Recruitment interactions can override catalytic interactions in determining the functional identity of a protein kinase.

The yeast Saccharomyces cerevisae has four distinct mitogen-activated protein kinase kinases (MAPKKs), each of which has a distinct functional identity characterized by communication with specific upstream and downstream partners to form distinct functional pathways. These four kinases belong to one family, sharing closely related catalytic domains. How have these four related kinases diverged to take on four distinct functional roles? The specificity of an enzyme for a particular substrate is often thought to reside in differences in the catalytic domain. However, many kinases, including MAPKKs, have modular interaction domains and motifs that have been shown to play an important role in determining the specificity of kinases through recruitment to specific partners and complexes. Here we probe the relative importance of catalytic domain interactions versus recruitment interactions in defining the functional identity of MAPKKs by asking whether we can use recruitment interactions to force other MAPKK catalytic domains to play the functional role of the mating MAPKK, Ste7. We find that two alternative MAPKKs, Pbs2 and Mkk2, can be forced to functionally replace the mating MAPKK Ste7, but only if the proper set of recruitment interactions are grafted onto their catalytic domains. These results show that within a family of kinases, recruitment interactions can play a dominant role in defining functional identity, and is consistent with a model in which new kinase functions can arise through recombination of existing catalytic domains with new interaction modules.

Rapid diversification of cell signaling phenotypes by modular domain recombination.

Cell signaling proteins are often modular, containing distinct catalytic and regulatory domains. Recombination of such biological modules has been proposed to be a major source of evolutionary innovation. We systematically analyzed the phenotypic diversity of a signaling response that results from domain recombination by using 11 proteins in the yeast mating pathway to construct a library of 66 chimeric domain recombinants. Domain recombination resulted in greater diversity in pathway response dynamics than did duplication of genes, of single domains, or of two unlinked domains. Domain recombination also led to changes in mating phenotype, including recombinants with increased mating efficiency over the wild type. Thus, novel linkages between preexisting domains may have a major role in the evolution of protein networks and novel phenotypic behaviors.

Structure and functional role of dynein's microtubule-binding domain.

Dynein motors move various cargos along microtubules within the cytoplasm and power the beating of cilia and flagella. An unusual feature of dynein is that its microtubule-binding domain (MTBD) is separated from its ring-shaped AAA+ adenosine triphosphatase (ATPase) domain by a 15-nanometer coiled-coil stalk. We report the crystal structure of the mouse cytoplasmic dynein MTBD and a portion of the coiled coil, which supports a mechanism by which the ATPase domain and MTBD may communicate through a shift in the heptad registry of the coiled coil. Surprisingly, functional data suggest that the MTBD, and not the ATPase domain, is the main determinant of the direction of dynein motility.

The affinity of the dynein microtubule-binding domain is modulated by the conformation of its coiled-coil stalk.

The microtubule-binding domain (MTBD) of dynein is separated from the AAA (ATPase with any other activity) core of the motor by an approximately 15-nm stalk that is predicted to consist of an antiparallel coiled coil. However, the structure of this coiled coil and the mechanism it uses to mediate communication between the MTBD and ATP-binding core are unknown. Here, we sought to identify the optimal alignment between the hydrophobic heptad repeats in the two strands of the stalk coiled coil. To do this, we fused the MTBD of mouse cytoplasmic dynein, together with 12-36 residues of its stalk, onto a stable coiled-coil base provided by Thermus thermophilus seryl-tRNA synthetase and tested these chimeric constructs for microtubule binding in vitro. The results identified one alignment that yielded a protein displaying high affinity for microtubules (2.2 microM). The effects of mutations applied to the MTBD of this construct paralleled those previously reported (Koonce, M. P., and Tikhonenko, I. (2000) Mol. Biol. Cell 11, 523-529) for an intact dynein motor unit in the absence of ATP, suggesting that it resembles the tight binding state of native intact dynein. All other alignments showed at least 10-fold lower affinity for microtubules with the exception of one, which had an intermediate affinity. Based on these results and on amino acid sequence analysis, we hypothesize that dynein utilizes small amounts of sliding displacement between the two strands of its coiled-coil stalk as a means of communication between the AAA core of the motor and the MTBD during the mechanochemical cycle.

Expression and genomic analysis of midasin, a novel and highly conserved AAA protein distantly related to dynein.

BACKGROUND: The largest open reading frame in the Saccharomyces genome encodes midasin (MDN1p, YLR106p), an AAA ATPase of 560 kDa that is essential for cell viability. Orthologs of midasin have been identified in the genome projects for Drosophila, Arabidopsis, and Schizosaccharomyces pombe. RESULTS: Midasin is present as a single-copy gene encoding a well-conserved protein of approximately 600 kDa in all eukaryotes for which data are available. In humans, the gene maps to 6q15 and encodes a predicted protein of 5596 residues (632 kDa). Sequence alignments of midasin from humans, yeast, Giardia and Encephalitozoon indicate that its domain structure comprises an N-terminal domain (35 kDa), followed by an AAA domain containing six tandem AAA protomers (approximately 30 kDa each), a linker domain (260 kDa), an acidic domain (approximately 70 kDa) containing 35-40% aspartate and glutamate, and a carboxy-terminal M-domain (30 kDa) that possesses MIDAS sequence motifs and is homologous to the I-domain of integrins. Expression of hemagglutamin-tagged midasin in yeast demonstrates a polypeptide of the anticipated size that is localized principally in the nucleus. CONCLUSIONS: The highly conserved structure of midasin in eukaryotes, taken in conjunction with its nuclear localization in yeast, suggests that midasin may function as a nuclear chaperone and be involved in the assembly/disassembly of macromolecular complexes in the nucleus. The AAA domain of midasin is evolutionarily related to that of dynein, but it appears to lack a microtubule-binding site.