (1-5)-Y-X (1-5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria.

Using synchrotron radiation as an ultrabright infrared source, we have been able to map the distributions of functional groups such as proteins, lipids, and nucleic acids inside a single living cell with a spatial resolution of a few microns. In particular, we have mapped the changes in the lipid and protein distributions in both the final stages of cell division and also during necrosis.

The DNA‐binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence‐specific DNA‐binding. Both are assumed to contain helix‐turn‐helix (HTH)‐related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147‐154]. We propose that the disordered region folds into a ‘recognition’ helix and generates a full HTH‐related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N‐ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA‐binding. Specific DNA‐binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA‐induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA‐binding on or off by controlling the DNA‐induced conformational change in R2.