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Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-theta, PKC-iota and PKC-betaII kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC-substrate complex has been determined. In the previously determined PKC-iota complex, residues 533-551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-iota kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0 A resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-iota are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-iota, which is different from the case of the prototypical AGC-branch kinase PKA.

One major goal of molecular recognition processes in biological systems is to understand how the chemical reaction is regulated by protein binding sites. For example, it is well known that the specific interaction of the cytochrome c-cytochrome c peroxidase complex plays an important role in longrange electron transfer (ET).' However, the noncovalent model systems in which fast ET occurs from donor to acceptor via intermolecular interaction remain limited.2 Here, we wish to report a nonmutagenic approach to the construction of the noncovalent ET model that is based on the complex of methyl viologen (2) and a myoglobin reconstituted with a modified zinc porphyrin (l*Zn) having an assembly of eight carboxylic acid groups as a specific interface. The present supramolecular system, designed to mimic the protein-protein complex of a reductoxidase, enables us to observe the fast singlet ET from zinc myoglobin to methyl viologen as a first example.Our strategy for the construction of rMb(l*Zn)-2 pairing is shown in Scheme 1.' Eight carboxylates attached to 6-and 7-propionates of mesoporphyrin IX were designed as an artificial recognition site for the cationic a~ceptor.~ Free base 1 was prepared by the coupling of mesoporphyrin and 5-aminoisophthalate condensed with two aspartic acids. The synthetic zinc porphyrin l*Zn was easily inserted into horse heart apomyoglobin by a routine m e t h~d .~-~ The characteristic visible absorption spectrum of the reconstituted protein was similar to that of reference protein reconstituted with mesoporphyrin zinc complex 3.231.Addition of 2 to the solution of rMb(l*Zn) brought about no changes in the visible absorption spectrum. In contrast, fluorescence quenching in rMb(l*Zn) clearly was observed upon addition of 2 in 10 mM phosphate buffer, pH 7.0, while reference reconstituted myoglobin, rMb(3*Zn), showed no (1) (a) Takano, T.; Dickerson, R. E. Throughout this paper, myoglobins reconstituted with l.Zn and 3*Zn are abbreviated as rMb( l-Zn) and rMb(3*Zn), respectively.(4) 5-[(tert-Butoxycarbonyl)amino]isophthaloyldiaspa~ic acid as a precursor interacts with 2 in pH 7.0 phosphate buffer solution. The affinity constant was determined to be (3.6 f 0.4) x IO2 M-' by 'H NMR measurement.(5) See supporting information for preparation details.(a) Teale, F. W. Bioclzim. Biophys. Acfu 1959,35,543. (b) Yonetani, T.; Asakura, T. J. B i d . Clzem.'1969, 244, 4580. (7) For examples of reconstituted semisynthetic myoglobin, cf.: (a) Suzuki, A.; Okuda, K.; Kawagoe, K.; Toi, H.; Aoyama, Y.; Ogoshi, H. Takimura, T.; Ohara, T.; Hitomi, Y.; Ogoshi, H. J. Chenz. Soc., Chem. Commun., in press. (e) Hayashi, T.; Hitomi, Y.; Suzuki, A.; Takimura, T.; Ogoshi, H. Chem. Lett. 1995, 91 1. 0002-7863/95/1517-11606$09.00/0 z I I,ll . a, C v) 2.5 i i .-> a, 1.5 L 0 3 c

The genetic code in a eukaryotic system has been expanded by the engineering of Escherichia coli tyrosyl-tRNA synthetase (TyrRS) with the Y37V and Q195C mutations (37V195C), which specifically recognize 3-iodo- l -tyrosine rather than l -tyrosine. In the present study, we determined the 3-iodo- l -tyrosine- and l -tyrosine-bound structures of the 37V195C mutant of the E. coli TyrRS catalytic domain at 2.0-Å resolution. The γ-methyl group of Val-37 and the sulfur atom of Cys-195 make van der Waals contacts with the iodine atom of 3-iodo- l -tyrosine. The Val-37 and Cys-195 side chains are rigidly fixed by the neighboring residues forming the hydrophobic core of the TyrRS. The major roles of the two mutations are different for the 3-iodo- l -tyrosine-selective recognition in the first step of the aminoacylation reaction (the amino acid activation step): the Y37V mutation eliminates the fatal steric repulsion with the iodine atom, and the Q195C mutation reduces the l -tyrosine misrecognition. The structure of the 37V195C mutant TyrRS complexed with an l -tyrosyladenylate analogue was also solved, indicating that the 3-iodo- l -tyrosine and l -tyrosine side chains are similarly discriminated in the second step (the aminoacyl transfer step). These results demonstrate that the amino acid-binding pocket on the 37V195C mutant is optimized for specific 3-iodo- l -tyrosine recognition.

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